Synthetic Protein-to-DNA Input Exchange for Protease Activity Detection Using CRISPR-Cas12aClick to copy article linkArticle link copied!
- Luca CapelliLuca CapelliDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Luca Capelli
- Federica PedriniFederica PedriniDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Federica Pedrini
- Andrea C. Di PedeAndrea C. Di PedeDepartment of Chemistry, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, ItalyMore by Andrea C. Di Pede
- Alejandro Chamorro-GarciaAlejandro Chamorro-GarciaDepartment of Chemistry, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, ItalyMore by Alejandro Chamorro-Garcia
- Neda BagheriNeda BagheriDepartment of Chemistry, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, ItalyMore by Neda Bagheri
- Simone FortunatiSimone FortunatiDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Simone Fortunati
- Marco GiannettoMarco GiannettoDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Marco Giannetto
- Monica MattarozziMonica MattarozziDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Monica Mattarozzi
- Roberto CorradiniRoberto CorradiniDepartment of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Roberto Corradini
- Alessandro PorchettaAlessandro PorchettaDepartment of Chemistry, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, ItalyMore by Alessandro Porchetta
- Alessandro Bertucci*Alessandro Bertucci*Email: [email protected]Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, ItalyMore by Alessandro Bertucci
Abstract
We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units─a peptide and a peptide nucleic acid (PNA), fused together. The peptide presents the substrate of MMP2, while the PNA serves as a nucleic acid output for subsequent processing. This chemical translator was immobilized on micrometer magnetic beads as a physical support for an activity-based assay. We incorporated into our design a single-stranded DNA partially hybridized with the PNA sequence and bearing a region complementary to the RNA guide of CRISPR-Cas12a. The target-induced nuclease activity of Cas12a results in the degradation of FRET-labeled DNA reporters and amplified fluorescence signal, enabling the detection of MMP2 in the low picomolar range, showing a limit of detection of 72 pg/mL. This study provides new design principles for a broader applicability of CRISPR-Cas-based biosensing.
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Attribution (BY): Credit must be given to the creator.
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Attribution (BY): Credit must be given to the creator.
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Introduction
Figure 1
Figure 1. Schematic illustration of the proposed MMP2 activity-based detection method, leveraging a synthetic protein-to-DNA input exchange. A peptide-PNA chemical translator is used to convert MMP2 enzymatic activity into an input for CRISPR-Cas12a-based signal amplification.
Experimental Section
Synthesis of the Peptide-PNA Chemical Translator
Analysis of MMP2-Mediated Proteolytic Cleavage of the Peptide-PNA Translator
Melting Measurement
Functionalization of Magnetic Beads with the Peptide-PNA Translator
Characterization of the Functionalization Steps
Loading Curve
Kinetic Analysis of MMP2-Mediated Proteolytic Cleavage
Specificity Assay
Quantitative Fluorescence-Based Assay
CRISPR-Cas12a-Assisted Activity-Based Assays
CRISPR-Cas12a-Assisted Activity-Based Assays in Cell Medium Culture
ELISA Protocol for MMP2 Detection
Results and Discussion
Figure 2
Figure 2. Design of the peptide-PNA chemical translator and HPLC-HRMS analysis of protease cleavage products. (a) Illustration of the molecular design of the chemical translator, reporting the amino acid sequence of the peptide unit and the nucleobase sequence of the PNA unit. An azide group is conjugated to the 5′-end of the probe to enable click chemistry for anchoring to solid supports. A fluorescent version of the translator was obtained by conjugating 5(6)-carboxy-tetramethylrhodamine (TAMRA) to additional lysine residue present at the 3′-end. (b) Schematic representation of the MMP2-mediated enzymatic cleavage of the chemical translator (red circle-line probe), highlighting the cleavage site and the formation of two reaction products, a small peptide fragment (blue circles) and a larger fragment containing the PNA unit (orange circles + orange line). (c) HPLC-ESI-HRMS chromatogram of the chemical translator after incubation with MMP2 (24 h, 37 °C, [MMP2]:[translator] 1:10). The three main peaks correspond to the PNA-containing fragment (rt 13.25 min, m/z = 528.7370 [M+6H]6+, 634.2828 [M+5H]5+, 792.6016 [M+4H]4+, 1056.4669 [M+3H]3+ orange box), to unreacted molecular translator present in large excess in the reaction mixture (rt 16.42 min, m/z = 620.9489 [M+6H]6+, 744.9370 [M+5H]5+, 930.6689 [M+4H]4+ red box), and to the peptide fragment (rt 21.45 min, m/z = 571.2845 [M + H]+, 593.2655 [M + Na]+ blue box).
