Solid-State Nanopore Real-Time Assay for Monitoring Cas9 Endonuclease ReactivityClick to copy article linkArticle link copied!
- Chalmers C. C. Chau*Chalmers C. C. Chau*Email: [email protected]Bragg Centre for Materials Research, School of Electronic and Electrical Engineering, University of Leeds, Leeds LS2 9JT, U.K.More by Chalmers C. C. Chau
- Nicole E. WeckmanNicole E. WeckmanInstitute for Studies in Transdisciplinary Engineering Education & Practice, Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto M5S 1A4, CanadaMore by Nicole E. Weckman
- Emma E. ThomsonEmma E. ThomsonSchool of Bioscience, University of Sheffield, Sheffield S10 2TN, U.K.More by Emma E. Thomson
- Paolo ActisPaolo ActisBragg Centre for Materials Research, School of Electronic and Electrical Engineering, University of Leeds, Leeds LS2 9JT, U.K.More by Paolo Actis
Abstract
The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.
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Introduction
Results and Discussion
Real-Time Quantitative Nanopore Analysis System for Endonuclease
Figure 1
Figure 1. Generation of the restriction site containing 3 kbp dsDNA. (A) Schematic illustration of the generation of the restriction site containing 3 kbp dsDNA (RS-dsDNA). (B) Agarose gel electrophoresis analysis of the undigested RS-dsDNA and the digested RS-dsDNA; the 3 kbp original fragment was digested into 1.5 kbp dsDNA.
buffer name | composition |
---|---|
buffer 3.1 | 50 mM Tris–HCl, 10 mM MgCl2, 100 mM NaCl, 100 μg/mL BSA at pH 7.9 |
buffer 4 | 20 mM Tris-OAc, 10 mM MgOAc, 50 mM KOAc, 1 mM DTT, pH 7.9 |
buffer 2.1 | 50 mM Tris–HCl, 10 mM MgCl2, 50 mM NaCl, 100 μg/mL BSA, pH 7.9 |
CutSmart | 20 mM Tris-OAc, 10 mM MgOAc, 50 mM KOAc, 100 μg/mL BSA, pH 7.9 |
Figure 2
Figure 2. Restriction enzyme SwaI cleavage activities. (A) Schematic illustration of the glass nanopore detection setup. The cis chamber of the nanopore is filled with the restriction enzyme SwaI, the RS-dsDNA, the trans chamber is composed of a polymer electrolyte mixture (0.1 M KCl, 50% (w/v) PEG 35K). Application of a negative voltage causes the dsDNA to migrate from the cis to the trans chamber. The RS-dsDNA was mixed with the SwaI restriction enzyme and digestion buffer to a final concentration of 9.24 nM RS-dsDNA, 5 units of enzyme, and 1× digestion buffer. (B) 20 random translocation event peaks plotted as overlay. The cis chamber of the glass nanopore was filled with 10 nM RS-dsDNA diluted with buffer 3.1 (Table 1) containing 5 units of SwaI enzyme. (C) Population distribution of the translocation events at 1, 10, 20, and 30 min. The arrowheads across the four plots point at the population centered at approximately 0.2 ms and 0.25 nA. This population is attributed to the larger RS-dsDNA prior to digestion. As the time progresses to 10, 20 min and finally at 30 min, this population gradually disappears and a secondary population centered at approximately 0.15 ms and 0.15 nA begins to emerge; the side histograms of the peak amplitude axes show the emergence of the 0.15 nA population. The color bar represents the count of events found in each hexagon.
Figure 3
Figure 3. Digestion of RS-dsDNA monitored over the course of one hour. The RS-dsDNA was mixed with the SwaI restriction enzyme and digestion buffer to a final concentration of 9.24 nM RS-dsDNA, 5 units of enzyme, and 1× digestion buffer. (A) The ridgeline plot shows the gradual population changes from 1 to 60 min. (B) The probability of detecting the translocation of RS-dsDNA drops from 60% to near 0%. Two boundaries were defined as the ±10% of the peak value of the RS-dsDNA using the 1 min data, and the same boundaries were applied across all the data. The probability value was calculated by integrating the area under the curves (AUC) between the boundaries shown in (A); the initial starting percentage changes according to the width of the boundaries. (C) Enzyme reaction rate (slope average) plotted against the concentration of the enzyme. The enzyme reaction rate was calculated by fitting a linear regression line at the first 15 min (initial velocity region) of digestion under different enzyme concentrations (n = 3); the reaction rate obtained from the linear regression line is thus defined as the changes in probability of detecting the RS-dsDNA over the changes in time. The coefficient of determination for the fit is R2 = 0.9822. Error bars represent standard error of the mean of the slope values between measurements. According to the manufacturer, a single unit of SwaI is defined as the amount of enzyme required to digest 1 μg of pXba DNA in 1 h at 25 °C in a total reaction volume of 50 μL.
Effects of Salt on SwaI Cleavage Activity
Figure 4
Figure 4. Buffer-dependent restriction enzyme kinetics. (A) Restriction digestion of the RS-dsDNA in different buffers. The optimal buffer for the restriction enzyme SwaI is the buffer 3.1, as recommended by the supplier. Three other buffers (4, 2.1 and CutSmart) were tested, and the activity of SwaI varied and resulted in lower digestion activity in buffer 4 and CutSmart. (B) The gel band intensity was quantified and calculated relative to the 1.5 kbp’s band intensity within the sample lane (self-reference). (C) Probability of detecting 3 kbp dsDNA as a function of the digestion time for all the buffer tested and controls. m is the slope after fitting with the linear fit to the experimental data. Error bars are standard error of the mean. (D) Box plot comparing the probability of detecting the RS-dsDNA at 1 min and at 30 min. The two-tailed unpaired t-test was used to test the differences between the distribution of the probabilities to detect RS-dsDNA at 1 min and at 30 min. There are significant differences for buffer 3.1, buffer 2.1, and CutSmart at 1 min and at 30 min. The calculated values for buffer 3.1 at 1 min is 69.35 ± 3.94% and 19.28 ± 2.95% at 30 min, respectively; for buffer 4 at 1 min, it is 78.51 ± 4.55% and 69.43 ± 4.4%, respectively; for buffer 2.1 at 1 min, it is 89.54 ± 4.77% and 25.6 ± 4.44%, respectively; for CutSmart at 1 min, it is 79.81 ± 4.23% and 59.56 ± 5.42%, respectively; for no digestion at 1 min, it is 93.31 ± 2.76% and 89.17 ± 3.62%, respectively; for digested at 1 min, it is 8.52 ± 1.72% and 7.26 ± 2.15%, respectively. (ns, not significant; ****P < 0.0001; **P < 0.005; data assume normal distribution; Levene’s test (P > 0.05) indicates data have homoscedasticity; N = 3). (E) Ridgeline plots for buffer 4, buffer 2.1, and CutSmart. The cis chamber of the glass nanopore was filled with 10 nM RS-dsDNA diluted with buffer 3.1, buffer 4, buffer 2.1, or CutSmart (Table 1) containing 5 units of SwaI enzyme.
Effects of RNA/DNA Mismatches on CRISPR-Cas9 Cleavage Activity
Figure 5
Figure 5. CRISPR-Cas9-mediated dsDNA cleavage. (A) Schematic illustrating the process of the Cas9 mediated cleavage on the 3 kbp RS-dsDNA. The Cas9 was mixed with tracrRNA and crRNA to form the RNP complex. The RNA guides the Cas9 RNP to the position next to the PAM site. The Cas9 RNP then carries out double stranded cleavage 3–4 bp upstream of the PAM site to cleave the dsDNA. This resulted in the formation of 2 × 1.5 kbp dsDNA. (B) On-target and off-target crRNA sequence. The full complementary sequence (On target) and the off target variations at different positions upstream of the PAM site and different mismatches. (C) Agarose gel electrophoresis following 30 min incubation at 25 °C and overnight incubation at 4 °C. Cas9 RNPs were formed with the on-target crRNA or off-target crRNA variants. The gel band intensity was quantified and calculated relative to the 1.5 kbp’s band intensity within the sample lane (self-reference).
Figure 6
Figure 6. Measuring the activity of the Cas9 endonuclease with the nanopore. (A) Ridgeline plot showing the KDE estimated PDFs of the translocation experiment at each min for the 5 tested on-target and off-target crRNA sequences. (B) Probability of detecting 3 kbp dsDNA as a function of the digestion time for all the variants tested and controls. m is the slope after fitting with the linear fit to the experimental data. Error bars are standard error of the mean. (C) Box plot comparing the probability of detecting the RS-dsDNA at 1 min and at 30 min. The two-tailed unpaired t-test was used to test the differences between the distribution of the probabilities to detect RS-dsDNA at 1 min and at 30 min. There are significant differences for the on-target, off-target 2, and off-target 3 at 1 min and at 30 min. The calculated values for on target at 1 min are 28.31 ± 8.55% and 8.01 ± 5.37% at 30 min, respectively; for off target 1 at 1 min, it is 68.67 ± 6.57% and 61.08 ± 0.94%, respectively; for off-target 2 at 1 min, it is 50.25 ± 5.93% and 25.79 ± 10.6%, respectively; for off-target 3 at 1 min, it is 39.76 ± 5.65% and 12.04 ± 3.67%, respectively; for off-target 4 at 1 min, it is 77.95 ± 1.67% and 69.36 ± 6.11%, respectively; for no digestion at 1 min, it is 84.22 ± 3.38% and 80.44 ± 7.56%, respectively; for digested at 1 min, it is 8.52 ± 1.72% and 4.39 ± 2.07%, respectively. (ns, not significant; *P < 0.05; **P < 0.005; data assume normal distribution; Levene’s test (P > 0.05) indicates data have homoscedasticity; N = 3, two-tailed t-test).
Conclusions
Methods
Solid-State Nanopore Fabrication and Measurement
Polymer Electrolyte Bath Generation
Kinetic Translocation Experiment
CRISPR-Cas9 Ribonucleoproteins Complex Assembly and Reaction
Data Availability
All the translocation trace data supporting this work can be freely accessed via the University of Leeds data repository: 10.5518/1454
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c15173.
Supporting note on polymer electrolyte-modified solid-state nanopore mechanism, generation of the RS-dsDNA and the sequence data, generation of the RS-dsDNA, restriction digestion of the RS-dsDNA, Cas9-related crRNA sequence and digestion, KDE and probability calculations, and relevant supporting figures (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
We thank the group of bioelectronics member and Professor Christoph Wälti of University of Leeds for providing insightful feedback. We thank Dr Alexander Kulak of University of Leeds for imaging the nanopore with the scanning electron microscopy. C.C.C.C. acknowledges the scientific contributions of Dame Jocelyn Bell Burnell, particularly her discovery of the “PSR B1919 + 21 (CP 1919)” pulsar, and the iconic design of Joy Division’s “Unknown Pleasures” album cover, both of which inspired the creation of the plots in Figures 3, 4, and 6, and the table of content graphic.
AUC | area under the curve |
KDE | kernel density estimation |
PEG | poly(ethylene) glycol |
PCR | polymerase chain reaction |
RS-dsDNA | restriction site containing dsDNA |
CRISPR | clustered regularly interspaced short palindromic repeats |
PAM | protospacer adjacent motif |
RNP | ribonucleoprotein |
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- 13Mayer, S. F.; Cao, C.; Dal Peraro, M. Biological nanopores for single-molecule sensing. iScience 2022, 25, 104145, DOI: 10.1016/j.isci.2022.104145Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsFOktL3N&md5=be04fb1d36349cea136fa0d297c82573Biological nanopores for single-molecule sensingMayer, Simon Finn; Cao, Chan; Dal Peraro, MatteoiScience (2022), 25 (4), 104145CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)A review. Evolution has found countless ways to transport material across cells and cellular compartments sepd. by membranes. Protein assemblies are the cornerstone for the formation of channels and pores that enable this regulated passage of mols. in and out of cells, contributing to maintaining most of the fundamental processes that sustain living organisms. As in several other occasions, we have borrowed from the natural properties of these biol. systems to push technol. forward and have been able to hijack these nano-scale proteinaceous pores to learn about the phys. and chem. features of mols. passing through them. Today, a large repertoire of biol. pores is exploited as mol. sensors for characterizing biomols. that are relevant for the advancement of life sciences and application to medicine. Although the technol. has quickly matured to enable nucleic acid sensing with transformative implications for genomics, biol. pores stand as some of the most promising candidates to drive the next developments in single-mol. proteomics.
- 14Ying, Y.-L. Nanopore-based technologies beyond DNA sequencing. Nat. Nanotechnol. 2022, 17, 1136– 1146, DOI: 10.1038/s41565-022-01193-2Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisV2nsrbM&md5=f11d8860754531fc1fe1fcf71f1097eeNanopore-based technologies beyond DNA sequencingYing, Yi-Lun; Hu, Zheng-Li; Zhang, Shengli; Qing, Yujia; Fragasso, Alessio; Maglia, Giovanni; Meller, Amit; Bayley, Hagan; Dekker, Cees; Long, Yi-TaoNature Nanotechnology (2022), 17 (11), 1136-1146CODEN: NNAABX; ISSN:1748-3387. (Nature Portfolio)Abstr.: Inspired by the biol. processes of mol. recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive anal. tools for individual mols. In particular, nanopore-based single-mol. DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in mol. sensing and sequencing, chem. catalysis and biophys. characterization. We highlight the prospects of applying nanopores for single-protein anal. and sequencing, single-mol. covalent chem., clin. sensing applications for single-mol. liq. biopsy, and the use of synthetic biomimetic nanopores as exptl. models for natural systems. We suggest that nanopore technologies will continue to be explored to address a no. of scientific challenges as control over pore design improves.
- 15Varongchayakul, N.; Song, J.; Meller, A.; Grinstaff, M. W. Single-molecule protein sensing in a nanopore: a tutorial. Chem. Soc. Rev. 2018, 47, 8512– 8524, DOI: 10.1039/C8CS00106EGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFalu7%252FL&md5=aaa171dcde2e09746529db53104492b5Single-molecule protein sensing in a nanopore: a tutorialVarongchayakul, Nitinun; Song, Jiaxi; Meller, Amit; Grinstaff, Mark W.Chemical Society Reviews (2018), 47 (23), 8512-8524CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Proteins are the structural elements and machinery of cells responsible for a functioning biol. architecture and homeostasis. Advances in nanotechnol. are catalyzing key breakthroughs in many areas, including the anal. and study of proteins at the single-mol. level. Nanopore sensing is at the forefront of this revolution. This tutorial review provides readers a guidebook and ref. for detecting and characterizing proteins at the single-mol. level using nanopores. Specifically, the review describes the key materials, nanoscale features, and design requirements of nanopores. It also discusses general design requirements as well as details on the anal. of protein translocation. Finally, the article provides the background necessary to understand current research trends and to encourage the identification of new biomedical applications for protein sensing using nanopores.
- 16Platnich, C. M.; Earle, M. K.; Keyser, U. F. Chemical Annealing Restructures RNA for Nanopore Detection. J. Am. Chem. Soc. 2024, 146, 12919– 12924, DOI: 10.1021/jacs.4c03753Google ScholarThere is no corresponding record for this reference.
- 17Bandara, Y. M. N. D. Y.; Farajpour, N.; Freedman, K. J. Nanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein’s Isoelectric Point. J. Am. Chem. Soc. 2022, 144, 3063– 3073, DOI: 10.1021/jacs.1c11540Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xjt1GmsL8%253D&md5=5e7c775eec3b97b1578098d668533dbbNanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein's Isoelectric PointBandara, Y. M. Nuwan D. Y.; Farajpour, Nasim; Freedman, Kevin J.Journal of the American Chemical Society (2022), 144 (7), 3063-3073CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the elec. sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concns. can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irresp. of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelec. point (pI) of Cas9 was found to be correlated and a crit. factor in our expts. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be crit. for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.
- 18Gao, T. Label-Free Resistance Cytometry at the Orifice of a Nanopipette. Anal. Chem. 2021, 93, 2942– 2949, DOI: 10.1021/acs.analchem.0c04585Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitV2rtrc%253D&md5=7884db87f67bae1dca19c624fcfbb645Label-Free Resistance Cytometry at the Orifice of a NanopipetteGao, Tienan; Gao, Xiangyi; Xu, Cong; Wang, Menglin; Chen, Mingli; Wang, Jianhua; Ma, Furong; Yu, Ping; Mao, LanqunAnalytical Chemistry (Washington, DC, United States) (2021), 93 (5), 2942-2949CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Development of new principles and techniques at the single-cell level is significantly important since cells as basic units of living organisms always bear large heterogeneity. Herein, we demonstrate a new electrochem. principle for single-cell anal. based on an ion current blockage at the orifice of a nanopipette, defined as resistance cytometry. The amplitude and the frequency of ion current transients show strong dependence on the size and the concn. of cells, which could be used for in situ cell sizing and counting. This technique shows good ability to detect the size change of RBCs under stimulations of different pH and osmotic pressure values. More importantly, the as-presented resistance cytometry can distinguish lymphoma blood cells from normal blood cells for patient blood samples. The as-presented resistance cytometry is label-free, non-invasive, and non-destructive, which not only opens new opportunities for single-cell anal. but also provides a new platform for cell-related medical diagnostic technologies.
- 19Shumyantseva, V. V. Enzymology on an Electrode and in a Nanopore: Analysis Algorithms, Enzyme Kinetics, and Perspectives. Bionanosci. 2022, 12, 1341– 1355, DOI: 10.1007/s12668-022-01037-2Google ScholarThere is no corresponding record for this reference.
- 20Willems, K.; Van Meervelt, V.; Wloka, C.; Maglia, G. Single-molecule nanopore enzymology. Philos. Trans. R. Soc., B 2017, 372, 20160230, DOI: 10.1098/rstb.2016.0230Google ScholarThere is no corresponding record for this reference.
- 21Sheng, Y.; Zhang, S.; Liu, L.; Wu, H. C. Measuring Enzymatic Activities with Nanopores. ChemBioChem 2020, 21, 2089– 2097, DOI: 10.1002/cbic.202000079Google ScholarThere is no corresponding record for this reference.
- 22Wloka, C. Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore. ACS Nano 2017, 11, 4387– 4394, DOI: 10.1021/acsnano.6b07760Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltVSisLw%253D&md5=ca0b3e1b28d90efa6a3bf94de79ccea7Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological NanoporeWloka, Carsten; Van Meervelt, Veerle; van Gelder, Dewi; Danda, Natasha; Jager, Nienke; Williams, Chris P.; Maglia, GiovanniACS Nano (2017), 11 (5), 4387-4394CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The covalent addn. of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biol. processes. Here, the authors report a fast, single-mol., and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. Ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiol. conditions. Using defined conjugates, also isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.
- 23Galenkamp, N. S.; Biesemans, A.; Maglia, G. Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings. Nat. Chem. 2020, 12, 481– 488, DOI: 10.1038/s41557-020-0437-0Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVyjs74%253D&md5=922cd1a0ecdc2a96faf98a98f7afc622Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordingsGalenkamp, Nicole Stephanie; Biesemans, Annemie; Maglia, GiovanniNature Chemistry (2020), 12 (5), 481-488CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Abstr.: Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodn. and kinetic characterization at the single-mol. level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the elec. recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by mols. that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymic reactions affected by product inhibition or when the release of products is the rate-limiting step.
- 24Robinson, P. K. Enzymes: principles and biotechnological applications. Essays Biochem. 2015, 59, 1– 41, DOI: 10.1042/bse0590001Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28zlvFSnsQ%253D%253D&md5=2f68b0d48239c71e0a4da84ae04bba3aEnzymes: principles and biotechnological applicationsRobinson Peter KEssays in biochemistry (2015), 59 (), 1-41 ISSN:.Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.
- 25Cheley, S.; Xie, H.; Bayley, H. A Genetically Encoded Pore for the Stochastic Detection of a Protein Kinase. ChemBioChem 2006, 7, 1923– 1927, DOI: 10.1002/cbic.200600274Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXltVM%253D&md5=fe2580df39f2b5ee0db138f51f350816A genetically encoded pore for the stochastic detection of a protein kinaseCheley, Stephen; Xie, Hongzhi; Bayley, HaganChemBioChem (2006), 7 (12), 1923-1927CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Stochastic sensing is an emerging approach for the detection of a wide variety of analytes at the level of individual mols. Detection is accomplished by observing the modulation of the current that flows through a single protein pore that has been engineered to bind an analyte of interest. Previously, protein analytes have been detected by using pores to which ligands have been appended at specific sites by targeted chem. modification. Here, we report the first genetically encoded stochastic sensor element for detecting a protein. A protein kinase inhibitor peptide sequence was incorporated into the α-hemolysin polypeptide, which was used to form a heteroheptameric pore contg. a single copy of the inhibitor sequence. With this pore, the successful detection of the catalytic subunit of protein kinase A was demonstrated. This development should greatly facilitate the detection of active kinase subunits by stochastic sensing and the rapid screening of kinase inhibitors by an approach that yields kinetic information.