Figure 3
Figure 3. Design of the sensing platform and characterization of the activity-based sensing mechanism. (a) Multistep functionalization scheme of the magnetic beads with the chemical translator. Bifunctional DBCO-NHS allows for conversion of the amine groups initially present on the bead surface into DBCO functionalities. The peptide-PNA translator was then grafted onto these beads through an SPAAC reaction between the azide moiety on the 5′-end of the peptide unit of the translator and DBCO. (b) Zeta potential measurements during the various functionalization steps. After functionalization with DBCO, the zeta potential of a dispersion of the magnetic beads in PBS buffer shifts from 33 ± 1 mV to −4 ± 1 mV. Conjugation with the chemical translator increases the zeta potential to 11 ± 1 mV (n = 3, mean + SD). (c) Illustration of the MMP2 cleavage assay using magnetic beads functionalized with a fluorophore-tagged chemical translator. When the sensing platform is incubated with MMP2, proteolytic cleavage followed by magnetic separation of the beads results in a supernatant enriched in the PNA-containing cleavage product. (d) Normalized fluorescence intensity obtained for the cleavage assay conducted using different MMP2 incubation times ([MMP2] 100 nM, magnetic beads 1 mg/mL, n = 3, mean + SD). (e) Normalized fluorescence intensity obtained with MMP2 (100 nM) and other nonspecific MMPs (100 nM) (n = 3, mean + SD). (f) Fluorescence intensity values obtained for the assay conducted using different concentrations of MMP2 in the range of 0.03–10 nM (cleavage time 2h), from which it was possible to calculate an LOD of 104 pM (n = 3, mean ± SD), calculated as the concentration of MMP2 leading to a signal change equal to 3 times the standard deviation of the background (absence of the target).
Figure 4
Figure 4. CRISPR-Cas12a-based amplification of MMP2-derived nucleic acid output. (a) Pictorial illustration of the proposed MMP2 activity-based assay using CRISPR-Cas12a-based signal amplification. The peptide-PNA translator on the magnetic bead surface is hybridized with an ssDNA sequence serving as the trigger input for target-induced CRISPR-Cas12 trans-cleavage nuclease activity. Following incubation with MMP2 (2 h) and magnetic separation of the beads, the nucleic acid-containing cleavage product enriches the supernatant. This is incubated with a preformed crRNA/Cas12a complex (20 nM) in the presence of an excess of ssDNA hairpin reporter (100 nM). (b) Fluorescence kinetic profiles of CRISPR-Cas12a trans-cleavage activity (i.e., Cas12a-mediated degradation of the DNA hairpin reporters) upon interaction with MMP2-derived ssDNA activator in the MMP2 concentration range 1–300 pM. (c) Calibration curve obtained through a linear fit of signal gain % values in the 3–100 pM MMP2 concentration range. The curve is described by the following equation: y = (−452.1 ± 48.7) + (174.6 ± 11.4)x, R2 = 0.96. (d) Signal gain % values, calculated at the 2 h end point, obtained for CRISPR-Cas12a-based (red bars) and unamplified fluorescence assays (blue bars) for different concentrations of MMP2 (n = 3, mean ± SD).
Figure 5
Figure 5. Analysis of MMP2 in undiluted cell culture medium. (a) Calibration curve obtained through a linear fit of fluorescence intensity values in the 30–300 pM MMP2 concentration range using spiked samples in undiluted DMEM cell culture medium. The curve is described by the following equation: y = (−1,660,085 ± 353,763) + (849,889 ± 71,517)x, R2 = 0.93. (b) Recovery values for different concentrations of MMP2 in undiluted DMEM cell culture medium when using the developed CRISPR-Cas assay or a commercially available standard ELISA (n = 3, mean ± SD).
Conclusions
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c02622.
Materials, nucleic acid sequences, including those of the PNA-peptide translator, a description of the experimental conditions for SPAAC reaction; FT-IR spectra, fluorescence emission spectra, zeta potential measurements, chromatogram for the peptide-PNA conjugate characterization, two-photon microscopy image of functionalized magnetic beads, and experimental results from ELISA (PDF)
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Acknowledgments
This research was granted by University of Parma through the action Bando di Ateneo 2022 per la ricerca cofunded by MUR-Italian Ministry of Universities and Research–D.M. 737/2021-PNR-PNRR-NextGenerationEU. This work was supported by the Guido Berlucchi Foundation Mini Grant codice progetto BERTUCCI_2022_FONDBERLUCCHI. A.B. and A.P. acknowledge financial support under the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.1, Call for tender No. 104 published on 2.2.2022 by the Italian Ministry of University and Research (MUR), funded by the European Union – NextGenerationEU– Project Title “CRISPR-Cas-based sensing platforms for the monitoring of clinically relevant antibodies”– CUP D53D23009090001- Project Code 2022FPYZ2N - Grant Assignment Decree No. 958 adopted on 30-06-2023 by the Italian Ministry of Ministry of University and Research (MUR). This work has benefited from the equipment and framework of the COMP-HUB and COMP-R Initiatives, funded by the “Departments of Excellence” program of the Italian Ministry for University and Research (MIUR, 2018-2022 and MUR, 2023-2027).
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- 15Pham, W.; Choi, Y.; Weissleder, R.; Tung, C. H. Developing a Peptide-Based near-Infrared Molecular Probe for Protease Sensing. Bioconjugate Chem. 2004, 15 (6), 1403– 1407, DOI: 10.1021/bc049924sGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnslGqsLs%253D&md5=9be677530b817fe8c7134f9dacc047ffDeveloping a peptide-based near-infrared molecular probe for protease sensingPham, Wellington; Choi, Yongdoo; Weissleder, Ralph; Tung, Ching-HsuanBioconjugate Chemistry (2004), 15 (6), 1403-1407CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Recently near-IR (NIR) mol. probes have become important reporter mols. for a no. of types of in vivo biomedical imaging. A peptide-based NIR fluorescence probe consisting of a NIR fluorescence emitter (Cy5.5), a NIR fluorescence absorber (NIRQ820), and a protease selective peptide sequence was designed to sense protease activity. Using a MMP-7 model, we showed that NIRQ820 efficiently absorbs the emission energy of Cy5.5 resulting in a low initial signal. Upon reacting with its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a Kcat/Km of 100 000 M-1 s-1. The described synthetic strategy should have wide application for other NIR probe prepns.
- 16Ryu, J. H.; Lee, A.; Lee, S.; Ahn, C. H.; Park, J. W.; Leary, J. F.; Park, S.; Kim, K.; Kwon, I. C.; Youn, I. C.; Choi, K. One-Step” Detection of Matrix Metalloproteinase Activity Using a Fluorogenic Peptide Probe-Immobilized Diagnostic Kit. Bioconjugate Chem. 2010, 21 (7), 1378– 1384, DOI: 10.1021/bc100008bGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvV2kurc%253D&md5=779eb471d5afec751a2fb5beed6e2b0c"One-Step" Detection of Matrix Metalloproteinase Activity Using a Fluorogenic Peptide Probe-Immobilized Diagnostic KitRyu, Ju Hee; Lee, Aeju; Lee, Seulki; Ahn, Cheol-Hee; Park, Jong Woong; Leary, James F.; Park, Sangjin; Kim, Kwangmeyung; Kwon, Ick Chan; Youn, In-Chan; Choi, KuiwonBioconjugate Chemistry (2010), 21 (7), 1378-1384CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Matrix metalloproteinases (MMPs) have been shown to be abundant in pathol. conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biol. samples provides important clin. information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96-well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-IR (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concns. of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concn. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Addnl., the MMP-D-KIT was used to detect MMP activity in biol. samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biol. samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.