- 26Harrington, L.; Cheley, S.; Alexander, L. T.; Knapp, S.; Bayley, H. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E4417– E4426, DOI: 10.1073/pnas.1312739110Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFCisL%252FF&md5=a56393697939130134dd120952116711Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrateHarrington, Leon; Cheley, Stephen; Alexander, Leila T.; Knapp, Stefan; Bayley, HaganProceedings of the National Academy of Sciences of the United States of America (2013), 110 (47), E4417-E4426,SE4417/1-SE4417/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In stochastic sensing, the assocn. and dissocn. of analyte mols. is obsd. as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free detn. of rate and equil. consts. with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-mol. detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we obsd. unusually high assocn. rate consts. for the interaction of Pim kinases with their consensus substrate Pimtide (∼107 to 108 M-1·s-1), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.
- 27Harrington, L.; Alexander, L. T.; Knapp, S.; Bayley, H. Single-Molecule Protein Phosphorylation and Dephosphorylation by Nanopore Enzymology. ACS Nano 2019, 13, 633– 641, DOI: 10.1021/acsnano.8b07697Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1SgtLnF&md5=b86fe4747ab1834504d4edc336ac70f3Single-molecule protein phosphorylation and dephosphorylation by nanopore enzymologyHarrington, Leon; Alexander, Leila T.; Knapp, Stefan; Bayley, HaganACS Nano (2019), 13 (1), 633-641CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Reversible protein phosphorylation plays a crucial and ubiquitous role in the control of almost all cellular processes. The interplay of protein kinases and phosphatases acting in opposition ensures tight dynamic control of protein phosphorylation states within the cell. Previously, engineered α-hemolysin pores bearing kinase substrate peptides have been developed as single-mol. stochastic sensors for protein kinases. Here, we used these pores to observe, label-free, the phosphorylation and dephosphorylation of a single substrate mol., pimtide. Further, we investigated the effect of Mg2+ and Mn2+ upon substrate and product binding and found that Mn2+ relaxed active site specificity toward nucleotides and enhanced product binding. In doing so, we demonstrated the power and versatility of nanopore enzymol. to scrutinize a crit. post-translational modification.
- 28Harrington, L.; Alexander, L. T.; Knapp, S.; Bayley, H. Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor. Angew. Chem., Int. Ed. 2015, 54, 8154– 8159, DOI: 10.1002/anie.201503141Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXps12rsL8%253D&md5=05527b50445ed619574e86b1981f877bPim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore SensorHarrington, Leon; Alexander, Leila T.; Knapp, Stefan; Bayley, HaganAngewandte Chemie, International Edition (2015), 54 (28), 8154-8159CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Protein kinases are crit. therapeutic targets. Pim kinases are implicated in several leukemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We obsd. synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free detn. of inhibition consts. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorometry. The approach is also amenable to implementation on high throughput chips.
- 29Nouri, R.; Jiang, Y.; Lian, X. L.; Guan, W. Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN). ACS Sensors 2020, 5, 1273– 1280, DOI: 10.1021/acssensors.0c00497Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosF2nsbY%253D&md5=a27c11eb4f9b6c1d6329610570bdc022Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN)Nouri, Reza; Jiang, Yuqian; Lian, Xiaojun Lance; Guan, WeihuaACS Sensors (2020), 5 (5), 1273-1280CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Nucleic acid detection methods are crucial for many fields such as pathogen detection and genotyping. Solid-state nanopore sensors represent a promising platform for nucleic acid detection due to its unique single mol. sensitivity and label-free electronic sensing. Here, we demonstrated the use of the glass nanopore for highly sensitive quantification of single-stranded circular DNAs (reporters), which could be degraded under the trans-cleavage activity of the target-specific CRISPR-Cas12a. We developed and optimized the Cas12a assay for HIV-1 anal. We validated the concept of the solid-state CRISPR-Cas12a-assisted nanopores (SCAN) to specifically detect the HIV-1 DNAs. We showed that the glass nanopore sensor is effective in monitoring the cleavage activity of the target DNA-activated Cas12a. We developed a model to predict the total exptl. time needed for making a statistically confident pos./neg. call in a qual. test. The SCAN concept combines the much-needed specificity and sensitivity into a single platform, and we anticipate that the SCAN would provide a compact, rapid, and low-cost method for nucleic acid detection at the point of care.
- 30Weckman, N. E. Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore Sensing. ACS Sensors 2019, 4, 2065– 2072, DOI: 10.1021/acssensors.9b00686Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsVertrzL&md5=292439fb275ad650facc06a8d2fe7c65Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore SensingWeckman, Nicole E.; Ermann, Niklas; Gutierrez, Richard; Chen, Kaikai; Graham, James; Tivony, Ran; Heron, Andrew; Keyser, Ulrich F.ACS Sensors (2019), 4 (8), 2065-2072CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific anal. of a DNA sample. Solid state nanopore sensors are esp. promising for this type of single-mol. sensing because of the ability to analyze patterns in the ionic current signatures of DNA mols. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify two different DNA targets in a background mixt. of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.
- 31Sandler, S. E. Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores. Nat. Biomed. Eng. 2024, 8, 325– 334, DOI: 10.1038/s41551-023-01078-2Google ScholarThere is no corresponding record for this reference.
- 32Yang, W. Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores. Nano Lett. 2018, 18, 6469– 6474, DOI: 10.1021/acs.nanolett.8b02968Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1KhurjL&md5=5c13dba2cb82df4d4d1f2958e5772cccDetection of CRISPR-dCas9 on DNA with Solid-State NanoporesYang, Wayne; Restrepo-Perez, Laura; Bengtson, Michel; Heerema, Stephanie J.; Birnie, Anthony; van der Torre, Jaco; Dekker, CeesNano Letters (2018), 18 (10), 6469-6474CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore expts. that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore expts., we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA mols. that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore expts., dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.
- 33Ma, H.; Wang, Y.; Li, Y. X.; Xie, B. K.; Hu, Z. L.; Yu, R. J.; Long, Y. T.; Ying, Y. L. Label-Free Mapping of Multivalent Binding Pathways with Ligand–Receptor-Anchored Nanopores. J. Am. Chem. Soc. 2024, 146, 28014– 28022, DOI: 10.1021/jacs.4c04934Google ScholarThere is no corresponding record for this reference.
- 34Kowalczyk, S. W.; Wells, D. B.; Aksimentiev, A.; Dekker, C. Slowing down DNA Translocation through a Nanopore in Lithium Chloride. Nano Lett. 2012, 12, 1038– 1044, DOI: 10.1021/nl204273hGoogle Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlsVCktg%253D%253D&md5=31be87d5508361ac5aba5a7120e51979Slowing down DNA Translocation through a Nanopore in Lithium ChlorideKowalczyk, Stefan W.; Wells, David B.; Aksimentiev, Aleksei; Dekker, CeesNano Letters (2012), 12 (2), 1038-1044CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The charge of a DNA mol. is a crucial parameter in many DNA detection and manipulation schemes such as gel electrophoresis and lab-on-a-chip applications. Here, we study the partial redn. of the DNA charge due to counterion binding by means of nanopore translocation expts. and all-atom mol. dynamics (MD) simulations. Surprisingly, we find that the translocation time of a DNA mol. through a solid-state nanopore strongly increases as the counterions decrease in size from K+ to Na+ to Li+, both for double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). MD simulations elucidate the microscopic origin of this effect: Li+ and Na+ bind DNA stronger than K+. These fundamental insights into the counterion binding to DNA also provide a practical method for achieving at least 10-fold enhanced resoln. in nanopore applications.
- 35Plesa, C. Fast Translocation of Proteins through Solid State Nanopores. Nano Lett. 2013, 13, 658– 663, DOI: 10.1021/nl3042678Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1KlsL0%253D&md5=bce7b51bbacf0f73d77dd4ead8d6ece2Fast Translocation of Proteins through Solid State NanoporesPlesa, Calin; Kowalczyk, Stefan W.; Zinsmeester, Ruben; Grosberg, Alexander Y.; Rabin, Yitzhak; Dekker, CeesNano Letters (2013), 13 (2), 658-663CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Measurements on protein translocation through solid-state nanopores reveal anomalous (non-Smoluchowski) transport behavior, as evidenced by extremely low detected event rates; i.e., the capture rates are orders of magnitude smaller than what is theor. expected. Systematic exptl. measurements of the event rate dependence on the diffusion const. are performed by translocating proteins ranging in size from 6 to 660 kDa. The discrepancy is obsd. to be significantly larger for smaller proteins, which move faster and have a lower signal-to-noise ratio. This is further confirmed by measuring the event rate dependence on the pore size and concn. for a large 540 kDa protein and a small 37 kDa protein, where only the large protein follows the expected behavior. We dismiss various possible causes for this phenomenon and conclude that it is due to a combination of the limited temporal resoln. and low signal-to-noise ratio. A one-dimensional first-passage time-distribution model supports this and suggests that the bulk of the proteins translocate on time scales faster than can be detected. We discuss the implications for protein characterization using solid-state nanopores and highlight several possible routes to address this problem.
- 36Chau, C. C.; Radford, S. E.; Hewitt, E. W.; Actis, P. Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore. Nano Lett. 2020, 20, 5553– 5561, DOI: 10.1021/acs.nanolett.0c02246Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1WnurrJ&md5=3d330bfaeae1011185b928d1ebc33a8eMacromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State NanoporeChau, Chalmers C.; Radford, Sheena E.; Hewitt, Eric W.; Actis, PaoloNano Letters (2020), 20 (7), 5553-5561CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Nanopore anal. of nucleic acid is now routine, but detection of proteins remains challenging. Here, the authors report the systematic characterization of the effect of macromol. crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). A remarkable ∼1000-fold increase in the mol. count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. were obsd. Also macromol. crowding facilitates the study of the topol. of DNA plasmids and the characterization of amyloid fibril prepns. with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addn. of a macromol. crowding agent to the electrolyte. The authors therefore envision that macromol. crowding can be applied to many applications in the anal. of biomols. by solid-state nanopores.
- 37Chau, C. Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore. ACS Nano 2022, 16, 20075– 20085, DOI: 10.1021/acsnano.2c08312Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1ygsbvP&md5=26c141887bfff87b6d709d9cd6f13de8Probing RNA Conformations Using a Polymer-Electrolyte Solid-State NanoporeChau, Chalmers; Marcuccio, Fabio; Soulias, Dimitrios; Edwards, Martin Andrew; Tuplin, Andrew; Radford, Sheena E.; Hewitt, Eric; Actis, PaoloACS Nano (2022), 16 (12), 20075-20085CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Nanopore systems have emerged as a leading platform for the anal. of biomol. complexes with single-mol. resoln. The conformation of biomols., such as RNA, is highly dependent on the electrolyte compn., but solid-state nanopore systems often require high salt concn. to operate, precluding anal. of macromol. conformations under physiol. relevant conditions. Here, we report the implementation of a polymer-electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer-electrolyte bath governs the translocation dynamics of the analyte which correlates with the phys. properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer-electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiol. relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer-electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomols. under physiol. relevant conditions.
- 38Confederat, S.; Sandei, I.; Mohanan, G.; Wälti, C.; Actis, P. Nanopore fingerprinting of supramolecular DNA nanostructures. Biophys. J. 2022, 121, 4882– 4891, DOI: 10.1016/j.bpj.2022.08.020Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1Clu7vM&md5=d9a9c0fc6dd9ab3cf249870223457960Nanopore fingerprinting of supramolecular DNA nanostructuresConfederat, Samuel; Sandei, Ilaria; Mohanan, Gayathri; Walti, Christoph; Actis, PaoloBiophysical Journal (2022), 121 (24), 4882-4891CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)DNA nanotechnol. has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resoln. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equiv. charge surplus as a discriminant. We compare the assembly yield of four supramol. DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and at. force microscopy imaging. We demonstrate that the nanopore system can provide anal. quantification of the complex supramol. nanostructures within minutes, without any need for labeling and with single-mol. resoln. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the anal. and manipulation of large DNA assemblies in real time.
- 39Confederat, S.; Lee, S.; Vang, D.; Soulias, D.; Marcuccio, F.; Peace, T. I.; Edwards, M. A.; Strobbia, P.; Samanta, D.; Wälti, C. Next-Generation Nanopore Sensors Based on Conductive Pulse Sensing for Enhanced Detection of Nanoparticles. Small 2024, 20, 2305186, DOI: 10.1002/smll.202305186Google ScholarThere is no corresponding record for this reference.
- 40Marcuccio, F. Mechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer–Electrolyte Nanopore. ACS Nanosci. Au 2023, 3, 172– 181, DOI: 10.1021/acsnanoscienceau.2c00050Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXmsFGrsg%253D%253D&md5=318e9bc708154debe2354af02b435dcdMechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer-Electrolyte NanoporeMarcuccio, Fabio; Soulias, Dimitrios; Chau, Chalmers C. C.; Radford, Sheena E.; Hewitt, Eric; Actis, Paolo; Edwards, Martin AndrewACS Nanoscience Au (2023), 3 (2), 172-181CODEN: ANACCX; ISSN:2694-2496. (American Chemical Society)Solid-state nanopores have been widely employed in the detection of biomols., but low signal-to-noise ratios still represent a major obstacle in the discrimination of nucleic acid and protein sequences substantially smaller than the nanopore diam. The addn. of 50% poly(ethylene) glycol (PEG) to the external soln. is a simple way to enhance the detection of such biomols. Here, we demonstrate with finite-element modeling and expts. that the addn. of PEG to the external soln. introduces a strong imbalance in the transport properties of cations and anions, drastically affecting the current response of the nanopore. We further show that the strong asym. current response is due to a polarity-dependent ion distribution and transport at the nanopipette tip region, leading to either ion depletion or enrichment for few tens of nanometers across its aperture. We provide evidence that a combination of the decreased/increased diffusion coeffs. of cations/anions in the bath outside the nanopore and the interaction between a translocating mol. and the nanopore-bath interface is responsible for the increase in the translocation signals. We expect this new mechanism to contribute to further developments in nanopore sensing by suggesting that tuning the diffusion coeffs. of ions could enhance the sensitivity of the system.
- 41Meyer, N.; Janot, J.-M.; Torrent, J.; Balme, S. Real-Time Fast Amyloid Seeding and Translocation of α-Synuclein with a Nanopipette. ACS Cent. Sci. 2022, 8, 441– 448, DOI: 10.1021/acscentsci.1c01404Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XksFKgtbg%253D&md5=f88378af6bfc37da61301120dfc36992Real-Time Fast Amyloid Seeding and Translocation of α-Synuclein with a NanopipetteMeyer, Nathan; Janot, Jean-Marc; Torrent, Joan; Balme, SebastienACS Central Science (2022), 8 (4), 441-448CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)The detection to α-synuclein (αS) assemblies as a biomarker of synucleinopathies is an important challenge for further development of an early diagnosis tool. Here, we present proof of concept real-time fast amyloid seeding and translocation (RT-FAST) based on a nanopipette that combines in one unique system a reaction vessel to accelerate the seed amplification and nanopore sensor for single-mol. αS assembly detection. RT-FAST allows the detection of the presence αS seeds WT and A53T variant in a given sample in only 90 min by adding a low quantity (35μL at 100 nM) of recombinant αS for amplification. It also shows cross-seeding aggregation by adding mixing seeds A53T with WT monomers. Finally, we establish the dependence between the capture rate of aggregates by the nanopore sensor and the initial seed concn. from 200 pM to 2 pM, which promises further development toward a quant. anal. of the initial seed concn.
- 42Meyer, N.; Arroyo, N.; Roustan, L.; Janot, J.; Charles-Achille, S.; Torrent, J.; Picaud, F.; Balme, S. Secondary Nucleation of Aβ Revealed by Single-Molecule and Computational Approaches. Advanced Science 2024, 11, 2404916, DOI: 10.1002/advs.202404916Google ScholarThere is no corresponding record for this reference.
- 43Sandler, S. E. Multiplexed Digital Characterization of Misfolded Protein Oligomers via Solid-State Nanopores. J. Am. Chem. Soc. 2023, 145, 25776– 25788, DOI: 10.1021/jacs.3c09335Google ScholarThere is no corresponding record for this reference.
- 44Shen, B. W.; Heiter, D. F.; Lunnen, K. D.; Wilson, G. G.; Stoddard, B. L. DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target. Nucleic Acids Res. 2017, 45, 1516– 1528, DOI: 10.1093/nar/gkw1200Google ScholarThere is no corresponding record for this reference.
- 45Barrangou, R. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science 2007, 315, 1709– 1712, DOI: 10.1126/science.1138140Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjtlWntb8%253D&md5=b88c0100d2c0469213afda20e47c39cdCRISPR Provides Acquired Resistance Against Viruses in ProkaryotesBarrangou, Rodolphe; Fremaux, Christophe; Deveau, Helene; Richards, Melissa; Boyaval, Patrick; Moineau, Sylvain; Romero, Dennis A.; Horvath, PhilippeScience (Washington, DC, United States) (2007), 315 (5819), 1709-1712CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addn. of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with assocd. cas genes, provided resistance against phages, and resistance specificity is detd. by spacer-phage sequence similarity.
- 46Wanunu, M.; Sutin, J.; McNally, B.; Chow, A.; Meller, A. DNA Translocation Governed by Interactions with Solid-State Nanopores. Biophys. J. 2008, 95, 4716– 4725, DOI: 10.1529/biophysj.108.140475Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlOqtLnM&md5=ed5c8c30545222c5864800f11f5f54fdDNA translocation governed by interactions with solid-state nanoporesWanunu, Meni; Sutin, Jason; McNally, Ben; Chow, Andrew; Meller, AmitBiophysical Journal (2008), 95 (10), 4716-4725CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)We investigate the voltage-driven translocation dynamics of individual DNA mols. through solid-state nanopores in the diam. range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diam. is decreased from 5 to 2.7 nm, and steep temp. dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA mols., in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer mols., an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA mols. to a regime where the longer the DNA, the more current is blocked. Temp. dependence studies reveal that for increasing DNA lengths, addnl. interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor detg. DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resoln. than obsd. with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.
- 47Li, J.; Talaga, D. S. The distribution of DNA translocation times in solid-state nanopores. J. Phys.: Condens. Matter 2010, 22, 454129, DOI: 10.1088/0953-8984/22/45/454129Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFartLzP&md5=70c79a7ae6896602196636ce65b04139The distribution of DNA translocation times in solid-state nanoporesLi, Jiali; Talaga, David S.Journal of Physics: Condensed Matter (2010), 22 (45), 454129/1-454129/8CODEN: JCOMEL; ISSN:0953-8984. (Institute of Physics Publishing)This paper systematically investigates the effects of soln. viscosity, applied voltage and DNA chain length on the distribution of DNA translocation times through 8±2 nm diam. silicon nitride nanopores. Linear dsDNA translocation events were selected based on the magnitude of current blockage and accumulated into scatter plots of current blockage and event duration (translocation time). The translocation time distribution was fitted to the soln. of a Smoluchowski-type equation for 1D biased diffusion to a sink. The DNA drifting speed under bias and diffusion const. were extd. from the fits as functions of soln. viscosity, applied voltage and DNA chain length. Combined with the Einstein-Smoluchowski relation, this model allowed evaluation of the viscous drag force on DNA mols. This model also allowed estn. of the uncertainty in detg. the DNA chain length due to the influence of friction on the spread of translocation times in a nanopore measurement. The data anal. suggests that the simple 1D biased diffusion model fits the exptl. data well for a wide range of conditions. Some deviations from predicted behavior were obsd. and show where addnl. phenomena are likely to contribute to the distribution of DNA translocation times.
- 48Kesselheim, S.; Müller, W.; Holm, C. Origin of Current Blockades in Nanopore Translocation Experiments. Phys. Rev. Lett. 2014, 112, 018101, DOI: 10.1103/PhysRevLett.112.018101Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitlehurk%253D&md5=c8bf5244f200101ff3a3e3075f3212c9Origin of current blockades in nanopore translocation experimentsKesselheim, Stefan; Mueller, Wojciech; Holm, ChristianPhysical Review Letters (2014), 112 (1), 018101/1-018101/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)We present a detailed investigation of the ionic current in a cylindrical model nanopore in the absence and the presence of a double stranded DNA homopolymer. Our atomistic simulations are capable of reproducing almost exactly the exptl. data obtained by Smeets et al., including notably the crossover salt concn. that yields equal current measurements in both situations. We can rule out that the obsd. current blockade is due to the steric exclusion of charge carriers from the DNA, since for all investigated salt concns. the charge carrier d. is higher when the DNA is present. Calcns. using a mean-field electrokinetic model proposed by van Dorp et al. fail quant. in predicting this effect. We can relate the shortcomings of the mean-field model to a surface related mol. drag that the ions feel in the presence of the DNA. This drag is independent of the salt concn. and originates from electrostatic, hydrodynamic, and excluded vol. interactions.
- 49Gibson, D. G. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343– 345, DOI: 10.1038/nmeth.1318Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVemsbw%253D&md5=46284924c7d73c47cfb490983338e480Enzymatic assembly of DNA molecules up to several hundred kilobasesGibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
- 50Li, Y.; Sandler, S. E.; Keyser, U. F.; Zhu, J. DNA Volume, Topology, and Flexibility Dictate Nanopore Current Signals. Nano Lett. 2023, 23, 7054– 7061, DOI: 10.1021/acs.nanolett.3c01823Google ScholarThere is no corresponding record for this reference.
- 51Cadinu, P.; Kang, M.; Nadappuram, B. P.; Ivanov, A. P.; Edel, J. B. Individually Addressable Multi-nanopores for Single-Molecule Targeted Operations. Nano Lett. 2020, 20, 2012– 2019, DOI: 10.1021/acs.nanolett.9b05307Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivVaqsLg%253D&md5=b2b715cc9a7162d0a23004b1c9f203d6Individually Addressable Multi-nanopores for Single-Molecule Targeted OperationsCadinu, Paolo; Kang, Minkyung; Nadappuram, Binoy Paulose; Ivanov, Aleksandar P.; Edel, Joshua B.Nano Letters (2020), 20 (3), 2012-2019CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The fine-tuning of mol. transport is a ubiquitous problem of single-mol. methods. The latter is evident even in powerful single-mol. techniques such as nanopore sensing, where the quest for resolving more detailed biomol. features is often limited by insufficient control of the dynamics of individual mols. within the detection vol. of the nanopore. In this work, we introduce and characterize a reconfigurable multi-nanopore architecture that enables addnl. channels to manipulate the dynamics of DNA mols. in a nanopore. We show that the fabrication process of this device, consisting of four adjacent, individually addressable nanopores located at the tip of a quartz nanopipette, is fast and highly reproducible. By individually tuning the elec. field across each nanopore, these devices can operate in several unique cooperative detection modes that allow moving, sensing, and trapping of DNA mols. with high efficiency and increased temporal resoln.
- 52Cadinu, P. Double Barrel Nanopores as a New Tool for Controlling Single-Molecule Transport. Nano Lett. 2018, 18, 2738– 2745, DOI: 10.1021/acs.nanolett.8b00860Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlvVKks7Y%253D&md5=e0603e993e4a1132e7e451d715671228Double Barrel Nanopores as a New Tool for Controlling Single-Molecule TransportCadinu, Paolo; Campolo, Giulia; Pud, Sergii; Yang, Wayne; Edel, Joshua B.; Dekker, Cees; Ivanov, Aleksandar P.Nano Letters (2018), 18 (4), 2738-2745CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The ability to control the motion of single biomols. is key to improving a wide range of biophys. and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, mol. control and the possibility of slow readouts of long polymer mols. are still limited due to fast analyte transport and low signal-to-noise ratios. Here, the authors report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are sepd. by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (1) pore-to-pore transfer, which can be controlled at ∼100% efficiency, and (2) DNA mols. bridging between the two nanopores, which enables detection with an enhanced temporal resoln. (e.g., an increase of >2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resoln. readout of biol. mols.
- 53Cadinu, P. Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge. Nano Lett. 2017, 17, 6376– 6384, DOI: 10.1021/acs.nanolett.7b03196Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVWmsrvF&md5=01bd2c208ce4d2cd46c5f45b4c728f04Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter NanobridgeCadinu, Paolo; Paulose Nadappuram, Binoy; Lee, Dominic J.; Sze, Jasmine Y. Y.; Campolo, Giulia; Zhang, Yanjun; Shevchuk, Andrew; Ladame, Sylvain; Albrecht, Tim; Korchev, Yuri; Ivanov, Aleksandar P.; Edel, Joshua B.Nano Letters (2017), 17 (10), 6376-6384CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)There is a growing realization, esp. within the diagnostic and therapeutic community, that the amt. of information enclosed in a single mol. can not only enable a better understanding of biophys. pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single mol. strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control mol. transport in terms of transport rate, resoln., and signal-to-noise ratio (SNR) is essential to take full advantage of the technol. benefits. Here the authors propose a novel soln. to these challenges based on a method that allows biomols. to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm sepn. Mols. that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resoln., sensitivity, and limit of detection. α-synuclein. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.
- 54Steinbock, L. J.; Otto, O.; Chimerel, C.; Gornall, J.; Keyser, U. F. Detecting DNA Folding with Nanocapillaries. Nano Lett. 2010, 10, 2493– 2497, DOI: 10.1021/nl100997sGoogle Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmvVSlsL8%253D&md5=b29d1d4606b361e6216d9e05e7df6cd8Detecting DNA Folding with NanocapillariesSteinbock, Lorenz J.; Otto, Oliver; Chimerel, Catalin; Gornall, Joanne; Keyser, Ulrich F.Nano Letters (2010), 10 (7), 2493-2497CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors demonstrate for the first time the detection of the folding state of double-stranded DNA in nanocapillaries with the resistive pulse technique. The authors show that glass capillaries can be pulled into nanocapillaries with diams. down to 45 nm. The authors study translocation of λ -DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ -DNA mols. The authors' expts. prove that nanocapillaries are suitable for label-free anal. of DNA in aq. solns. and viable alternatives to solid-state nanopores made by silicon nanotechnol.
- 55Smeets, R. M. M. Salt Dependence of Ion Transport and DNA Translocation through Solid-State Nanopores. Nano Lett. 2006, 6, 89– 95, DOI: 10.1021/nl052107wGoogle Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht12rtbfK&md5=74e23d4453737c1063d0f0a9a75d9447Salt Dependence of Ion Transport and DNA Translocation through Solid-State NanoporesSmeets, Ralph M. M.; Keyser, Ulrich F.; Krapf, Diego; Wu, Meng-Yue; Dekker, Nynke H.; Dekker, CeesNano Letters (2006), 6 (1), 89-95CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)We report exptl. measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concns. from 1 M to 1 μM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-μm-long dsDNA for 50 mM to 1 M salt concns. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concn. of 370 ± 40 mM.
- 56Bošković, F.; Zhu, J.; Chen, K.; Keyser, U. F. Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State Nanopores. Nano Lett. 2019, 19, 7996– 8001, DOI: 10.1021/acs.nanolett.9b03184Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVylsL3K&md5=f05ce915e55deb46f55280a82eecfb72Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State NanoporesBoskovic, Filip; Zhu, Jinbo; Chen, Kaikai; Keyser, Ulrich F.Nano Letters (2019), 19 (11), 7996-8001CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)G-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-mol. methods require fluorescent labels, which can affect Gq folding kinetics. Here the authors introduce, a single-mol. Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. The authors use ∼5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addn., smGNA allows for discrimination of (un-)folded Gq structures, provides insights into single-mol. kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-mol. level without labeling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.
- 57Lastra, L. S.; Bandara, Y. M. N. D. Y.; Sharma, V.; Freedman, K. J. Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance. ACS Sensors 2022, 7, 1883– 1893, DOI: 10.1021/acssensors.2c00479Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsFWnsbzP&md5=4f204344814bc243903917f76ff80f40Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt ImbalanceLastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Sharma, Vinay; Freedman, Kevin J.ACS Sensors (2022), 7 (7), 1883-1893CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Nanopores are a promising single-mol. sensing device class that captures mol.-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a mol. A systematic study was conducted here to study the CPS-based mol. discrimination and its voltage-dependent characteristics. CPS was obsd. when the cation movement along both elec. and chem. gradients was favored, which led to an ∼3x improvement in SNR (i.e., signal-to-noise ratio) and an ∼8x increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asym. salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other mol. systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10a-a protein with broad utilities in genetic engineering and immunol.-bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically obsd. for proteins.
- 58Charron, M.; Briggs, K.; King, S.; Waugh, M.; Tabard-Cossa, V. Precise DNA Concentration Measurements with Nanopores by Controlled Counting. Anal. Chem. 2019, 91, 12228– 12237, DOI: 10.1021/acs.analchem.9b01900Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1egu7bM&md5=1f2a6c31a33b900129c87acb6cbd1e9dPrecise DNA Concentration Measurements with Nanopores by Controlled CountingCharron, Martin; Briggs, Kyle; King, Simon; Waugh, Matthew; Tabard-Cossa, VincentAnalytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12228-12237CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Using a solid-state nanopore to measure the concn. of clin. relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theor. electrophoretic capture models. We exptl. reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based mol. concn. detn. insufficiently precise to meet the stds. of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-mol. nanopore-based concn. measurements. We demonstrate controlled counting, a concn. measurement technique, which involves measuring the simultaneous capture rates of a mixt. of both the target mol. and an internal calibrator of precisely known concn. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge d. or distribution, are removed while allowing for higher-precision measurements. The quant. tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomol. concn.
- 59Karau, P.; Tabard-Cossa, V. Capture and Translocation Characteristics of Short Branched DNA Labels in Solid-State Nanopores. ACS Sensors 2018, 3, 1308– 1315, DOI: 10.1021/acssensors.8b00165Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVOru7jK&md5=36766810f7cabb5796d4eed5ce4f64c4Capture and Translocation Characteristics of Short Branched DNA Labels in Solid-State NanoporesKarau, Philipp; Tabard-Cossa, VincentACS Sensors (2018), 3 (7), 1308-1315CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The challenge when employing solid-state nanopores as single-mol. sensors in a given assay is the specificity of the ionic current signal during the translocation of target mols. Here the authors present the capture and translocation characteristics of short structurally defined DNA mols. that could serve as effective surrogate labels in biosensing applications. The authors produced T-shaped or Y-shaped DNA mols. with a 50 bp double-stranded DNA (dsDNA) backbone and a 25 bp dsDNA branch in the middle, as improved labels over short linear DNA fragments. The authors show that mol. topologies can be distinguished from linear DNA by analyzing ionic current blockades produced as these DNA labels translocate through nanopores fabricated by controlled breakdown on 10-nm-thick SiN membranes and ranging in diam. from 4 to 10 nm. Event signatures are shown to be a direct result of the structure of the label and lead to an increased signal-to-noise ratio over that of short linear dsDNA, in addn. to well resolved dwell times for the pore size in this range. These results show that structurally defined branched DNA mols. can be robustly detected for a broad range of pore size, and thus represent promising candidates as surrogate labels in a variety of nanopore-based mol. or immunoassay schemes.
- 60Kong, J.; Bell, N. A. W.; Keyser, U. F. Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores. Nano Lett. 2016, 16, 3557– 3562, DOI: 10.1021/acs.nanolett.6b00627Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvVKlsLo%253D&md5=2ce28ab30d6655e9af9c2fa84137d8cdQuantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State NanoporesKong, Jinglin; Bell, Nicholas A. W.; Keyser, Ulrich F.Nano Letters (2016), 16 (6), 3557-3562CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein mols. bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concns. in the nanomolar range. After incubation with target protein at different concns., the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concn. For our proof-of-principle expts. we use two std. binding systems, biotin-streptavidin and digoxigenin-antidigoxigenin, that allow for measurements of the concn. down to the low nanomolar range. The results demonstrate the potential for a novel quant. and specific protein detection scheme using the DNA carrier method.
- 61Chen, K. Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores. Nano Lett. 2017, 17, 5199– 5205, DOI: 10.1021/acs.nanolett.7b01009Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlKisbzE&md5=2a172c6bacb68355ddf0c3501088fbb5Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State NanoporesChen, Kaikai; Juhasz, Matyas; Gularek, Felix; Weinhold, Elmar; Tian, Yu; Keyser, Ulrich F.; Bell, Nicholas A. W.Nano Letters (2017), 17 (9), 5199-5205CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Nanopore sensors show great potential for rapid, single-mol. detn. of DNA sequence information. Here, we develop an ionic current-based method for detg. the positions of short sequence motifs in double-stranded DNA mols. with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-L-methionine cofactor analog we create covalently attached biotin labels at 5'-TCGA-3' sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to addnl. current blockade signals when the DNA passes through a conical quartz nanopore. We det. the relationship between translocation time and position along the DNA contour and find a min. resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA mols. by detg. the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixt. Our method provides a simple, generic single-mol. detection platform enabling DNA characterization in an elec. format suited for portable devices for potential diagnostic applications.
- 62Wang, V.; Ermann, N.; Keyser, U. F. Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA Structure. Nano Lett. 2019, 19, 5661– 5666, DOI: 10.1021/acs.nanolett.9b02219Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlyis7zP&md5=f1ebf842f945a11f4c9070840f27d128Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA StructureWang, Vivian; Ermann, Niklas; Keyser, Ulrich F.Nano Letters (2019), 19 (8), 5661-5666CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The translocation of double-stranded DNA through a solid-state nanopore may either decrease or increase the ionic current depending on the ionic concn. of the surrounding soln. Below a certain crossover ionic concn., the current change inverts from a current blockade to current enhancement. In this paper, we show that the crossover concn. for bundled DNA nanostructures composed of multiple connected DNA double-helixes is lower than that of double-stranded DNA. Our measurements suggest that counterion mobility in the vicinity of DNA is reduced depending on the three-dimensional structure of the mol. We further demonstrate that introducing neutral polymers such as polyethylene glycol into the measurement soln. reduces electroosmotic outflow from the nanopore, allowing translocation of large DNA structures at low salt concns. Our expts. contribute to an improved understanding of ion transport in confined DNA environments, which is crit. for the development of nanopore sensing techniques as well as synthetic membrane channels. Our salt-dependent measurements of model DNA nanostructures will guide the development of computational models of DNA translocation through nanopores.
- 63Lastra, L. S.; Bandara, Y. M. N. D. Y.; Nguyen, M.; Farajpour, N.; Freedman, K. J. On the origins of conductive pulse sensing inside a nanopore. Nat. Commun. 2022, 13, 2186, DOI: 10.1038/s41467-022-29758-8Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht12mu7vM&md5=577a2bf5b893a71ff43767cc9969e475On the origins of conductive pulse sensing inside a nanoporeLastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Nguyen, Michelle; Farajpour, Nasim; Freedman, Kevin J.Nature Communications (2022), 13 (1), 2186CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concns. Contrarily, conductive pulses are obsd. under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a mol.-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and expts. underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chem., phys., and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).
- 64Ivanov, A. P. On-Demand Delivery of Single DNA Molecules Using Nanopipets. ACS Nano 2015, 9, 3587– 3595, DOI: 10.1021/acsnano.5b00911Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvF2htL8%253D&md5=0405a261544828d3b653ba3b7b692590On-Demand Delivery of Single DNA Molecules Using NanopipetsIvanov, Aleksandar P.; Actis, Paolo; Jonsson, Peter; Klenerman, David; Korchev, Yuri; Edel, Joshua B.ACS Nano (2015), 9 (4), 3587-3595CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Understanding the behavioral properties of single mols. or larger scale populations interacting with single mols. is currently a hotly pursued topic in nanotechnol. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single mol. detection strategies have existed for a no. of years, currently lacking are efficient methods for the controllable delivery of single mols. in aq. environments. In this article we show both exptl. and from simulations that nanopipets in conjunction with asym. voltage pulses can be used for label-free detection and delivery of single mols. through the tip of a nanopipet with "on-demand" timing resoln. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA mols. from solns. with concns. as low as 3 pM.
- 65Bhattacharya, S.; Satpati, P. Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations. ACS Omega 2023, 8, 1817– 1837, DOI: 10.1021/acsomega.2c05583Google ScholarThere is no corresponding record for this reference.
- 66Pickar-Oliver, A.; Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 2019, 20, 490– 507, DOI: 10.1038/s41580-019-0131-5Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVOju7jM&md5=35a684f4388b64c543d4dcc37c7543ceThe next generation of CRISPR-Cas technologies and applicationsPickar-Oliver, Adrian; Gersbach, Charles A.Nature Reviews Molecular Cell Biology (2019), 20 (8), 490-507CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)A review. The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technol. have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homol.-directed repair for DNA repair, as well as single-base editing enzymes. In addn. to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biol. processes and are propelling CRISPR-Cas-based tools toward clin. use in gene and cell therapies.
- 67Wiedenheft, B. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10092– 10097, DOI: 10.1073/pnas.1102716108Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXot1egtbs%253D&md5=c43f31a58f29e3e3222b9826129f861dRNA-guide complex from a bacterial immune system enhances target recognition through seed sequence interactionsWiedenheft, Blake; van Duijn, Esther; Bultema, Jelle; Waghmare, Sakharam; Zhou, Kaihong; Barendregt, Arjan; Westphal, Wiebke; Heck, Albert; Boekem, Egbert; Dickman, Mark; Doudn, Jennifer A.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (25), 10092-10097, S10092/1-S10092/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-assocd. (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural anal. of the complex by small-angle x-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-assocd. complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.
- 68Pacesa, M. R-loop formation and conformational activation mechanisms of Cas9. Nature 2022, 609, 191– 196, DOI: 10.1038/s41586-022-05114-0Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1amu77N&md5=6d2d51ab4d1d69b8380e93bd8b29d5fcR-loop formation and conformational activation mechanisms of Cas9Pacesa, Martin; Loeff, Luuk; Querques, Irma; Muckenfuss, Lena M.; Sawicka, Marta; Jinek, MartinNature (London, United Kingdom) (2022), 609 (7925), 191-196CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Cas9 is a CRISPR-assocd. endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 was widely used for genome editing applications4-6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here the authors report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a pos. charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.
- 69Sternberg, S. H.; Redding, S.; Jinek, M.; Greene, E. C.; Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 2014, 507, 62– 67, DOI: 10.1038/nature13011Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjs1GgtL0%253D&md5=e66bf209c17f583057ea2ce0d8a23a23DNA interrogation by the CRISPR RNA-guided endonuclease Cas9Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.Nature (London, United Kingdom) (2014), 507 (7490), 62-67CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The clustered regularly interspaced short palindromic repeats (CRISPR)-assocd. enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-mol. and bulk biochem. expts. to det. how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM d., and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand sepn. and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA mols., and to regulate scission of double-stranded DNA.
- 70Li, T.; Yang, Y.; Qi, H.; Cui, W.; Zhang, L.; Fu, X.; He, X.; Liu, M.; Li, P. f.; Yu, T. CRISPR/Cas9 therapeutics: progress and prospects. Signal Transduction Targeted Ther. 2023, 8, 36, DOI: 10.1038/s41392-023-01309-7Google ScholarThere is no corresponding record for this reference.
- 71Cameron, P. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 2017, 14, 600– 606, DOI: 10.1038/nmeth.4284Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVCms7o%253D&md5=16b95f82fa2694bc366bac620914b9eaMapping the genomic landscape of CRISPR-Cas9 cleavageCameron, Peter; Fuller, Chris K.; Donohoue, Paul D.; Jones, Brittnee N.; Thompson, Matthew S.; Carter, Matthew M.; Gradia, Scott; Vidal, Bastien; Garner, Elizabeth; Slorach, Euan M.; Lau, Elaine; Banh, Lynda M.; Lied, Alexandra M.; Edwards, Leslie S.; Settle, Alexander H.; Capurso, Daniel; Llaca, Victor; Deschamps, Stephane; Cigan, Mark; Young, Joshua K.; May, Andrew P.Nature Methods (2017), 14 (6), 600-606CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochem. method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexs are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The no. of sites identified depended on sgRNA sequence and nuclease concn. Sites identified at lower concns. showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochem. identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
- 72Tsai, S. Q. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 2017, 14, 607– 614, DOI: 10.1038/nmeth.4278Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVCms70%253D&md5=9f36a171aba1728a693d260cf7151c33CIRCLE-seq: a highly sensit13ive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targetsTsai, Shengdar Q.; Nguyen, Nhu T.; Malagon-Lopez, Jose; Topkar, Ved V.; Aryee, Martin J.; Joung, J. KeithNature Methods (2017), 14 (6), 607-614CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochem. methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations assocd. with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochem. approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technol. and does not require ref. genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations assocd. with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.
- 73Zhang, L. Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes. Nucleic Acids Res. 2020, 48, 5037– 5053, DOI: 10.1093/nar/gkaa231Google ScholarThere is no corresponding record for this reference.