- 17Li, X.; Deng, D.; Xue, J.; Qu, L.; Achilefu, S.; Gu, Y. Quantum Dots Based Molecular Beacons for in Vitro and in Vivo Detection of MMP-2 on Tumor. Biosens. Bioelectron. 2014, 61, 512– 518, DOI: 10.1016/j.bios.2014.05.035Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFSgs77F&md5=b72dd49e0341a5a20eda8bcf4e6aa2cbQuantum dots based molecular beacons for in vitro and in vivo detection of MMP-2 on tumorLi, Xin; Deng, Dawei; Xue, Jianpeng; Qu, Lingzhi; Achilefu, Samuel; Gu, YueqingBiosensors & Bioelectronics (2014), 61 (), 512-518CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Matrix metalloproteinase-2 (MMP-2) is a protease related to tumor invasion and metastasis. It is heavily secreted by malignant tumor cells, allowing the protease to serve as an imaging biomarker of cancer. In this study, a novel sensing system based on fluorescence resonance energy transfer (FRET) from quantum dot (QD, the donor) to org. dye (the acceptor) was constructed for the in vitro and in vivo detection of matrix metalloproteinases-2 via a MMP-2-specific peptide substrate (GPLGVRGKGG). Specifically, 535 nm-emitting CdTe QD were bound to Rhodamine B (RB) through the peptide for in vitro detection of MMP-2, while 720 nm-emitting CdTeS QDs was linked to near IR dye ICG-Der-02 (MPA) by the peptide for measurement in vivo. When these probes were exposed to MMP-2, the selective cleavage of the peptide resulted in the recovery of fluorescence from QDs. By using the produced 540QD-peptide-RB and 720QD-peptide-MPA probes, we successfully examd. MMP-2 in live cells and tumor on nude mouse, resp. Due to the tunable fluorescence of Qds, this nanosensor can be fine-tuned for a wide range of applications such as the detection of different biomarkers and early diagnosis of disease.
- 18Feng, D.; Zhang, Y.; Feng, T.; Shi, W.; Li, X.; Ma, H. A Graphene Oxide-Peptide Fluorescence Sensor Tailor-Made for Simple and Sensitive Detection of Matrix Metalloproteinase 2. Chem. Commun. 2011, 47 (38), 10680– 10682, DOI: 10.1039/c1cc13975dGoogle Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFyqtLfP&md5=481c624299ce90d5e041ff7518844af7A graphene oxide-peptide fluorescence sensor tailor-made for simple and sensitive detection of matrix metalloproteinase 2Feng, Duan; Zhang, Yangyang; Feng, Tingting; Shi, Wen; Li, Xiaohua; Ma, HuiminChemical Communications (Cambridge, United Kingdom) (2011), 47 (38), 10680-10682CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A graphene oxide-peptide based fluorescence sensor has been developed for matrix metalloproteinase 2 (MMP2), and its applicability has been demonstrated by monitoring the concn. of MMP2 secreted by HeLa cells, revealing that HeLa cells with a d. of 5.48 × 105 cells per mL can produce 22 nM in cell culture media in 24 h.
- 19Welser, K.; Adsley, R.; Moore, B. M.; Chan, W. C.; Aylott, J. W. Protease Sensing with Nanoparticle Based Platforms. Analyst 2011, 136 (1), 29– 41, DOI: 10.1039/C0AN00429DGoogle ScholarThere is no corresponding record for this reference.