- 74Pacesa, M. Structural basis for Cas9 off-target activity. Cell 2022, 185, 4067, DOI: 10.1016/j.cell.2022.09.026Google ScholarThere is no corresponding record for this reference.
- 75Boyle, E. A.; Becker, W. R.; Bai, H. B.; Chen, J. S.; Doudna, J. A.; Greenleaf, W. J. Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement. Sci. Adv. 2021, 7, eabe5496 DOI: 10.1126/sciadv.abe5496Google ScholarThere is no corresponding record for this reference.
- 76Ivanov, I. E. Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 5853– 5860, DOI: 10.1073/pnas.1913445117Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Wksr4%253D&md5=363156f6b639f86f5002ba36e43157a5Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoilingIvanov, Ivan E.; Wright, Addison V.; Cofsky, Joshua C.; Palacio Aris, Kevin D.; Doudna, Jennifer A.; Bryant, ZevProceedings of the National Academy of Sciences of the United States of America (2020), 117 (11), 5853-5860CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The CRISPR-Cas9 nuclease has been widely repurposed as a mol. and cell biol. tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its assocd. guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mech. description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-mol. technique that can simultaneously monitor DNA unwinding with base-pair resoln. and binding of fluorescently labeled macromols. in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mech. perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk expts. we observe promiscuous cleavage under physiol. neg. supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.
- 77Jones, S. K. Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nat. Biotechnol. 2021, 39, 84– 93, DOI: 10.1038/s41587-020-0646-5Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslKhu7jP&md5=04276aac1ce05ba8f74f72b9875d490fMassively parallel kinetic profiling of natural and engineered CRISPR nucleasesJones Jr, Stephen K.; Hawkins, John A.; Johnson, Nicole V.; Jung, Cheulhee; Hu, Kuang; Rybarski, James R.; Chen, Janice S.; Doudna, Jennifer A.; Press, William H.; Finkelstein, Ilya J.Nature Biotechnology (2021), 39 (1), 84-93CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets contg. mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophys. model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, esp. Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quant. and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.
- 78Samanta, D.; Ebrahimi, S. B.; Ramani, N.; Mirkin, C. A. Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters. J. Am. Chem. Soc. 2022, 144, 16310– 16315, DOI: 10.1021/jacs.2c07625Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1CktrzK&md5=2985152044d0e4df316d0ed2769cc130Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled ReportersSamanta, Devleena; Ebrahimi, Sasha B.; Ramani, Namrata; Mirkin, Chad A.Journal of the American Chemical Society (2022), 144 (36), 16310-16315CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into soln. can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ~ 75-fold turn-on signal and limits of detection (LODs) as low as ~ 10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ~ 30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temp. (25°C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clin. relevant concns. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show ATP (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the anal. of ATP in human serum samples with sensitivity on par with that of several com. available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.
- 79Kong, D. Direct SARS-CoV-2 Nucleic Acid Detection by Y-Shaped DNA Dual-Probe Transistor Assay. J. Am. Chem. Soc. 2021, 143, 17004– 17014, DOI: 10.1021/jacs.1c06325Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitF2nu73M&md5=eda26ee7dfe0625f88e0e64b41da8d70Direct SARS-CoV-2 Nucleic Acid Detection by Y-Shaped DNA Dual-Probe Transistor AssayKong, Derong; Wang, Xuejun; Gu, Chenjian; Guo, Mingquan; Wang, Yao; Ai, Zhaolin; Zhang, Shen; Chen, Yiheng; Liu, Wentao; Wu, Yungen; Dai, Changhao; Guo, Qianying; Qu, Di; Zhu, Zhaoqin; Xie, Youhua; Liu, Yunqi; Wei, DachengJournal of the American Chemical Society (2021), 143 (41), 17004-17014CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Rapid screening of infected individuals from a large population is an effective means in epidemiol., esp. to contain outbreaks such as COVID-19. The gold std. assays for COVID-19 diagnostics are mainly based on the reverse transcription polymerase chain reaction, which mismatches the requirements for wide-population screening due to time-consuming nucleic acid extn. and amplification procedures. Here, we report a direct nucleic acid assay by using a graphene field-effect transistor (g-FET) with Y-shaped DNA dual probes (Y-dual probes). The assay relies on Y-dual probes modified on g-FET simultaneously targeting ORF1ab and N genes of SARS-CoV-2 nucleic acid, enabling high a recognition ratio and a limit of detection (0.03 copy μL-1) 1-2 orders of magnitude lower than existing nucleic acid assays. The assay realizes the fastest nucleic acid testing (~ 1 min) and achieves direct 5-in-1 pooled testing for the first time. Owing to its rapid, ultrasensitive, easily operated features as well as capability in pooled testing, it holds great promise as a comprehensive tool for population-wide screening of COVID-19 and other epidemics.
- 80Gebala, M. Cation–Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting. J. Am. Chem. Soc. 2015, 137, 14705– 14715, DOI: 10.1021/jacs.5b08395Google Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGms7%252FK&md5=3207df4e121730c93aabd4f60be67cecCation-anion interactions within the nucleic acid ion atmosphere revealed by ion countingGebala, Magdalena; Giambasu, George M.; Lipfert, Jan; Bisaria, Namita; Bonilla, Steve; Li, Guangchao; York, Darrin M.; Herschlag, DanielJournal of the American Chemical Society (2015), 137 (46), 14705-14715CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The ion atm. is a crit. structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Exptl. methods that "count" the no. of ions thermodynamically assocd. with the ion atm. allow dissection of energetic properties of the ion atm., and thus provide direct comparison to theor. results. Previous expts. have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atm. Here, the authors systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, the authors found, surprisingly, that the extent of anion exclusion and cation inclusion significantly depended on salt identity. The differences were prominent at higher concns. and mirrored trends in mean activity coeffs. of the electrolyte solns. Salts with lower activity coeffs. exhibited greater accumulation of both cations and anions within the ion atm., strongly suggesting that cation-anion correlation effects are present in the ion atm. and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation-anion correlations extend to nucleic acid kinetics and thermodn., the authors followed the folding of P4-P6, a domain of the Tetrahymena group I ribozyme, via single-mol. FRET in solns. with different salts. Solns. of identical concn. but lower activity gave slower and less favorable folding. The results revealed hitherto unknown properties of the ion atm. and suggested possible roles of oriented ion pairs or anion-bridged cations in the ion atm. for electrolyte solns. of salts with reduced activity. Consideration of these new results led to a re-evaluation of the strengths and limitations of Poisson-Boltzmann theory and highlighted the need for next-generation at.-level models of the ion atm.
- 81Gebala, M.; Bonilla, S.; Bisaria, N.; Herschlag, D. Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?. J. Am. Chem. Soc. 2016, 138, 10925– 10934, DOI: 10.1021/jacs.6b04289Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1KktL3E&md5=3b6117f074e95f14805dee1e52ec18a0Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?Gebala, Magdalena; Bonilla, Steve; Bisaria, Namita; Herschlag, DanielJournal of the American Chemical Society (2016), 138 (34), 10925-10934CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other mols., and the energetics of these processes are profoundly influenced by the ion atm. that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atm., understanding its properties and effects will require synergy between computational modeling and expt. Prior computational models and expts. suggested that cation occupancy in the ion atm. depends on the size of the cation. However, the computational models were not independently tested and the exptl. obsd. effects were small. Here, the authors evaluated a computational model of ion size effects by exptl. testing a blind prediction made from that model, and they present addnl. exptl. results that extend the understanding of the ion atm. G. M. Giambasu et al. (2015) developed and implemented a 3-dimensional ref. interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helixes and this model predicted that Na+ would outcompete Cs+ by 1.8-2.1-fold; i.e., with Cs+ in 2-fold excess of Na+ the ion atm. would contain equal no. of each cation. However, the authors' ion counting expts. indicated that there was no significant preference for Na+ over Cs+. There was an ∼25% preferential occupancy of Li+ over larger cations in the ion atm., but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, the authors followed the folding of the P4-P6 RNA domain, and showed that differences in folding with different alkali metal ions obsd. at high concn. arose from cation-anion interactions and not cation size effects. Overall, these results provide a crit. test of a computational prediction, fundamental information about ion atm. properties, and parameters that will aid in the development of next-generation nucleic acid computational models.
- 82Cruz-León, S. Twisting DNA by salt. Nucleic Acids Res. 2022, 50, 5726– 5738, DOI: 10.1093/nar/gkac445Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhslSjsrnO&md5=56939d2cc0f7646ba4191197bab98fa8Twisting DNA by saltCruz-Leon, Sergio; Vanderlinden, Willem; Muller, Peter; Forster, Tobias; Staudt, Georgina; Lin, Yi-Yun; Lipfert, Jan; Schwierz, NadineNucleic Acids Research (2022), 50 (10), 5726-5738CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)The structure and properties of DNA depend on the environment, in particular the ion atm. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concn. and identity of the surrounding ions. To resolve how cations influence the twist, we combine single-mol. magnetic tweezer expts. and extensive all-atom mol. dynamics simulations. Two interconnected trends are obsd. for monovalent alkali and divalent alk. earth cations. First, DNA twist increases monotonously with increasing concn. for all ions investigated. Second, for a given salt concn., DNA twist strongly depends on cation identity. At 100 mM concn., DNA twist increases as Na+ < K+ < Rb+ < Ba2+ < Li+ ≈ Cs+ < Sr2+ < Mg2+ < Ca2+. Our mol. dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the obsd. ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs+ and Sr2+. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnol.
- 83SantaLucia, J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1460– 1465, DOI: 10.1073/pnas.95.4.1460Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXht1Wqsbc%253D&md5=1a4e89f9f0caa91aecd5944add0aaf83A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamicsSantalucia, John, Jr.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (4), 1460-1465CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A unified view of polymer, dumbbell, and oligonucleotide nearest-neighbor (NN) thermodn. is presented. DNA NN ΔG37° parameters from seven labs. are presented in the same format so that careful comparisons can be made. The seven studies used data from natural polymers, synthetic polymers, oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN parameters; used different methods of data anal.; used different salt concns.; and presented the NN thermodn. in different formats. As a result of these differences, there has been much confusion regarding the NN thermodn. of DNA polymers and oligomers. Herein I show that six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies remain. Further, a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodn. Empirical salt dependencies are also derived for oligonucleotides and polymers.
- 84Li, Y. CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid. Biochemistry 2023, 62, 2541– 2548, DOI: 10.1021/acs.biochem.3c00250Google ScholarThere is no corresponding record for this reference.
- 85Gong, S.; Yu, H. H.; Johnson, K. A.; Taylor, D. W. DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity. Cell Rep. 2018, 22, 359– 371, DOI: 10.1016/j.celrep.2017.12.041Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzpsVSrsQ%253D%253D&md5=4edeb52aece481f5b53e37c3167918c7DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 ActivityGong Shanzhong; Yu Helen Hong; Johnson Kenneth A; Taylor David WCell reports (2018), 22 (2), 359-371 ISSN:.Bacterial adaptive immunity utilizes RNA-guided surveillance complexes comprising Cas proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single-guide RNA that base pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and the RuvC nuclease domains cleave opposing strands. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, and R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.
- 86Kimsey, I. J.; Petzold, K.; Sathyamoorthy, B.; Stein, Z. W.; Al-Hashimi, H. M. Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes. Nature 2015, 519, 315– 320, DOI: 10.1038/nature14227Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXks1yqsL4%253D&md5=27816c16fe99c08b4c036c8611c6dae1Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexesKimsey, Isaac J.; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W.; Al-Hashimi, Hashim M.Nature (London, United Kingdom) (2015), 519 (7543), 315-320CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Rare tautomeric and anionic nucleobases are believed to have fundamental biol. roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equil. with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10-3 to 10-5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.
- 87Leontis, N. B. The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res. 2002, 30, 3497– 3531, DOI: 10.1093/nar/gkf481Google Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xms1Klsb8%253D&md5=d6f4bc813e7d72e1a06a8b3658d08aebThe non-Watson-Crick base pairs and their associated isostericity matricesLeontis, Neocles B.; Stombaugh, Jesse; Westhof, EricNucleic Acids Research (2002), 30 (16), 3497-3531CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)RNA mols. exhibit complex structures in which a large fraction of the bases engage in non-Watson-Crick base pairing, forming motifs that mediate long-range RNA-RNA interactions and create binding sites for proteins and small mol. ligands. The rapidly growing no. of three-dimensional RNA structures at. resoln. requires that databases contain the annotation of such base pairs. An unambiguous and descriptive nomenclature was proposed recently in which RNA base pairs were classified by the base edges participating in the interaction (Watson-Crick, Hoogsteen/CH or sugar edge) and the orientation of the glycosidic bonds relative to the hydrogen bonds (cis or trans). Twelve basic geometric families were identified and all 12 have been obsd. in crystal structures. For each base pairing family, we present here the 4×4 isostericity matrixes' summarizing the geometric relationships between the 16 pairwise combinations of the four std. bases, A, C, G and U. Whenever available, a representative example of each obsd. base pair from X-ray crystal structures (3.0 Å resoln. or better) is provided or, otherwise, theor. plausible models. This format makes apparent the recurrent geometric patterns that are obsd. and helps identify isosteric pairs that co-vary or interchange in sequences of homologous mols. while maintaining conserved three-dimensional motifs.
- 88Maity, H.; Baidya, L.; Reddy, G. Salt-Induced Transitions in the Conformational Ensembles of Intrinsically Disordered Proteins. J. Phys. Chem. B 2022, 126, 5959– 5971, DOI: 10.1021/acs.jpcb.2c03476Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitVKkt77P&md5=8e7f72a55f4245a2dfeb5133798f30bcSalt-Induced Transitions in the Conformational Ensembles of Intrinsically Disordered ProteinsMaity, Hiranmay; Baidya, Lipika; Reddy, GovardhanJournal of Physical Chemistry B (2022), 126 (32), 5959-5971CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Salts modulate the behavior of intrinsically disordered proteins (IDPs) and influence the formation of membraneless organelles through liq.-liq. phase sepn. (LLPS). In low ionic strength solns., IDP conformations are perturbed by the screening of electrostatic interactions, independent of the salt identity. In this regime, insight into the IDP behavior can be obtained using the theory for salt-induced transitions in charged polymers. However, salt specific interactions with the charged and uncharged residues, known as the Hofmeister effect, influence IDP behavior in high ionic strength solns. There is a lack of reliable theor. models in high salt concn. regimes to predict the salt effect on IDPs. The authors propose a simulation methodol. using a coarse-grained IDP model and exptl. measured water to salt soln. transfer free energies of various chem. groups that allowed the authors to study the salt specific transitions induced in the IDPs conformational ensemble. The authors probed the effect of three different monovalent salts on five IDPs belonging to various polymer classes based on charged residue content. All the IDPs of different polymer classes behave as self-avoiding walks (SAW) at physiol. salt concn. In high salt concns., the transitions obsd. in the IDP conformational ensembles are dependent on the salt used and the IDP sequence and compn. Changing the anion with the cation fixed can result in the IDP transition from a SAW like-behavior to a collapsed globule. An important implication of these results is that a suitable salt can be identified to induce condensation of an IDP through LLPS.
- 89Beveridge, R. Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins. J. Am. Chem. Soc. 2019, 141, 4908– 4918, DOI: 10.1021/jacs.8b13483Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktV2mtb0%253D&md5=9877691dbb9c1737e10e1643befff8baIon Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered ProteinsBeveridge, Rebecca; Migas, Lukasz G.; Das, Rahul K.; Pappu, Rohit V.; Kriwacki, Richard W.; Barran, Perdita E.Journal of the American Chemical Society (2019), 141 (12), 4908-4918CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation vs. clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from soln. on the basis of charge and shape. Here, the authors use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. The authors report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compns., κ14 and κ56, distinguished by the extent of linear mixing vs. segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in soln. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase mol. dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concn., with the wild-type sequence showing the most sensitivity to changes in salt concn. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.
- 90Liu, Y. Single-Molecule Detection of α-Synuclein Oligomers in Parkinson’s Disease Patients Using Nanopores. ACS Nano 2023, 17, 22999– 23009, DOI: 10.1021/acsnano.3c08456Google ScholarThere is no corresponding record for this reference.
- 91Byrd, E. J.; Wilkinson, M.; Radford, S. E.; Sobott, F. Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein. J. Am. Soc. Mass Spectrom. 2023, 34, 493– 504, DOI: 10.1021/jasms.2c00379Google ScholarThere is no corresponding record for this reference.
- 92Wang, H.; Wu, J.; Sternke-Hoffmann, R.; Zheng, W.; Mörman, C.; Luo, J. Multivariate effects of pH, salt, and Zn2+ ions on Aβ40 fibrillation. Commun. Chem. 2022, 5, 171, DOI: 10.1038/s42004-022-00786-1Google ScholarThere is no corresponding record for this reference.
- 93Yoshimura, Y. Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 14446– 14451, DOI: 10.1073/pnas.1208228109Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVaqurfE&md5=860aa8e3ba70b377b56cade4c8753a17Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formationYoshimura, Yuichi; Lin, Yuxi; Yagi, Hisashi; Lee, Young-Ho; Kitayama, Hiroki; Sakurai, Kazumasa; So, Masatomo; Ogi, Hirotsugu; Naiki, Hironobu; Goto, YujiProceedings of the National Academy of Sciences of the United States of America (2012), 109 (36), 14446-14451, S14446/1-S14446/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates assocd. with protein misfolding diseases. Although they differ in morphol., the two forms are often treated indiscriminately. β2-Microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concn. at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersatd. soln., analogous to crystn. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradn., and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Soly. and supersatn. will be key factors for further understanding the aberrant aggregation of proteins.
- 94Gaspar, R.; Lund, M.; Sparr, E.; Linse, S. Anomalous Salt Dependence Reveals an Interplay of Attractive and Repulsive Electrostatic Interactions in α-synuclein Fibril Formation. QRB discov. 2020, 1, e2 DOI: 10.1017/qrd.2020.7Google ScholarThere is no corresponding record for this reference.
- 95Yusko, E. C. Single-Particle Characterization of Aβ Oligomers in Solution. ACS Nano 2012, 6, 5909– 5919, DOI: 10.1021/nn300542qGoogle Scholar95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotlShu7Y%253D&md5=24c8e5c564399c975d2389c1c94a20d2Single-Particle Characterization of Aβ Oligomers in SolutionYusko, Erik C.; Prangkio, Panchika; Sept, David; Rollings, Ryan C.; Li, Jiali; Mayer, MichaelACS Nano (2012), 6 (7), 5909-5919CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Detg. the pathol. role of amyloids in amyloid-assocd. diseases will require a method for characterizing the dynamic distributions in size and shape of amyloid oligomers with high resoln. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloid-β oligomers directly in soln. and without chem. modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β aggregate sizes. The approach revealed the distribution of protofibrillar lengths (12- to 155-mer) as well as the av. cross-sectional area of protofibrils and fibers.
- 96Awasthi, S.; Ying, C.; Li, J.; Mayer, M. Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution. ACS Nano 2023, 17, 12325– 12335, DOI: 10.1021/acsnano.3c01393Google ScholarThere is no corresponding record for this reference.
- 97Zhao, C. DNase-targeted natural product screening based on a sensitive and selective DNase I detecting system. RSC Adv. 2017, 7, 30911– 30918, DOI: 10.1039/C7RA04911KGoogle ScholarThere is no corresponding record for this reference.
- 98Chau, C. C.; Weckman, N. E.; Thomson, E. E.; Actis, P. Solid-State Nanopore Real Time Assay for Monitoring Cas9 Endonuclease Reactivity. bioRxiv 2024, bioRxiv:2024.09.20.612695, DOI: 10.1101/2024.09.20.612695Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. Generation of the restriction site containing 3 kbp dsDNA. (A) Schematic illustration of the generation of the restriction site containing 3 kbp dsDNA (RS-dsDNA). (B) Agarose gel electrophoresis analysis of the undigested RS-dsDNA and the digested RS-dsDNA; the 3 kbp original fragment was digested into 1.5 kbp dsDNA.
Figure 2
Figure 2. Restriction enzyme SwaI cleavage activities. (A) Schematic illustration of the glass nanopore detection setup. The cis chamber of the nanopore is filled with the restriction enzyme SwaI, the RS-dsDNA, the trans chamber is composed of a polymer electrolyte mixture (0.1 M KCl, 50% (w/v) PEG 35K). Application of a negative voltage causes the dsDNA to migrate from the cis to the trans chamber. The RS-dsDNA was mixed with the SwaI restriction enzyme and digestion buffer to a final concentration of 9.24 nM RS-dsDNA, 5 units of enzyme, and 1× digestion buffer. (B) 20 random translocation event peaks plotted as overlay. The cis chamber of the glass nanopore was filled with 10 nM RS-dsDNA diluted with buffer 3.1 (Table 1) containing 5 units of SwaI enzyme. (C) Population distribution of the translocation events at 1, 10, 20, and 30 min. The arrowheads across the four plots point at the population centered at approximately 0.2 ms and 0.25 nA. This population is attributed to the larger RS-dsDNA prior to digestion. As the time progresses to 10, 20 min and finally at 30 min, this population gradually disappears and a secondary population centered at approximately 0.15 ms and 0.15 nA begins to emerge; the side histograms of the peak amplitude axes show the emergence of the 0.15 nA population. The color bar represents the count of events found in each hexagon.
Figure 3
Figure 3. Digestion of RS-dsDNA monitored over the course of one hour. The RS-dsDNA was mixed with the SwaI restriction enzyme and digestion buffer to a final concentration of 9.24 nM RS-dsDNA, 5 units of enzyme, and 1× digestion buffer. (A) The ridgeline plot shows the gradual population changes from 1 to 60 min. (B) The probability of detecting the translocation of RS-dsDNA drops from 60% to near 0%. Two boundaries were defined as the ±10% of the peak value of the RS-dsDNA using the 1 min data, and the same boundaries were applied across all the data. The probability value was calculated by integrating the area under the curves (AUC) between the boundaries shown in (A); the initial starting percentage changes according to the width of the boundaries. (C) Enzyme reaction rate (slope average) plotted against the concentration of the enzyme. The enzyme reaction rate was calculated by fitting a linear regression line at the first 15 min (initial velocity region) of digestion under different enzyme concentrations (n = 3); the reaction rate obtained from the linear regression line is thus defined as the changes in probability of detecting the RS-dsDNA over the changes in time. The coefficient of determination for the fit is R2 = 0.9822. Error bars represent standard error of the mean of the slope values between measurements. According to the manufacturer, a single unit of SwaI is defined as the amount of enzyme required to digest 1 μg of pXba DNA in 1 h at 25 °C in a total reaction volume of 50 μL.
Figure 4
Figure 4. Buffer-dependent restriction enzyme kinetics. (A) Restriction digestion of the RS-dsDNA in different buffers. The optimal buffer for the restriction enzyme SwaI is the buffer 3.1, as recommended by the supplier. Three other buffers (4, 2.1 and CutSmart) were tested, and the activity of SwaI varied and resulted in lower digestion activity in buffer 4 and CutSmart. (B) The gel band intensity was quantified and calculated relative to the 1.5 kbp’s band intensity within the sample lane (self-reference). (C) Probability of detecting 3 kbp dsDNA as a function of the digestion time for all the buffer tested and controls. m is the slope after fitting with the linear fit to the experimental data. Error bars are standard error of the mean. (D) Box plot comparing the probability of detecting the RS-dsDNA at 1 min and at 30 min. The two-tailed unpaired t-test was used to test the differences between the distribution of the probabilities to detect RS-dsDNA at 1 min and at 30 min. There are significant differences for buffer 3.1, buffer 2.1, and CutSmart at 1 min and at 30 min. The calculated values for buffer 3.1 at 1 min is 69.35 ± 3.94% and 19.28 ± 2.95% at 30 min, respectively; for buffer 4 at 1 min, it is 78.51 ± 4.55% and 69.43 ± 4.4%, respectively; for buffer 2.1 at 1 min, it is 89.54 ± 4.77% and 25.6 ± 4.44%, respectively; for CutSmart at 1 min, it is 79.81 ± 4.23% and 59.56 ± 5.42%, respectively; for no digestion at 1 min, it is 93.31 ± 2.76% and 89.17 ± 3.62%, respectively; for digested at 1 min, it is 8.52 ± 1.72% and 7.26 ± 2.15%, respectively. (ns, not significant; ****P < 0.0001; **P < 0.005; data assume normal distribution; Levene’s test (P > 0.05) indicates data have homoscedasticity; N = 3). (E) Ridgeline plots for buffer 4, buffer 2.1, and CutSmart. The cis chamber of the glass nanopore was filled with 10 nM RS-dsDNA diluted with buffer 3.1, buffer 4, buffer 2.1, or CutSmart (Table 1) containing 5 units of SwaI enzyme.
Figure 5
Figure 5. CRISPR-Cas9-mediated dsDNA cleavage. (A) Schematic illustrating the process of the Cas9 mediated cleavage on the 3 kbp RS-dsDNA. The Cas9 was mixed with tracrRNA and crRNA to form the RNP complex. The RNA guides the Cas9 RNP to the position next to the PAM site. The Cas9 RNP then carries out double stranded cleavage 3–4 bp upstream of the PAM site to cleave the dsDNA. This resulted in the formation of 2 × 1.5 kbp dsDNA. (B) On-target and off-target crRNA sequence. The full complementary sequence (On target) and the off target variations at different positions upstream of the PAM site and different mismatches. (C) Agarose gel electrophoresis following 30 min incubation at 25 °C and overnight incubation at 4 °C. Cas9 RNPs were formed with the on-target crRNA or off-target crRNA variants. The gel band intensity was quantified and calculated relative to the 1.5 kbp’s band intensity within the sample lane (self-reference).
Figure 6
Figure 6. Measuring the activity of the Cas9 endonuclease with the nanopore. (A) Ridgeline plot showing the KDE estimated PDFs of the translocation experiment at each min for the 5 tested on-target and off-target crRNA sequences. (B) Probability of detecting 3 kbp dsDNA as a function of the digestion time for all the variants tested and controls. m is the slope after fitting with the linear fit to the experimental data. Error bars are standard error of the mean. (C) Box plot comparing the probability of detecting the RS-dsDNA at 1 min and at 30 min. The two-tailed unpaired t-test was used to test the differences between the distribution of the probabilities to detect RS-dsDNA at 1 min and at 30 min. There are significant differences for the on-target, off-target 2, and off-target 3 at 1 min and at 30 min. The calculated values for on target at 1 min are 28.31 ± 8.55% and 8.01 ± 5.37% at 30 min, respectively; for off target 1 at 1 min, it is 68.67 ± 6.57% and 61.08 ± 0.94%, respectively; for off-target 2 at 1 min, it is 50.25 ± 5.93% and 25.79 ± 10.6%, respectively; for off-target 3 at 1 min, it is 39.76 ± 5.65% and 12.04 ± 3.67%, respectively; for off-target 4 at 1 min, it is 77.95 ± 1.67% and 69.36 ± 6.11%, respectively; for no digestion at 1 min, it is 84.22 ± 3.38% and 80.44 ± 7.56%, respectively; for digested at 1 min, it is 8.52 ± 1.72% and 4.39 ± 2.07%, respectively. (ns, not significant; *P < 0.05; **P < 0.005; data assume normal distribution; Levene’s test (P > 0.05) indicates data have homoscedasticity; N = 3, two-tailed t-test).
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- 8Ying, Y.-L. Single-entity electrochemistry at confined sensing interfaces. Sci. China:Chem. 2020, 63, 589– 618, DOI: 10.1007/s11426-020-9716-28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVOqtbk%253D&md5=a7c9d126321f182076948ba8846c230fSingle-entity electrochemistry at confined sensing interfacesYing, Yi-Lun; Wang, Jiajun; Leach, Anna Rose; Jiang, Ying; Gao, Rui; Xu, Cong; Edwards, Martin A.; Pendergast, Andrew D.; Ren, Hang; Weatherly, Connor K. Terry; Wang, Wei; Actis, Paolo; Mao, Lanqun; White, Henry S.; Long, Yi-TaoScience China: Chemistry (2020), 63 (5), 589-618CODEN: SCCCCS; ISSN:1869-1870. (Science China Press)A review. Absract: Measurements at the single-entity level provide more precise diagnosis and understanding of basic biol. and chem. processes. Recent advances in the chem. measurement provide a means for ultra-sensitive anal. Confining the single analyte and electrons near the sensing interface can greatly enhance the sensitivity and selectivity. In this review, we summarize the recent progress in single-entity electrochem. of single mols., single particles, single cells and even brain anal. The benefits of confining these entities to a compatible size sensing interface are exemplified. Finally, the opportunities and challenges of single entity electrochem. are addressed.
- 9Ahmed, S. A. Iontronic Sensing Based on Confined Ion Transport. Anal. Chem. 2024, 96, 8056– 8077, DOI: 10.1021/acs.analchem.4c01354There is no corresponding record for this reference.
- 10Wang, Y.; Zhao, Y.; Bollas, A.; Wang, Y.; Au, K. F. Nanopore sequencing technology, bioinformatics and applications. Nat. Biotechnol. 2021, 39, 1348– 1365, DOI: 10.1038/s41587-021-01108-x10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVChu7bK&md5=e8e31d95498d099fd7d1ace564f3e433Nanopore sequencing technology, bioinformatics and applicationsWang, Yunhao; Zhao, Yue; Bollas, Audrey; Wang, Yuru; Au, Kin FaiNature Biotechnology (2021), 39 (11), 1348-1365CODEN: NABIF9; ISSN:1087-0156. (Nature Portfolio)Abstr.: Rapid advances in nanopore technologies for sequencing single long DNA and RNA mols. have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of exptl. and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clin. diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and anal. approaches through the development of new nanopores, base-calling methods and exptl. protocols tailored to particular applications.
- 11Xue, L. Solid-state nanopore sensors. Nat. Rev. Mater. 2020, 5, 931– 951, DOI: 10.1038/s41578-020-0229-611https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlWks7bM&md5=f9836bb89501dd6210512501c23790fcSolid-state nanopore sensorsXue, Liang; Yamazaki, Hirohito; Ren, Ren; Wanunu, Meni; Ivanov, Aleksandar P.; Edel, Joshua B.Nature Reviews Materials (2020), 5 (12), 931-951CODEN: NRMADL; ISSN:2058-8437. (Nature Research)A review. Abstr.: Nanopore-based sensors have established themselves as a prominent tool for soln.-based, single-mol. anal. of the key building blocks of life, including nucleic acids, proteins, glycans and a large pool of biomols. that have an essential role in life and healthcare. The predominant mol. readout method is based on measuring the temporal fluctuations in the ionic current through the pore. Recent advances in materials science and surface chemistries have not only enabled more robust and sensitive devices but also facilitated alternative detection modalities based on field-effect transistors, quantum tunnelling and optical methods such as fluorescence and plasmonic sensing. In this Review, we discuss recent advances in nanopore fabrication and sensing strategies that endow nanopores not only with sensitivity but also with selectivity and high throughput, and highlight some of the challenges that still need to be addressed.
- 12Albrecht, T. Single-Molecule Analysis with Solid-State Nanopores. Annu. Rev. Anal. Chem. 2019, 12, 371– 387, DOI: 10.1146/annurev-anchem-061417-12590312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cjnvVOjsQ%253D%253D&md5=01b2499b53cb6faef3d8314b2467bc9dSingle-Molecule Analysis with Solid-State NanoporesAlbrecht TimAnnual review of analytical chemistry (Palo Alto, Calif.) (2019), 12 (1), 371-387 ISSN:.Solid-state nanopores and nanopipettes are an exciting class of single-molecule sensors that has grown enormously over the last two decades. They offer a platform for testing fundamental concepts of stochasticity and transport at the nanoscale, for studying single-molecule biophysics and, increasingly, also for new analytical applications and in biomedical sensing. This review covers some fundamental aspects underpinning sensor operation and transport and, at the same time, it aims to put these into context as an analytical technique. It highlights new and recent developments and discusses some of the challenges lying ahead.
- 13Mayer, S. F.; Cao, C.; Dal Peraro, M. Biological nanopores for single-molecule sensing. iScience 2022, 25, 104145, DOI: 10.1016/j.isci.2022.10414513https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsFOktL3N&md5=be04fb1d36349cea136fa0d297c82573Biological nanopores for single-molecule sensingMayer, Simon Finn; Cao, Chan; Dal Peraro, MatteoiScience (2022), 25 (4), 104145CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)A review. Evolution has found countless ways to transport material across cells and cellular compartments sepd. by membranes. Protein assemblies are the cornerstone for the formation of channels and pores that enable this regulated passage of mols. in and out of cells, contributing to maintaining most of the fundamental processes that sustain living organisms. As in several other occasions, we have borrowed from the natural properties of these biol. systems to push technol. forward and have been able to hijack these nano-scale proteinaceous pores to learn about the phys. and chem. features of mols. passing through them. Today, a large repertoire of biol. pores is exploited as mol. sensors for characterizing biomols. that are relevant for the advancement of life sciences and application to medicine. Although the technol. has quickly matured to enable nucleic acid sensing with transformative implications for genomics, biol. pores stand as some of the most promising candidates to drive the next developments in single-mol. proteomics.
- 14Ying, Y.-L. Nanopore-based technologies beyond DNA sequencing. Nat. Nanotechnol. 2022, 17, 1136– 1146, DOI: 10.1038/s41565-022-01193-214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisV2nsrbM&md5=f11d8860754531fc1fe1fcf71f1097eeNanopore-based technologies beyond DNA sequencingYing, Yi-Lun; Hu, Zheng-Li; Zhang, Shengli; Qing, Yujia; Fragasso, Alessio; Maglia, Giovanni; Meller, Amit; Bayley, Hagan; Dekker, Cees; Long, Yi-TaoNature Nanotechnology (2022), 17 (11), 1136-1146CODEN: NNAABX; ISSN:1748-3387. (Nature Portfolio)Abstr.: Inspired by the biol. processes of mol. recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive anal. tools for individual mols. In particular, nanopore-based single-mol. DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in mol. sensing and sequencing, chem. catalysis and biophys. characterization. We highlight the prospects of applying nanopores for single-protein anal. and sequencing, single-mol. covalent chem., clin. sensing applications for single-mol. liq. biopsy, and the use of synthetic biomimetic nanopores as exptl. models for natural systems. We suggest that nanopore technologies will continue to be explored to address a no. of scientific challenges as control over pore design improves.
- 15Varongchayakul, N.; Song, J.; Meller, A.; Grinstaff, M. W. Single-molecule protein sensing in a nanopore: a tutorial. Chem. Soc. Rev. 2018, 47, 8512– 8524, DOI: 10.1039/C8CS00106E15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFalu7%252FL&md5=aaa171dcde2e09746529db53104492b5Single-molecule protein sensing in a nanopore: a tutorialVarongchayakul, Nitinun; Song, Jiaxi; Meller, Amit; Grinstaff, Mark W.Chemical Society Reviews (2018), 47 (23), 8512-8524CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Proteins are the structural elements and machinery of cells responsible for a functioning biol. architecture and homeostasis. Advances in nanotechnol. are catalyzing key breakthroughs in many areas, including the anal. and study of proteins at the single-mol. level. Nanopore sensing is at the forefront of this revolution. This tutorial review provides readers a guidebook and ref. for detecting and characterizing proteins at the single-mol. level using nanopores. Specifically, the review describes the key materials, nanoscale features, and design requirements of nanopores. It also discusses general design requirements as well as details on the anal. of protein translocation. Finally, the article provides the background necessary to understand current research trends and to encourage the identification of new biomedical applications for protein sensing using nanopores.
- 16Platnich, C. M.; Earle, M. K.; Keyser, U. F. Chemical Annealing Restructures RNA for Nanopore Detection. J. Am. Chem. Soc. 2024, 146, 12919– 12924, DOI: 10.1021/jacs.4c03753There is no corresponding record for this reference.
- 17Bandara, Y. M. N. D. Y.; Farajpour, N.; Freedman, K. J. Nanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein’s Isoelectric Point. J. Am. Chem. Soc. 2022, 144, 3063– 3073, DOI: 10.1021/jacs.1c1154017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xjt1GmsL8%253D&md5=5e7c775eec3b97b1578098d668533dbbNanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein's Isoelectric PointBandara, Y. M. Nuwan D. Y.; Farajpour, Nasim; Freedman, Kevin J.Journal of the American Chemical Society (2022), 144 (7), 3063-3073CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the elec. sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concns. can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irresp. of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelec. point (pI) of Cas9 was found to be correlated and a crit. factor in our expts. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be crit. for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.
- 18Gao, T. Label-Free Resistance Cytometry at the Orifice of a Nanopipette. Anal. Chem. 2021, 93, 2942– 2949, DOI: 10.1021/acs.analchem.0c0458518https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitV2rtrc%253D&md5=7884db87f67bae1dca19c624fcfbb645Label-Free Resistance Cytometry at the Orifice of a NanopipetteGao, Tienan; Gao, Xiangyi; Xu, Cong; Wang, Menglin; Chen, Mingli; Wang, Jianhua; Ma, Furong; Yu, Ping; Mao, LanqunAnalytical Chemistry (Washington, DC, United States) (2021), 93 (5), 2942-2949CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Development of new principles and techniques at the single-cell level is significantly important since cells as basic units of living organisms always bear large heterogeneity. Herein, we demonstrate a new electrochem. principle for single-cell anal. based on an ion current blockage at the orifice of a nanopipette, defined as resistance cytometry. The amplitude and the frequency of ion current transients show strong dependence on the size and the concn. of cells, which could be used for in situ cell sizing and counting. This technique shows good ability to detect the size change of RBCs under stimulations of different pH and osmotic pressure values. More importantly, the as-presented resistance cytometry can distinguish lymphoma blood cells from normal blood cells for patient blood samples. The as-presented resistance cytometry is label-free, non-invasive, and non-destructive, which not only opens new opportunities for single-cell anal. but also provides a new platform for cell-related medical diagnostic technologies.
- 19Shumyantseva, V. V. Enzymology on an Electrode and in a Nanopore: Analysis Algorithms, Enzyme Kinetics, and Perspectives. Bionanosci. 2022, 12, 1341– 1355, DOI: 10.1007/s12668-022-01037-2There is no corresponding record for this reference.
- 20Willems, K.; Van Meervelt, V.; Wloka, C.; Maglia, G. Single-molecule nanopore enzymology. Philos. Trans. R. Soc., B 2017, 372, 20160230, DOI: 10.1098/rstb.2016.0230There is no corresponding record for this reference.
- 21Sheng, Y.; Zhang, S.; Liu, L.; Wu, H. C. Measuring Enzymatic Activities with Nanopores. ChemBioChem 2020, 21, 2089– 2097, DOI: 10.1002/cbic.202000079There is no corresponding record for this reference.
- 22Wloka, C. Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore. ACS Nano 2017, 11, 4387– 4394, DOI: 10.1021/acsnano.6b0776022https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXltVSisLw%253D&md5=ca0b3e1b28d90efa6a3bf94de79ccea7Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological NanoporeWloka, Carsten; Van Meervelt, Veerle; van Gelder, Dewi; Danda, Natasha; Jager, Nienke; Williams, Chris P.; Maglia, GiovanniACS Nano (2017), 11 (5), 4387-4394CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The covalent addn. of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biol. processes. Here, the authors report a fast, single-mol., and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. Ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiol. conditions. Using defined conjugates, also isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.
- 23Galenkamp, N. S.; Biesemans, A.; Maglia, G. Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings. Nat. Chem. 2020, 12, 481– 488, DOI: 10.1038/s41557-020-0437-023https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVyjs74%253D&md5=922cd1a0ecdc2a96faf98a98f7afc622Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordingsGalenkamp, Nicole Stephanie; Biesemans, Annemie; Maglia, GiovanniNature Chemistry (2020), 12 (5), 481-488CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Abstr.: Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodn. and kinetic characterization at the single-mol. level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the elec. recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by mols. that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymic reactions affected by product inhibition or when the release of products is the rate-limiting step.
- 24Robinson, P. K. Enzymes: principles and biotechnological applications. Essays Biochem. 2015, 59, 1– 41, DOI: 10.1042/bse059000124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28zlvFSnsQ%253D%253D&md5=2f68b0d48239c71e0a4da84ae04bba3aEnzymes: principles and biotechnological applicationsRobinson Peter KEssays in biochemistry (2015), 59 (), 1-41 ISSN:.Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.
- 25Cheley, S.; Xie, H.; Bayley, H. A Genetically Encoded Pore for the Stochastic Detection of a Protein Kinase. ChemBioChem 2006, 7, 1923– 1927, DOI: 10.1002/cbic.20060027425https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXltVM%253D&md5=fe2580df39f2b5ee0db138f51f350816A genetically encoded pore for the stochastic detection of a protein kinaseCheley, Stephen; Xie, Hongzhi; Bayley, HaganChemBioChem (2006), 7 (12), 1923-1927CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Stochastic sensing is an emerging approach for the detection of a wide variety of analytes at the level of individual mols. Detection is accomplished by observing the modulation of the current that flows through a single protein pore that has been engineered to bind an analyte of interest. Previously, protein analytes have been detected by using pores to which ligands have been appended at specific sites by targeted chem. modification. Here, we report the first genetically encoded stochastic sensor element for detecting a protein. A protein kinase inhibitor peptide sequence was incorporated into the α-hemolysin polypeptide, which was used to form a heteroheptameric pore contg. a single copy of the inhibitor sequence. With this pore, the successful detection of the catalytic subunit of protein kinase A was demonstrated. This development should greatly facilitate the detection of active kinase subunits by stochastic sensing and the rapid screening of kinase inhibitors by an approach that yields kinetic information.
- 26Harrington, L.; Cheley, S.; Alexander, L. T.; Knapp, S.; Bayley, H. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E4417– E4426, DOI: 10.1073/pnas.131273911026https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFCisL%252FF&md5=a56393697939130134dd120952116711Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrateHarrington, Leon; Cheley, Stephen; Alexander, Leila T.; Knapp, Stefan; Bayley, HaganProceedings of the National Academy of Sciences of the United States of America (2013), 110 (47), E4417-E4426,SE4417/1-SE4417/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In stochastic sensing, the assocn. and dissocn. of analyte mols. is obsd. as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free detn. of rate and equil. consts. with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-mol. detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we obsd. unusually high assocn. rate consts. for the interaction of Pim kinases with their consensus substrate Pimtide (∼107 to 108 M-1·s-1), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.
- 27Harrington, L.; Alexander, L. T.; Knapp, S.; Bayley, H. Single-Molecule Protein Phosphorylation and Dephosphorylation by Nanopore Enzymology. ACS Nano 2019, 13, 633– 641, DOI: 10.1021/acsnano.8b0769727https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1SgtLnF&md5=b86fe4747ab1834504d4edc336ac70f3Single-molecule protein phosphorylation and dephosphorylation by nanopore enzymologyHarrington, Leon; Alexander, Leila T.; Knapp, Stefan; Bayley, HaganACS Nano (2019), 13 (1), 633-641CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Reversible protein phosphorylation plays a crucial and ubiquitous role in the control of almost all cellular processes. The interplay of protein kinases and phosphatases acting in opposition ensures tight dynamic control of protein phosphorylation states within the cell. Previously, engineered α-hemolysin pores bearing kinase substrate peptides have been developed as single-mol. stochastic sensors for protein kinases. Here, we used these pores to observe, label-free, the phosphorylation and dephosphorylation of a single substrate mol., pimtide. Further, we investigated the effect of Mg2+ and Mn2+ upon substrate and product binding and found that Mn2+ relaxed active site specificity toward nucleotides and enhanced product binding. In doing so, we demonstrated the power and versatility of nanopore enzymol. to scrutinize a crit. post-translational modification.
- 28Harrington, L.; Alexander, L. T.; Knapp, S.; Bayley, H. Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor. Angew. Chem., Int. Ed. 2015, 54, 8154– 8159, DOI: 10.1002/anie.20150314128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXps12rsL8%253D&md5=05527b50445ed619574e86b1981f877bPim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore SensorHarrington, Leon; Alexander, Leila T.; Knapp, Stefan; Bayley, HaganAngewandte Chemie, International Edition (2015), 54 (28), 8154-8159CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Protein kinases are crit. therapeutic targets. Pim kinases are implicated in several leukemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We obsd. synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free detn. of inhibition consts. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorometry. The approach is also amenable to implementation on high throughput chips.
- 29Nouri, R.; Jiang, Y.; Lian, X. L.; Guan, W. Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN). ACS Sensors 2020, 5, 1273– 1280, DOI: 10.1021/acssensors.0c0049729https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosF2nsbY%253D&md5=a27c11eb4f9b6c1d6329610570bdc022Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN)Nouri, Reza; Jiang, Yuqian; Lian, Xiaojun Lance; Guan, WeihuaACS Sensors (2020), 5 (5), 1273-1280CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Nucleic acid detection methods are crucial for many fields such as pathogen detection and genotyping. Solid-state nanopore sensors represent a promising platform for nucleic acid detection due to its unique single mol. sensitivity and label-free electronic sensing. Here, we demonstrated the use of the glass nanopore for highly sensitive quantification of single-stranded circular DNAs (reporters), which could be degraded under the trans-cleavage activity of the target-specific CRISPR-Cas12a. We developed and optimized the Cas12a assay for HIV-1 anal. We validated the concept of the solid-state CRISPR-Cas12a-assisted nanopores (SCAN) to specifically detect the HIV-1 DNAs. We showed that the glass nanopore sensor is effective in monitoring the cleavage activity of the target DNA-activated Cas12a. We developed a model to predict the total exptl. time needed for making a statistically confident pos./neg. call in a qual. test. The SCAN concept combines the much-needed specificity and sensitivity into a single platform, and we anticipate that the SCAN would provide a compact, rapid, and low-cost method for nucleic acid detection at the point of care.
- 30Weckman, N. E. Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore Sensing. ACS Sensors 2019, 4, 2065– 2072, DOI: 10.1021/acssensors.9b0068630https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsVertrzL&md5=292439fb275ad650facc06a8d2fe7c65Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore SensingWeckman, Nicole E.; Ermann, Niklas; Gutierrez, Richard; Chen, Kaikai; Graham, James; Tivony, Ran; Heron, Andrew; Keyser, Ulrich F.ACS Sensors (2019), 4 (8), 2065-2072CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific anal. of a DNA sample. Solid state nanopore sensors are esp. promising for this type of single-mol. sensing because of the ability to analyze patterns in the ionic current signatures of DNA mols. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify two different DNA targets in a background mixt. of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.
- 31Sandler, S. E. Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores. Nat. Biomed. Eng. 2024, 8, 325– 334, DOI: 10.1038/s41551-023-01078-2There is no corresponding record for this reference.
- 32Yang, W. Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores. Nano Lett. 2018, 18, 6469– 6474, DOI: 10.1021/acs.nanolett.8b0296832https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1KhurjL&md5=5c13dba2cb82df4d4d1f2958e5772cccDetection of CRISPR-dCas9 on DNA with Solid-State NanoporesYang, Wayne; Restrepo-Perez, Laura; Bengtson, Michel; Heerema, Stephanie J.; Birnie, Anthony; van der Torre, Jaco; Dekker, CeesNano Letters (2018), 18 (10), 6469-6474CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore expts. that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore expts., we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA mols. that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore expts., dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.
- 33Ma, H.; Wang, Y.; Li, Y. X.; Xie, B. K.; Hu, Z. L.; Yu, R. J.; Long, Y. T.; Ying, Y. L. Label-Free Mapping of Multivalent Binding Pathways with Ligand–Receptor-Anchored Nanopores. J. Am. Chem. Soc. 2024, 146, 28014– 28022, DOI: 10.1021/jacs.4c04934There is no corresponding record for this reference.
- 34Kowalczyk, S. W.; Wells, D. B.; Aksimentiev, A.; Dekker, C. Slowing down DNA Translocation through a Nanopore in Lithium Chloride. Nano Lett. 2012, 12, 1038– 1044, DOI: 10.1021/nl204273h34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlsVCktg%253D%253D&md5=31be87d5508361ac5aba5a7120e51979Slowing down DNA Translocation through a Nanopore in Lithium ChlorideKowalczyk, Stefan W.; Wells, David B.; Aksimentiev, Aleksei; Dekker, CeesNano Letters (2012), 12 (2), 1038-1044CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The charge of a DNA mol. is a crucial parameter in many DNA detection and manipulation schemes such as gel electrophoresis and lab-on-a-chip applications. Here, we study the partial redn. of the DNA charge due to counterion binding by means of nanopore translocation expts. and all-atom mol. dynamics (MD) simulations. Surprisingly, we find that the translocation time of a DNA mol. through a solid-state nanopore strongly increases as the counterions decrease in size from K+ to Na+ to Li+, both for double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). MD simulations elucidate the microscopic origin of this effect: Li+ and Na+ bind DNA stronger than K+. These fundamental insights into the counterion binding to DNA also provide a practical method for achieving at least 10-fold enhanced resoln. in nanopore applications.
- 35Plesa, C. Fast Translocation of Proteins through Solid State Nanopores. Nano Lett. 2013, 13, 658– 663, DOI: 10.1021/nl304267835https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1KlsL0%253D&md5=bce7b51bbacf0f73d77dd4ead8d6ece2Fast Translocation of Proteins through Solid State NanoporesPlesa, Calin; Kowalczyk, Stefan W.; Zinsmeester, Ruben; Grosberg, Alexander Y.; Rabin, Yitzhak; Dekker, CeesNano Letters (2013), 13 (2), 658-663CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Measurements on protein translocation through solid-state nanopores reveal anomalous (non-Smoluchowski) transport behavior, as evidenced by extremely low detected event rates; i.e., the capture rates are orders of magnitude smaller than what is theor. expected. Systematic exptl. measurements of the event rate dependence on the diffusion const. are performed by translocating proteins ranging in size from 6 to 660 kDa. The discrepancy is obsd. to be significantly larger for smaller proteins, which move faster and have a lower signal-to-noise ratio. This is further confirmed by measuring the event rate dependence on the pore size and concn. for a large 540 kDa protein and a small 37 kDa protein, where only the large protein follows the expected behavior. We dismiss various possible causes for this phenomenon and conclude that it is due to a combination of the limited temporal resoln. and low signal-to-noise ratio. A one-dimensional first-passage time-distribution model supports this and suggests that the bulk of the proteins translocate on time scales faster than can be detected. We discuss the implications for protein characterization using solid-state nanopores and highlight several possible routes to address this problem.
- 36Chau, C. C.; Radford, S. E.; Hewitt, E. W.; Actis, P. Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore. Nano Lett. 2020, 20, 5553– 5561, DOI: 10.1021/acs.nanolett.0c0224636https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1WnurrJ&md5=3d330bfaeae1011185b928d1ebc33a8eMacromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State NanoporeChau, Chalmers C.; Radford, Sheena E.; Hewitt, Eric W.; Actis, PaoloNano Letters (2020), 20 (7), 5553-5561CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Nanopore anal. of nucleic acid is now routine, but detection of proteins remains challenging. Here, the authors report the systematic characterization of the effect of macromol. crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). A remarkable ∼1000-fold increase in the mol. count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. were obsd. Also macromol. crowding facilitates the study of the topol. of DNA plasmids and the characterization of amyloid fibril prepns. with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addn. of a macromol. crowding agent to the electrolyte. The authors therefore envision that macromol. crowding can be applied to many applications in the anal. of biomols. by solid-state nanopores.
- 37Chau, C. Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore. ACS Nano 2022, 16, 20075– 20085, DOI: 10.1021/acsnano.2c0831237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1ygsbvP&md5=26c141887bfff87b6d709d9cd6f13de8Probing RNA Conformations Using a Polymer-Electrolyte Solid-State NanoporeChau, Chalmers; Marcuccio, Fabio; Soulias, Dimitrios; Edwards, Martin Andrew; Tuplin, Andrew; Radford, Sheena E.; Hewitt, Eric; Actis, PaoloACS Nano (2022), 16 (12), 20075-20085CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Nanopore systems have emerged as a leading platform for the anal. of biomol. complexes with single-mol. resoln. The conformation of biomols., such as RNA, is highly dependent on the electrolyte compn., but solid-state nanopore systems often require high salt concn. to operate, precluding anal. of macromol. conformations under physiol. relevant conditions. Here, we report the implementation of a polymer-electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer-electrolyte bath governs the translocation dynamics of the analyte which correlates with the phys. properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer-electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiol. relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer-electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomols. under physiol. relevant conditions.
- 38Confederat, S.; Sandei, I.; Mohanan, G.; Wälti, C.; Actis, P. Nanopore fingerprinting of supramolecular DNA nanostructures. Biophys. J. 2022, 121, 4882– 4891, DOI: 10.1016/j.bpj.2022.08.02038https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1Clu7vM&md5=d9a9c0fc6dd9ab3cf249870223457960Nanopore fingerprinting of supramolecular DNA nanostructuresConfederat, Samuel; Sandei, Ilaria; Mohanan, Gayathri; Walti, Christoph; Actis, PaoloBiophysical Journal (2022), 121 (24), 4882-4891CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)DNA nanotechnol. has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resoln. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equiv. charge surplus as a discriminant. We compare the assembly yield of four supramol. DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and at. force microscopy imaging. We demonstrate that the nanopore system can provide anal. quantification of the complex supramol. nanostructures within minutes, without any need for labeling and with single-mol. resoln. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the anal. and manipulation of large DNA assemblies in real time.
- 39Confederat, S.; Lee, S.; Vang, D.; Soulias, D.; Marcuccio, F.; Peace, T. I.; Edwards, M. A.; Strobbia, P.; Samanta, D.; Wälti, C. Next-Generation Nanopore Sensors Based on Conductive Pulse Sensing for Enhanced Detection of Nanoparticles. Small 2024, 20, 2305186, DOI: 10.1002/smll.202305186There is no corresponding record for this reference.
- 40Marcuccio, F. Mechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer–Electrolyte Nanopore. ACS Nanosci. Au 2023, 3, 172– 181, DOI: 10.1021/acsnanoscienceau.2c0005040https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXmsFGrsg%253D%253D&md5=318e9bc708154debe2354af02b435dcdMechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer-Electrolyte NanoporeMarcuccio, Fabio; Soulias, Dimitrios; Chau, Chalmers C. C.; Radford, Sheena E.; Hewitt, Eric; Actis, Paolo; Edwards, Martin AndrewACS Nanoscience Au (2023), 3 (2), 172-181CODEN: ANACCX; ISSN:2694-2496. (American Chemical Society)Solid-state nanopores have been widely employed in the detection of biomols., but low signal-to-noise ratios still represent a major obstacle in the discrimination of nucleic acid and protein sequences substantially smaller than the nanopore diam. The addn. of 50% poly(ethylene) glycol (PEG) to the external soln. is a simple way to enhance the detection of such biomols. Here, we demonstrate with finite-element modeling and expts. that the addn. of PEG to the external soln. introduces a strong imbalance in the transport properties of cations and anions, drastically affecting the current response of the nanopore. We further show that the strong asym. current response is due to a polarity-dependent ion distribution and transport at the nanopipette tip region, leading to either ion depletion or enrichment for few tens of nanometers across its aperture. We provide evidence that a combination of the decreased/increased diffusion coeffs. of cations/anions in the bath outside the nanopore and the interaction between a translocating mol. and the nanopore-bath interface is responsible for the increase in the translocation signals. We expect this new mechanism to contribute to further developments in nanopore sensing by suggesting that tuning the diffusion coeffs. of ions could enhance the sensitivity of the system.
- 41Meyer, N.; Janot, J.-M.; Torrent, J.; Balme, S. Real-Time Fast Amyloid Seeding and Translocation of α-Synuclein with a Nanopipette. ACS Cent. Sci. 2022, 8, 441– 448, DOI: 10.1021/acscentsci.1c0140441https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XksFKgtbg%253D&md5=f88378af6bfc37da61301120dfc36992Real-Time Fast Amyloid Seeding and Translocation of α-Synuclein with a NanopipetteMeyer, Nathan; Janot, Jean-Marc; Torrent, Joan; Balme, SebastienACS Central Science (2022), 8 (4), 441-448CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)The detection to α-synuclein (αS) assemblies as a biomarker of synucleinopathies is an important challenge for further development of an early diagnosis tool. Here, we present proof of concept real-time fast amyloid seeding and translocation (RT-FAST) based on a nanopipette that combines in one unique system a reaction vessel to accelerate the seed amplification and nanopore sensor for single-mol. αS assembly detection. RT-FAST allows the detection of the presence αS seeds WT and A53T variant in a given sample in only 90 min by adding a low quantity (35μL at 100 nM) of recombinant αS for amplification. It also shows cross-seeding aggregation by adding mixing seeds A53T with WT monomers. Finally, we establish the dependence between the capture rate of aggregates by the nanopore sensor and the initial seed concn. from 200 pM to 2 pM, which promises further development toward a quant. anal. of the initial seed concn.
- 42Meyer, N.; Arroyo, N.; Roustan, L.; Janot, J.; Charles-Achille, S.; Torrent, J.; Picaud, F.; Balme, S. Secondary Nucleation of Aβ Revealed by Single-Molecule and Computational Approaches. Advanced Science 2024, 11, 2404916, DOI: 10.1002/advs.202404916There is no corresponding record for this reference.
- 43Sandler, S. E. Multiplexed Digital Characterization of Misfolded Protein Oligomers via Solid-State Nanopores. J. Am. Chem. Soc. 2023, 145, 25776– 25788, DOI: 10.1021/jacs.3c09335There is no corresponding record for this reference.
- 44Shen, B. W.; Heiter, D. F.; Lunnen, K. D.; Wilson, G. G.; Stoddard, B. L. DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target. Nucleic Acids Res. 2017, 45, 1516– 1528, DOI: 10.1093/nar/gkw1200There is no corresponding record for this reference.
- 45Barrangou, R. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science 2007, 315, 1709– 1712, DOI: 10.1126/science.113814045https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjtlWntb8%253D&md5=b88c0100d2c0469213afda20e47c39cdCRISPR Provides Acquired Resistance Against Viruses in ProkaryotesBarrangou, Rodolphe; Fremaux, Christophe; Deveau, Helene; Richards, Melissa; Boyaval, Patrick; Moineau, Sylvain; Romero, Dennis A.; Horvath, PhilippeScience (Washington, DC, United States) (2007), 315 (5819), 1709-1712CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addn. of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with assocd. cas genes, provided resistance against phages, and resistance specificity is detd. by spacer-phage sequence similarity.
- 46Wanunu, M.; Sutin, J.; McNally, B.; Chow, A.; Meller, A. DNA Translocation Governed by Interactions with Solid-State Nanopores. Biophys. J. 2008, 95, 4716– 4725, DOI: 10.1529/biophysj.108.14047546https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlOqtLnM&md5=ed5c8c30545222c5864800f11f5f54fdDNA translocation governed by interactions with solid-state nanoporesWanunu, Meni; Sutin, Jason; McNally, Ben; Chow, Andrew; Meller, AmitBiophysical Journal (2008), 95 (10), 4716-4725CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)We investigate the voltage-driven translocation dynamics of individual DNA mols. through solid-state nanopores in the diam. range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diam. is decreased from 5 to 2.7 nm, and steep temp. dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA mols., in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer mols., an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA mols. to a regime where the longer the DNA, the more current is blocked. Temp. dependence studies reveal that for increasing DNA lengths, addnl. interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor detg. DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resoln. than obsd. with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.
- 47Li, J.; Talaga, D. S. The distribution of DNA translocation times in solid-state nanopores. J. Phys.: Condens. Matter 2010, 22, 454129, DOI: 10.1088/0953-8984/22/45/45412947https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFartLzP&md5=70c79a7ae6896602196636ce65b04139The distribution of DNA translocation times in solid-state nanoporesLi, Jiali; Talaga, David S.Journal of Physics: Condensed Matter (2010), 22 (45), 454129/1-454129/8CODEN: JCOMEL; ISSN:0953-8984. (Institute of Physics Publishing)This paper systematically investigates the effects of soln. viscosity, applied voltage and DNA chain length on the distribution of DNA translocation times through 8±2 nm diam. silicon nitride nanopores. Linear dsDNA translocation events were selected based on the magnitude of current blockage and accumulated into scatter plots of current blockage and event duration (translocation time). The translocation time distribution was fitted to the soln. of a Smoluchowski-type equation for 1D biased diffusion to a sink. The DNA drifting speed under bias and diffusion const. were extd. from the fits as functions of soln. viscosity, applied voltage and DNA chain length. Combined with the Einstein-Smoluchowski relation, this model allowed evaluation of the viscous drag force on DNA mols. This model also allowed estn. of the uncertainty in detg. the DNA chain length due to the influence of friction on the spread of translocation times in a nanopore measurement. The data anal. suggests that the simple 1D biased diffusion model fits the exptl. data well for a wide range of conditions. Some deviations from predicted behavior were obsd. and show where addnl. phenomena are likely to contribute to the distribution of DNA translocation times.
- 48Kesselheim, S.; Müller, W.; Holm, C. Origin of Current Blockades in Nanopore Translocation Experiments. Phys. Rev. Lett. 2014, 112, 018101, DOI: 10.1103/PhysRevLett.112.01810148https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitlehurk%253D&md5=c8bf5244f200101ff3a3e3075f3212c9Origin of current blockades in nanopore translocation experimentsKesselheim, Stefan; Mueller, Wojciech; Holm, ChristianPhysical Review Letters (2014), 112 (1), 018101/1-018101/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)We present a detailed investigation of the ionic current in a cylindrical model nanopore in the absence and the presence of a double stranded DNA homopolymer. Our atomistic simulations are capable of reproducing almost exactly the exptl. data obtained by Smeets et al., including notably the crossover salt concn. that yields equal current measurements in both situations. We can rule out that the obsd. current blockade is due to the steric exclusion of charge carriers from the DNA, since for all investigated salt concns. the charge carrier d. is higher when the DNA is present. Calcns. using a mean-field electrokinetic model proposed by van Dorp et al. fail quant. in predicting this effect. We can relate the shortcomings of the mean-field model to a surface related mol. drag that the ions feel in the presence of the DNA. This drag is independent of the salt concn. and originates from electrostatic, hydrodynamic, and excluded vol. interactions.
- 49Gibson, D. G. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343– 345, DOI: 10.1038/nmeth.131849https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXksVemsbw%253D&md5=46284924c7d73c47cfb490983338e480Enzymatic assembly of DNA molecules up to several hundred kilobasesGibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.Nature Methods (2009), 6 (5), 343-345CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors describe an isothermal, single-reaction method for assembling multiple overlapping DNA mols. by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First they recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful mol. engineering tool.
- 50Li, Y.; Sandler, S. E.; Keyser, U. F.; Zhu, J. DNA Volume, Topology, and Flexibility Dictate Nanopore Current Signals. Nano Lett. 2023, 23, 7054– 7061, DOI: 10.1021/acs.nanolett.3c01823There is no corresponding record for this reference.
- 51Cadinu, P.; Kang, M.; Nadappuram, B. P.; Ivanov, A. P.; Edel, J. B. Individually Addressable Multi-nanopores for Single-Molecule Targeted Operations. Nano Lett. 2020, 20, 2012– 2019, DOI: 10.1021/acs.nanolett.9b0530751https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivVaqsLg%253D&md5=b2b715cc9a7162d0a23004b1c9f203d6Individually Addressable Multi-nanopores for Single-Molecule Targeted OperationsCadinu, Paolo; Kang, Minkyung; Nadappuram, Binoy Paulose; Ivanov, Aleksandar P.; Edel, Joshua B.Nano Letters (2020), 20 (3), 2012-2019CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The fine-tuning of mol. transport is a ubiquitous problem of single-mol. methods. The latter is evident even in powerful single-mol. techniques such as nanopore sensing, where the quest for resolving more detailed biomol. features is often limited by insufficient control of the dynamics of individual mols. within the detection vol. of the nanopore. In this work, we introduce and characterize a reconfigurable multi-nanopore architecture that enables addnl. channels to manipulate the dynamics of DNA mols. in a nanopore. We show that the fabrication process of this device, consisting of four adjacent, individually addressable nanopores located at the tip of a quartz nanopipette, is fast and highly reproducible. By individually tuning the elec. field across each nanopore, these devices can operate in several unique cooperative detection modes that allow moving, sensing, and trapping of DNA mols. with high efficiency and increased temporal resoln.
- 52Cadinu, P. Double Barrel Nanopores as a New Tool for Controlling Single-Molecule Transport. Nano Lett. 2018, 18, 2738– 2745, DOI: 10.1021/acs.nanolett.8b0086052https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlvVKks7Y%253D&md5=e0603e993e4a1132e7e451d715671228Double Barrel Nanopores as a New Tool for Controlling Single-Molecule TransportCadinu, Paolo; Campolo, Giulia; Pud, Sergii; Yang, Wayne; Edel, Joshua B.; Dekker, Cees; Ivanov, Aleksandar P.Nano Letters (2018), 18 (4), 2738-2745CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The ability to control the motion of single biomols. is key to improving a wide range of biophys. and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, mol. control and the possibility of slow readouts of long polymer mols. are still limited due to fast analyte transport and low signal-to-noise ratios. Here, the authors report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are sepd. by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (1) pore-to-pore transfer, which can be controlled at ∼100% efficiency, and (2) DNA mols. bridging between the two nanopores, which enables detection with an enhanced temporal resoln. (e.g., an increase of >2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resoln. readout of biol. mols.
- 53Cadinu, P. Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge. Nano Lett. 2017, 17, 6376– 6384, DOI: 10.1021/acs.nanolett.7b0319653https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVWmsrvF&md5=01bd2c208ce4d2cd46c5f45b4c728f04Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter NanobridgeCadinu, Paolo; Paulose Nadappuram, Binoy; Lee, Dominic J.; Sze, Jasmine Y. Y.; Campolo, Giulia; Zhang, Yanjun; Shevchuk, Andrew; Ladame, Sylvain; Albrecht, Tim; Korchev, Yuri; Ivanov, Aleksandar P.; Edel, Joshua B.Nano Letters (2017), 17 (10), 6376-6384CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)There is a growing realization, esp. within the diagnostic and therapeutic community, that the amt. of information enclosed in a single mol. can not only enable a better understanding of biophys. pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single mol. strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control mol. transport in terms of transport rate, resoln., and signal-to-noise ratio (SNR) is essential to take full advantage of the technol. benefits. Here the authors propose a novel soln. to these challenges based on a method that allows biomols. to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm sepn. Mols. that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resoln., sensitivity, and limit of detection. α-synuclein. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.
- 54Steinbock, L. J.; Otto, O.; Chimerel, C.; Gornall, J.; Keyser, U. F. Detecting DNA Folding with Nanocapillaries. Nano Lett. 2010, 10, 2493– 2497, DOI: 10.1021/nl100997s54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmvVSlsL8%253D&md5=b29d1d4606b361e6216d9e05e7df6cd8Detecting DNA Folding with NanocapillariesSteinbock, Lorenz J.; Otto, Oliver; Chimerel, Catalin; Gornall, Joanne; Keyser, Ulrich F.Nano Letters (2010), 10 (7), 2493-2497CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors demonstrate for the first time the detection of the folding state of double-stranded DNA in nanocapillaries with the resistive pulse technique. The authors show that glass capillaries can be pulled into nanocapillaries with diams. down to 45 nm. The authors study translocation of λ -DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ -DNA mols. The authors' expts. prove that nanocapillaries are suitable for label-free anal. of DNA in aq. solns. and viable alternatives to solid-state nanopores made by silicon nanotechnol.
- 55Smeets, R. M. M. Salt Dependence of Ion Transport and DNA Translocation through Solid-State Nanopores. Nano Lett. 2006, 6, 89– 95, DOI: 10.1021/nl052107w55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht12rtbfK&md5=74e23d4453737c1063d0f0a9a75d9447Salt Dependence of Ion Transport and DNA Translocation through Solid-State NanoporesSmeets, Ralph M. M.; Keyser, Ulrich F.; Krapf, Diego; Wu, Meng-Yue; Dekker, Nynke H.; Dekker, CeesNano Letters (2006), 6 (1), 89-95CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)We report exptl. measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concns. from 1 M to 1 μM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-μm-long dsDNA for 50 mM to 1 M salt concns. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concn. of 370 ± 40 mM.
- 56Bošković, F.; Zhu, J.; Chen, K.; Keyser, U. F. Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State Nanopores. Nano Lett. 2019, 19, 7996– 8001, DOI: 10.1021/acs.nanolett.9b0318456https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVylsL3K&md5=f05ce915e55deb46f55280a82eecfb72Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State NanoporesBoskovic, Filip; Zhu, Jinbo; Chen, Kaikai; Keyser, Ulrich F.Nano Letters (2019), 19 (11), 7996-8001CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)G-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-mol. methods require fluorescent labels, which can affect Gq folding kinetics. Here the authors introduce, a single-mol. Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. The authors use ∼5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addn., smGNA allows for discrimination of (un-)folded Gq structures, provides insights into single-mol. kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-mol. level without labeling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.
- 57Lastra, L. S.; Bandara, Y. M. N. D. Y.; Sharma, V.; Freedman, K. J. Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance. ACS Sensors 2022, 7, 1883– 1893, DOI: 10.1021/acssensors.2c0047957https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsFWnsbzP&md5=4f204344814bc243903917f76ff80f40Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt ImbalanceLastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Sharma, Vinay; Freedman, Kevin J.ACS Sensors (2022), 7 (7), 1883-1893CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Nanopores are a promising single-mol. sensing device class that captures mol.-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a mol. A systematic study was conducted here to study the CPS-based mol. discrimination and its voltage-dependent characteristics. CPS was obsd. when the cation movement along both elec. and chem. gradients was favored, which led to an ∼3x improvement in SNR (i.e., signal-to-noise ratio) and an ∼8x increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asym. salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other mol. systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10a-a protein with broad utilities in genetic engineering and immunol.-bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically obsd. for proteins.
- 58Charron, M.; Briggs, K.; King, S.; Waugh, M.; Tabard-Cossa, V. Precise DNA Concentration Measurements with Nanopores by Controlled Counting. Anal. Chem. 2019, 91, 12228– 12237, DOI: 10.1021/acs.analchem.9b0190058https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1egu7bM&md5=1f2a6c31a33b900129c87acb6cbd1e9dPrecise DNA Concentration Measurements with Nanopores by Controlled CountingCharron, Martin; Briggs, Kyle; King, Simon; Waugh, Matthew; Tabard-Cossa, VincentAnalytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12228-12237CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Using a solid-state nanopore to measure the concn. of clin. relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theor. electrophoretic capture models. We exptl. reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based mol. concn. detn. insufficiently precise to meet the stds. of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-mol. nanopore-based concn. measurements. We demonstrate controlled counting, a concn. measurement technique, which involves measuring the simultaneous capture rates of a mixt. of both the target mol. and an internal calibrator of precisely known concn. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge d. or distribution, are removed while allowing for higher-precision measurements. The quant. tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomol. concn.
- 59Karau, P.; Tabard-Cossa, V. Capture and Translocation Characteristics of Short Branched DNA Labels in Solid-State Nanopores. ACS Sensors 2018, 3, 1308– 1315, DOI: 10.1021/acssensors.8b0016559https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVOru7jK&md5=36766810f7cabb5796d4eed5ce4f64c4Capture and Translocation Characteristics of Short Branched DNA Labels in Solid-State NanoporesKarau, Philipp; Tabard-Cossa, VincentACS Sensors (2018), 3 (7), 1308-1315CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)The challenge when employing solid-state nanopores as single-mol. sensors in a given assay is the specificity of the ionic current signal during the translocation of target mols. Here the authors present the capture and translocation characteristics of short structurally defined DNA mols. that could serve as effective surrogate labels in biosensing applications. The authors produced T-shaped or Y-shaped DNA mols. with a 50 bp double-stranded DNA (dsDNA) backbone and a 25 bp dsDNA branch in the middle, as improved labels over short linear DNA fragments. The authors show that mol. topologies can be distinguished from linear DNA by analyzing ionic current blockades produced as these DNA labels translocate through nanopores fabricated by controlled breakdown on 10-nm-thick SiN membranes and ranging in diam. from 4 to 10 nm. Event signatures are shown to be a direct result of the structure of the label and lead to an increased signal-to-noise ratio over that of short linear dsDNA, in addn. to well resolved dwell times for the pore size in this range. These results show that structurally defined branched DNA mols. can be robustly detected for a broad range of pore size, and thus represent promising candidates as surrogate labels in a variety of nanopore-based mol. or immunoassay schemes.
- 60Kong, J.; Bell, N. A. W.; Keyser, U. F. Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores. Nano Lett. 2016, 16, 3557– 3562, DOI: 10.1021/acs.nanolett.6b0062760https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvVKlsLo%253D&md5=2ce28ab30d6655e9af9c2fa84137d8cdQuantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State NanoporesKong, Jinglin; Bell, Nicholas A. W.; Keyser, Ulrich F.Nano Letters (2016), 16 (6), 3557-3562CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein mols. bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concns. in the nanomolar range. After incubation with target protein at different concns., the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concn. For our proof-of-principle expts. we use two std. binding systems, biotin-streptavidin and digoxigenin-antidigoxigenin, that allow for measurements of the concn. down to the low nanomolar range. The results demonstrate the potential for a novel quant. and specific protein detection scheme using the DNA carrier method.
- 61Chen, K. Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores. Nano Lett. 2017, 17, 5199– 5205, DOI: 10.1021/acs.nanolett.7b0100961https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlKisbzE&md5=2a172c6bacb68355ddf0c3501088fbb5Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State NanoporesChen, Kaikai; Juhasz, Matyas; Gularek, Felix; Weinhold, Elmar; Tian, Yu; Keyser, Ulrich F.; Bell, Nicholas A. W.Nano Letters (2017), 17 (9), 5199-5205CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Nanopore sensors show great potential for rapid, single-mol. detn. of DNA sequence information. Here, we develop an ionic current-based method for detg. the positions of short sequence motifs in double-stranded DNA mols. with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-L-methionine cofactor analog we create covalently attached biotin labels at 5'-TCGA-3' sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to addnl. current blockade signals when the DNA passes through a conical quartz nanopore. We det. the relationship between translocation time and position along the DNA contour and find a min. resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA mols. by detg. the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixt. Our method provides a simple, generic single-mol. detection platform enabling DNA characterization in an elec. format suited for portable devices for potential diagnostic applications.
- 62Wang, V.; Ermann, N.; Keyser, U. F. Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA Structure. Nano Lett. 2019, 19, 5661– 5666, DOI: 10.1021/acs.nanolett.9b0221962https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlyis7zP&md5=f1ebf842f945a11f4c9070840f27d128Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA StructureWang, Vivian; Ermann, Niklas; Keyser, Ulrich F.Nano Letters (2019), 19 (8), 5661-5666CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The translocation of double-stranded DNA through a solid-state nanopore may either decrease or increase the ionic current depending on the ionic concn. of the surrounding soln. Below a certain crossover ionic concn., the current change inverts from a current blockade to current enhancement. In this paper, we show that the crossover concn. for bundled DNA nanostructures composed of multiple connected DNA double-helixes is lower than that of double-stranded DNA. Our measurements suggest that counterion mobility in the vicinity of DNA is reduced depending on the three-dimensional structure of the mol. We further demonstrate that introducing neutral polymers such as polyethylene glycol into the measurement soln. reduces electroosmotic outflow from the nanopore, allowing translocation of large DNA structures at low salt concns. Our expts. contribute to an improved understanding of ion transport in confined DNA environments, which is crit. for the development of nanopore sensing techniques as well as synthetic membrane channels. Our salt-dependent measurements of model DNA nanostructures will guide the development of computational models of DNA translocation through nanopores.
- 63Lastra, L. S.; Bandara, Y. M. N. D. Y.; Nguyen, M.; Farajpour, N.; Freedman, K. J. On the origins of conductive pulse sensing inside a nanopore. Nat. Commun. 2022, 13, 2186, DOI: 10.1038/s41467-022-29758-863https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht12mu7vM&md5=577a2bf5b893a71ff43767cc9969e475On the origins of conductive pulse sensing inside a nanoporeLastra, Lauren S.; Bandara, Y. M. Nuwan D. Y.; Nguyen, Michelle; Farajpour, Nasim; Freedman, Kevin J.Nature Communications (2022), 13 (1), 2186CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concns. Contrarily, conductive pulses are obsd. under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a mol.-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and expts. underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chem., phys., and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).
- 64Ivanov, A. P. On-Demand Delivery of Single DNA Molecules Using Nanopipets. ACS Nano 2015, 9, 3587– 3595, DOI: 10.1021/acsnano.5b0091164https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvF2htL8%253D&md5=0405a261544828d3b653ba3b7b692590On-Demand Delivery of Single DNA Molecules Using NanopipetsIvanov, Aleksandar P.; Actis, Paolo; Jonsson, Peter; Klenerman, David; Korchev, Yuri; Edel, Joshua B.ACS Nano (2015), 9 (4), 3587-3595CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Understanding the behavioral properties of single mols. or larger scale populations interacting with single mols. is currently a hotly pursued topic in nanotechnol. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single mol. detection strategies have existed for a no. of years, currently lacking are efficient methods for the controllable delivery of single mols. in aq. environments. In this article we show both exptl. and from simulations that nanopipets in conjunction with asym. voltage pulses can be used for label-free detection and delivery of single mols. through the tip of a nanopipet with "on-demand" timing resoln. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA mols. from solns. with concns. as low as 3 pM.
- 65Bhattacharya, S.; Satpati, P. Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations. ACS Omega 2023, 8, 1817– 1837, DOI: 10.1021/acsomega.2c05583There is no corresponding record for this reference.
- 66Pickar-Oliver, A.; Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 2019, 20, 490– 507, DOI: 10.1038/s41580-019-0131-566https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVOju7jM&md5=35a684f4388b64c543d4dcc37c7543ceThe next generation of CRISPR-Cas technologies and applicationsPickar-Oliver, Adrian; Gersbach, Charles A.Nature Reviews Molecular Cell Biology (2019), 20 (8), 490-507CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)A review. The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technol. have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homol.-directed repair for DNA repair, as well as single-base editing enzymes. In addn. to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biol. processes and are propelling CRISPR-Cas-based tools toward clin. use in gene and cell therapies.
- 67Wiedenheft, B. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10092– 10097, DOI: 10.1073/pnas.110271610867https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXot1egtbs%253D&md5=c43f31a58f29e3e3222b9826129f861dRNA-guide complex from a bacterial immune system enhances target recognition through seed sequence interactionsWiedenheft, Blake; van Duijn, Esther; Bultema, Jelle; Waghmare, Sakharam; Zhou, Kaihong; Barendregt, Arjan; Westphal, Wiebke; Heck, Albert; Boekem, Egbert; Dickman, Mark; Doudn, Jennifer A.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (25), 10092-10097, S10092/1-S10092/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-assocd. (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural anal. of the complex by small-angle x-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-assocd. complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.
- 68Pacesa, M. R-loop formation and conformational activation mechanisms of Cas9. Nature 2022, 609, 191– 196, DOI: 10.1038/s41586-022-05114-068https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1amu77N&md5=6d2d51ab4d1d69b8380e93bd8b29d5fcR-loop formation and conformational activation mechanisms of Cas9Pacesa, Martin; Loeff, Luuk; Querques, Irma; Muckenfuss, Lena M.; Sawicka, Marta; Jinek, MartinNature (London, United Kingdom) (2022), 609 (7925), 191-196CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Cas9 is a CRISPR-assocd. endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 was widely used for genome editing applications4-6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here the authors report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a pos. charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.
- 69Sternberg, S. H.; Redding, S.; Jinek, M.; Greene, E. C.; Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 2014, 507, 62– 67, DOI: 10.1038/nature1301169https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjs1GgtL0%253D&md5=e66bf209c17f583057ea2ce0d8a23a23DNA interrogation by the CRISPR RNA-guided endonuclease Cas9Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.Nature (London, United Kingdom) (2014), 507 (7490), 62-67CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The clustered regularly interspaced short palindromic repeats (CRISPR)-assocd. enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-mol. and bulk biochem. expts. to det. how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM d., and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand sepn. and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA mols., and to regulate scission of double-stranded DNA.
- 70Li, T.; Yang, Y.; Qi, H.; Cui, W.; Zhang, L.; Fu, X.; He, X.; Liu, M.; Li, P. f.; Yu, T. CRISPR/Cas9 therapeutics: progress and prospects. Signal Transduction Targeted Ther. 2023, 8, 36, DOI: 10.1038/s41392-023-01309-7There is no corresponding record for this reference.
- 71Cameron, P. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 2017, 14, 600– 606, DOI: 10.1038/nmeth.428471https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVCms7o%253D&md5=16b95f82fa2694bc366bac620914b9eaMapping the genomic landscape of CRISPR-Cas9 cleavageCameron, Peter; Fuller, Chris K.; Donohoue, Paul D.; Jones, Brittnee N.; Thompson, Matthew S.; Carter, Matthew M.; Gradia, Scott; Vidal, Bastien; Garner, Elizabeth; Slorach, Euan M.; Lau, Elaine; Banh, Lynda M.; Lied, Alexandra M.; Edwards, Leslie S.; Settle, Alexander H.; Capurso, Daniel; Llaca, Victor; Deschamps, Stephane; Cigan, Mark; Young, Joshua K.; May, Andrew P.Nature Methods (2017), 14 (6), 600-606CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochem. method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexs are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The no. of sites identified depended on sgRNA sequence and nuclease concn. Sites identified at lower concns. showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochem. identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
- 72Tsai, S. Q. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 2017, 14, 607– 614, DOI: 10.1038/nmeth.427872https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVCms70%253D&md5=9f36a171aba1728a693d260cf7151c33CIRCLE-seq: a highly sensit13ive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targetsTsai, Shengdar Q.; Nguyen, Nhu T.; Malagon-Lopez, Jose; Topkar, Ved V.; Aryee, Martin J.; Joung, J. KeithNature Methods (2017), 14 (6), 607-614CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochem. methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations assocd. with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochem. approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technol. and does not require ref. genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations assocd. with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.
- 73Zhang, L. Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes. Nucleic Acids Res. 2020, 48, 5037– 5053, DOI: 10.1093/nar/gkaa231There is no corresponding record for this reference.
- 74Pacesa, M. Structural basis for Cas9 off-target activity. Cell 2022, 185, 4067, DOI: 10.1016/j.cell.2022.09.026There is no corresponding record for this reference.
- 75Boyle, E. A.; Becker, W. R.; Bai, H. B.; Chen, J. S.; Doudna, J. A.; Greenleaf, W. J. Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement. Sci. Adv. 2021, 7, eabe5496 DOI: 10.1126/sciadv.abe5496There is no corresponding record for this reference.
- 76Ivanov, I. E. Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 5853– 5860, DOI: 10.1073/pnas.191344511776https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Wksr4%253D&md5=363156f6b639f86f5002ba36e43157a5Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoilingIvanov, Ivan E.; Wright, Addison V.; Cofsky, Joshua C.; Palacio Aris, Kevin D.; Doudna, Jennifer A.; Bryant, ZevProceedings of the National Academy of Sciences of the United States of America (2020), 117 (11), 5853-5860CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The CRISPR-Cas9 nuclease has been widely repurposed as a mol. and cell biol. tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its assocd. guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mech. description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-mol. technique that can simultaneously monitor DNA unwinding with base-pair resoln. and binding of fluorescently labeled macromols. in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mech. perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk expts. we observe promiscuous cleavage under physiol. neg. supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.
- 77Jones, S. K. Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nat. Biotechnol. 2021, 39, 84– 93, DOI: 10.1038/s41587-020-0646-577https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslKhu7jP&md5=04276aac1ce05ba8f74f72b9875d490fMassively parallel kinetic profiling of natural and engineered CRISPR nucleasesJones Jr, Stephen K.; Hawkins, John A.; Johnson, Nicole V.; Jung, Cheulhee; Hu, Kuang; Rybarski, James R.; Chen, Janice S.; Doudna, Jennifer A.; Press, William H.; Finkelstein, Ilya J.Nature Biotechnology (2021), 39 (1), 84-93CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets contg. mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophys. model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, esp. Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quant. and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.
- 78Samanta, D.; Ebrahimi, S. B.; Ramani, N.; Mirkin, C. A. Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters. J. Am. Chem. Soc. 2022, 144, 16310– 16315, DOI: 10.1021/jacs.2c0762578https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit1CktrzK&md5=2985152044d0e4df316d0ed2769cc130Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled ReportersSamanta, Devleena; Ebrahimi, Sasha B.; Ramani, Namrata; Mirkin, Chad A.Journal of the American Chemical Society (2022), 144 (36), 16310-16315CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into soln. can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ~ 75-fold turn-on signal and limits of detection (LODs) as low as ~ 10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ~ 30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temp. (25°C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clin. relevant concns. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show ATP (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the anal. of ATP in human serum samples with sensitivity on par with that of several com. available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.
- 79Kong, D. Direct SARS-CoV-2 Nucleic Acid Detection by Y-Shaped DNA Dual-Probe Transistor Assay. J. Am. Chem. Soc. 2021, 143, 17004– 17014, DOI: 10.1021/jacs.1c0632579https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitF2nu73M&md5=eda26ee7dfe0625f88e0e64b41da8d70Direct SARS-CoV-2 Nucleic Acid Detection by Y-Shaped DNA Dual-Probe Transistor AssayKong, Derong; Wang, Xuejun; Gu, Chenjian; Guo, Mingquan; Wang, Yao; Ai, Zhaolin; Zhang, Shen; Chen, Yiheng; Liu, Wentao; Wu, Yungen; Dai, Changhao; Guo, Qianying; Qu, Di; Zhu, Zhaoqin; Xie, Youhua; Liu, Yunqi; Wei, DachengJournal of the American Chemical Society (2021), 143 (41), 17004-17014CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Rapid screening of infected individuals from a large population is an effective means in epidemiol., esp. to contain outbreaks such as COVID-19. The gold std. assays for COVID-19 diagnostics are mainly based on the reverse transcription polymerase chain reaction, which mismatches the requirements for wide-population screening due to time-consuming nucleic acid extn. and amplification procedures. Here, we report a direct nucleic acid assay by using a graphene field-effect transistor (g-FET) with Y-shaped DNA dual probes (Y-dual probes). The assay relies on Y-dual probes modified on g-FET simultaneously targeting ORF1ab and N genes of SARS-CoV-2 nucleic acid, enabling high a recognition ratio and a limit of detection (0.03 copy μL-1) 1-2 orders of magnitude lower than existing nucleic acid assays. The assay realizes the fastest nucleic acid testing (~ 1 min) and achieves direct 5-in-1 pooled testing for the first time. Owing to its rapid, ultrasensitive, easily operated features as well as capability in pooled testing, it holds great promise as a comprehensive tool for population-wide screening of COVID-19 and other epidemics.
- 80Gebala, M. Cation–Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting. J. Am. Chem. Soc. 2015, 137, 14705– 14715, DOI: 10.1021/jacs.5b0839580https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslGms7%252FK&md5=3207df4e121730c93aabd4f60be67cecCation-anion interactions within the nucleic acid ion atmosphere revealed by ion countingGebala, Magdalena; Giambasu, George M.; Lipfert, Jan; Bisaria, Namita; Bonilla, Steve; Li, Guangchao; York, Darrin M.; Herschlag, DanielJournal of the American Chemical Society (2015), 137 (46), 14705-14715CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The ion atm. is a crit. structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Exptl. methods that "count" the no. of ions thermodynamically assocd. with the ion atm. allow dissection of energetic properties of the ion atm., and thus provide direct comparison to theor. results. Previous expts. have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atm. Here, the authors systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, the authors found, surprisingly, that the extent of anion exclusion and cation inclusion significantly depended on salt identity. The differences were prominent at higher concns. and mirrored trends in mean activity coeffs. of the electrolyte solns. Salts with lower activity coeffs. exhibited greater accumulation of both cations and anions within the ion atm., strongly suggesting that cation-anion correlation effects are present in the ion atm. and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation-anion correlations extend to nucleic acid kinetics and thermodn., the authors followed the folding of P4-P6, a domain of the Tetrahymena group I ribozyme, via single-mol. FRET in solns. with different salts. Solns. of identical concn. but lower activity gave slower and less favorable folding. The results revealed hitherto unknown properties of the ion atm. and suggested possible roles of oriented ion pairs or anion-bridged cations in the ion atm. for electrolyte solns. of salts with reduced activity. Consideration of these new results led to a re-evaluation of the strengths and limitations of Poisson-Boltzmann theory and highlighted the need for next-generation at.-level models of the ion atm.
- 81Gebala, M.; Bonilla, S.; Bisaria, N.; Herschlag, D. Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?. J. Am. Chem. Soc. 2016, 138, 10925– 10934, DOI: 10.1021/jacs.6b0428981https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1KktL3E&md5=3b6117f074e95f14805dee1e52ec18a0Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?Gebala, Magdalena; Bonilla, Steve; Bisaria, Namita; Herschlag, DanielJournal of the American Chemical Society (2016), 138 (34), 10925-10934CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other mols., and the energetics of these processes are profoundly influenced by the ion atm. that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atm., understanding its properties and effects will require synergy between computational modeling and expt. Prior computational models and expts. suggested that cation occupancy in the ion atm. depends on the size of the cation. However, the computational models were not independently tested and the exptl. obsd. effects were small. Here, the authors evaluated a computational model of ion size effects by exptl. testing a blind prediction made from that model, and they present addnl. exptl. results that extend the understanding of the ion atm. G. M. Giambasu et al. (2015) developed and implemented a 3-dimensional ref. interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helixes and this model predicted that Na+ would outcompete Cs+ by 1.8-2.1-fold; i.e., with Cs+ in 2-fold excess of Na+ the ion atm. would contain equal no. of each cation. However, the authors' ion counting expts. indicated that there was no significant preference for Na+ over Cs+. There was an ∼25% preferential occupancy of Li+ over larger cations in the ion atm., but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, the authors followed the folding of the P4-P6 RNA domain, and showed that differences in folding with different alkali metal ions obsd. at high concn. arose from cation-anion interactions and not cation size effects. Overall, these results provide a crit. test of a computational prediction, fundamental information about ion atm. properties, and parameters that will aid in the development of next-generation nucleic acid computational models.
- 82Cruz-León, S. Twisting DNA by salt. Nucleic Acids Res. 2022, 50, 5726– 5738, DOI: 10.1093/nar/gkac44582https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhslSjsrnO&md5=56939d2cc0f7646ba4191197bab98fa8Twisting DNA by saltCruz-Leon, Sergio; Vanderlinden, Willem; Muller, Peter; Forster, Tobias; Staudt, Georgina; Lin, Yi-Yun; Lipfert, Jan; Schwierz, NadineNucleic Acids Research (2022), 50 (10), 5726-5738CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)The structure and properties of DNA depend on the environment, in particular the ion atm. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concn. and identity of the surrounding ions. To resolve how cations influence the twist, we combine single-mol. magnetic tweezer expts. and extensive all-atom mol. dynamics simulations. Two interconnected trends are obsd. for monovalent alkali and divalent alk. earth cations. First, DNA twist increases monotonously with increasing concn. for all ions investigated. Second, for a given salt concn., DNA twist strongly depends on cation identity. At 100 mM concn., DNA twist increases as Na+ < K+ < Rb+ < Ba2+ < Li+ ≈ Cs+ < Sr2+ < Mg2+ < Ca2+. Our mol. dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the obsd. ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs+ and Sr2+. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnol.
- 83SantaLucia, J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1460– 1465, DOI: 10.1073/pnas.95.4.146083https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXht1Wqsbc%253D&md5=1a4e89f9f0caa91aecd5944add0aaf83A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamicsSantalucia, John, Jr.Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (4), 1460-1465CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A unified view of polymer, dumbbell, and oligonucleotide nearest-neighbor (NN) thermodn. is presented. DNA NN ΔG37° parameters from seven labs. are presented in the same format so that careful comparisons can be made. The seven studies used data from natural polymers, synthetic polymers, oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN parameters; used different methods of data anal.; used different salt concns.; and presented the NN thermodn. in different formats. As a result of these differences, there has been much confusion regarding the NN thermodn. of DNA polymers and oligomers. Herein I show that six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies remain. Further, a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodn. Empirical salt dependencies are also derived for oligonucleotides and polymers.
- 84Li, Y. CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid. Biochemistry 2023, 62, 2541– 2548, DOI: 10.1021/acs.biochem.3c00250There is no corresponding record for this reference.
- 85Gong, S.; Yu, H. H.; Johnson, K. A.; Taylor, D. W. DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity. Cell Rep. 2018, 22, 359– 371, DOI: 10.1016/j.celrep.2017.12.04185https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MzpsVSrsQ%253D%253D&md5=4edeb52aece481f5b53e37c3167918c7DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 ActivityGong Shanzhong; Yu Helen Hong; Johnson Kenneth A; Taylor David WCell reports (2018), 22 (2), 359-371 ISSN:.Bacterial adaptive immunity utilizes RNA-guided surveillance complexes comprising Cas proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single-guide RNA that base pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and the RuvC nuclease domains cleave opposing strands. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, and R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.
- 86Kimsey, I. J.; Petzold, K.; Sathyamoorthy, B.; Stein, Z. W.; Al-Hashimi, H. M. Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes. Nature 2015, 519, 315– 320, DOI: 10.1038/nature1422786https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXks1yqsL4%253D&md5=27816c16fe99c08b4c036c8611c6dae1Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexesKimsey, Isaac J.; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W.; Al-Hashimi, Hashim M.Nature (London, United Kingdom) (2015), 519 (7543), 315-320CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Rare tautomeric and anionic nucleobases are believed to have fundamental biol. roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equil. with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10-3 to 10-5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.
- 87Leontis, N. B. The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res. 2002, 30, 3497– 3531, DOI: 10.1093/nar/gkf48187https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xms1Klsb8%253D&md5=d6f4bc813e7d72e1a06a8b3658d08aebThe non-Watson-Crick base pairs and their associated isostericity matricesLeontis, Neocles B.; Stombaugh, Jesse; Westhof, EricNucleic Acids Research (2002), 30 (16), 3497-3531CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)RNA mols. exhibit complex structures in which a large fraction of the bases engage in non-Watson-Crick base pairing, forming motifs that mediate long-range RNA-RNA interactions and create binding sites for proteins and small mol. ligands. The rapidly growing no. of three-dimensional RNA structures at. resoln. requires that databases contain the annotation of such base pairs. An unambiguous and descriptive nomenclature was proposed recently in which RNA base pairs were classified by the base edges participating in the interaction (Watson-Crick, Hoogsteen/CH or sugar edge) and the orientation of the glycosidic bonds relative to the hydrogen bonds (cis or trans). Twelve basic geometric families were identified and all 12 have been obsd. in crystal structures. For each base pairing family, we present here the 4×4 isostericity matrixes' summarizing the geometric relationships between the 16 pairwise combinations of the four std. bases, A, C, G and U. Whenever available, a representative example of each obsd. base pair from X-ray crystal structures (3.0 Å resoln. or better) is provided or, otherwise, theor. plausible models. This format makes apparent the recurrent geometric patterns that are obsd. and helps identify isosteric pairs that co-vary or interchange in sequences of homologous mols. while maintaining conserved three-dimensional motifs.
- 88Maity, H.; Baidya, L.; Reddy, G. Salt-Induced Transitions in the Conformational Ensembles of Intrinsically Disordered Proteins. J. Phys. Chem. B 2022, 126, 5959– 5971, DOI: 10.1021/acs.jpcb.2c0347688https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitVKkt77P&md5=8e7f72a55f4245a2dfeb5133798f30bcSalt-Induced Transitions in the Conformational Ensembles of Intrinsically Disordered ProteinsMaity, Hiranmay; Baidya, Lipika; Reddy, GovardhanJournal of Physical Chemistry B (2022), 126 (32), 5959-5971CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Salts modulate the behavior of intrinsically disordered proteins (IDPs) and influence the formation of membraneless organelles through liq.-liq. phase sepn. (LLPS). In low ionic strength solns., IDP conformations are perturbed by the screening of electrostatic interactions, independent of the salt identity. In this regime, insight into the IDP behavior can be obtained using the theory for salt-induced transitions in charged polymers. However, salt specific interactions with the charged and uncharged residues, known as the Hofmeister effect, influence IDP behavior in high ionic strength solns. There is a lack of reliable theor. models in high salt concn. regimes to predict the salt effect on IDPs. The authors propose a simulation methodol. using a coarse-grained IDP model and exptl. measured water to salt soln. transfer free energies of various chem. groups that allowed the authors to study the salt specific transitions induced in the IDPs conformational ensemble. The authors probed the effect of three different monovalent salts on five IDPs belonging to various polymer classes based on charged residue content. All the IDPs of different polymer classes behave as self-avoiding walks (SAW) at physiol. salt concn. In high salt concns., the transitions obsd. in the IDP conformational ensembles are dependent on the salt used and the IDP sequence and compn. Changing the anion with the cation fixed can result in the IDP transition from a SAW like-behavior to a collapsed globule. An important implication of these results is that a suitable salt can be identified to induce condensation of an IDP through LLPS.
- 89Beveridge, R. Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins. J. Am. Chem. Soc. 2019, 141, 4908– 4918, DOI: 10.1021/jacs.8b1348389https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktV2mtb0%253D&md5=9877691dbb9c1737e10e1643befff8baIon Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered ProteinsBeveridge, Rebecca; Migas, Lukasz G.; Das, Rahul K.; Pappu, Rohit V.; Kriwacki, Richard W.; Barran, Perdita E.Journal of the American Chemical Society (2019), 141 (12), 4908-4918CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation vs. clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from soln. on the basis of charge and shape. Here, the authors use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. The authors report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compns., κ14 and κ56, distinguished by the extent of linear mixing vs. segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in soln. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase mol. dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concn., with the wild-type sequence showing the most sensitivity to changes in salt concn. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.
- 90Liu, Y. Single-Molecule Detection of α-Synuclein Oligomers in Parkinson’s Disease Patients Using Nanopores. ACS Nano 2023, 17, 22999– 23009, DOI: 10.1021/acsnano.3c08456There is no corresponding record for this reference.
- 91Byrd, E. J.; Wilkinson, M.; Radford, S. E.; Sobott, F. Taking Charge: Metal Ions Accelerate Amyloid Aggregation in Sequence Variants of α-Synuclein. J. Am. Soc. Mass Spectrom. 2023, 34, 493– 504, DOI: 10.1021/jasms.2c00379There is no corresponding record for this reference.
- 92Wang, H.; Wu, J.; Sternke-Hoffmann, R.; Zheng, W.; Mörman, C.; Luo, J. Multivariate effects of pH, salt, and Zn2+ ions on Aβ40 fibrillation. Commun. Chem. 2022, 5, 171, DOI: 10.1038/s42004-022-00786-1There is no corresponding record for this reference.
- 93Yoshimura, Y. Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 14446– 14451, DOI: 10.1073/pnas.120822810993https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVaqurfE&md5=860aa8e3ba70b377b56cade4c8753a17Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formationYoshimura, Yuichi; Lin, Yuxi; Yagi, Hisashi; Lee, Young-Ho; Kitayama, Hiroki; Sakurai, Kazumasa; So, Masatomo; Ogi, Hirotsugu; Naiki, Hironobu; Goto, YujiProceedings of the National Academy of Sciences of the United States of America (2012), 109 (36), 14446-14451, S14446/1-S14446/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates assocd. with protein misfolding diseases. Although they differ in morphol., the two forms are often treated indiscriminately. β2-Microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concn. at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersatd. soln., analogous to crystn. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradn., and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Soly. and supersatn. will be key factors for further understanding the aberrant aggregation of proteins.
- 94Gaspar, R.; Lund, M.; Sparr, E.; Linse, S. Anomalous Salt Dependence Reveals an Interplay of Attractive and Repulsive Electrostatic Interactions in α-synuclein Fibril Formation. QRB discov. 2020, 1, e2 DOI: 10.1017/qrd.2020.7There is no corresponding record for this reference.
- 95Yusko, E. C. Single-Particle Characterization of Aβ Oligomers in Solution. ACS Nano 2012, 6, 5909– 5919, DOI: 10.1021/nn300542q95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotlShu7Y%253D&md5=24c8e5c564399c975d2389c1c94a20d2Single-Particle Characterization of Aβ Oligomers in SolutionYusko, Erik C.; Prangkio, Panchika; Sept, David; Rollings, Ryan C.; Li, Jiali; Mayer, MichaelACS Nano (2012), 6 (7), 5909-5919CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Detg. the pathol. role of amyloids in amyloid-assocd. diseases will require a method for characterizing the dynamic distributions in size and shape of amyloid oligomers with high resoln. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloid-β oligomers directly in soln. and without chem. modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β aggregate sizes. The approach revealed the distribution of protofibrillar lengths (12- to 155-mer) as well as the av. cross-sectional area of protofibrils and fibers.
- 96Awasthi, S.; Ying, C.; Li, J.; Mayer, M. Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution. ACS Nano 2023, 17, 12325– 12335, DOI: 10.1021/acsnano.3c01393There is no corresponding record for this reference.
- 97Zhao, C. DNase-targeted natural product screening based on a sensitive and selective DNase I detecting system. RSC Adv. 2017, 7, 30911– 30918, DOI: 10.1039/C7RA04911KThere is no corresponding record for this reference.
- 98Chau, C. C.; Weckman, N. E.; Thomson, E. E.; Actis, P. Solid-State Nanopore Real Time Assay for Monitoring Cas9 Endonuclease Reactivity. bioRxiv 2024, bioRxiv:2024.09.20.612695, DOI: 10.1101/2024.09.20.612695There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.4c15173.
Supporting note on polymer electrolyte-modified solid-state nanopore mechanism, generation of the RS-dsDNA and the sequence data, generation of the RS-dsDNA, restriction digestion of the RS-dsDNA, Cas9-related crRNA sequence and digestion, KDE and probability calculations, and relevant supporting figures (PDF)
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