- 20Jin, Z.; Dridi, N.; Palui, G.; Palomo, V.; Jokerst, J. V.; Dawson, P. E.; Sang, Q. X. A.; Mattoussi, H. Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14. Anal. Chem. 2023, 95 (5), 2713– 2722, DOI: 10.1021/acs.analchem.2c03400Google ScholarThere is no corresponding record for this reference.
- 21Lei, Z.; Zhang, H.; Wang, Y.; Meng, X.; Wang, Z. Peptide Microarray-Based Metal Enhanced Fluorescence Assay for Multiple Profiling of Matrix Metalloproteinases Activities. Anal. Chem. 2017, 89 (12), 6749– 6757, DOI: 10.1021/acs.analchem.7b01037Google ScholarThere is no corresponding record for this reference.
- 22Biela, A.; Watkinson, M.; Meier, U. C.; Baker, D.; Giovannoni, G.; Becer, C. R.; Krause, S. Disposable MMP-9 Sensor Based on the Degradation of Peptide Cross-Linked Hydrogel Films Using Electrochemical Impedance Spectroscopy. Biosens. Bioelectron. 2015, 68, 660– 667, DOI: 10.1016/j.bios.2015.01.060Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsleqsL8%253D&md5=e896e54c77078ddcae50c289e108555aDisposable MMP-9 sensor based on the degradation of peptide crosslinked hydrogel films using electrochemical impedance spectroscopyBiela, Anna; Watkinson, Michael; Meier, Ute C.; Baker, David; Giovannoni, Gavin; Becer, C. Remzi; Krause, SteffiBiosensors & Bioelectronics (2015), 68 (), 660-667CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Matrix metalloproteinase-9 (MMP-9) plays an important role in both physiol. and pathol. processes. This enzyme is a peripheral biomarker of neuroinflammation in multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system. Presently, expensive magnetic resonance imaging (MRI) studies are used to monitor subclin. disease activity in MS. An alternative to costly MRI scans could be the detection of MMP-9, using a low-cost, disposable sensor system for MMP-9 suitable for home-monitoring of inflammation. This would allow an early prediction of the failure of anti-inflammatory therapies and more timely clin. intervention to limit neuronal damage and prevent disability. Here, the authors present the development of a disposable sensor for fast and straightforward detection of MMP-9. Biosensors were produced by coating electrodes with oxidized dextran and subsequent crosslinking with peptides contg. specific cleavage sites for MMP-9. Exposure of the films to the enzyme resulted in the degrdn. of the films, which was monitored using impedance measurements. The sensor response was rapid, a significant impedance change was usually obsd. within 5 min after the addn. of MMP-9. Sensors showed a negligible response to matrix metalloproteinase-2 (MMP-2), a protease which may interfere with MMP-9 detection. The peptide sequence with the highest sensitivity and selectivity Leu-Gly-Arg-Met-Gly-Leu-Pro-Gly-Lys was selected to construct calibration curves. MMP-9 was successfully detected in a clin. relevant range from 50 to 400 ng/mL. Two different processes of hydrogel degrdn. were obsd. on electrode surfaces with different roughness, and both appeared suitable to monitor MMP-9 activity. The sensor materials are generic and can be easily adopted to respond to other proteases by selecting peptide crosslinkers with suitable cleavage sites.
- 23Kou, B. B.; Chai, Y. Q.; Yuan, Y. L.; Yuan, R. PtNPs as Scaffolds to Regulate Interenzyme Distance for Construction of Efficient Enzyme Cascade Amplification for Ultrasensitive Electrochemical Detection of MMP-2. Anal. Chem. 2017, 89 (17), 9383– 9387, DOI: 10.1021/acs.analchem.7b02210Google ScholarThere is no corresponding record for this reference.
- 24Li, Y.; Liu, W.; Xu, Q.; Hu, J.; Zhang, C.-y. Construction of a Sensitive Protease Sensor with DNA-Peptide Conjugates for Single-Molecule Detection of Multiple Matrix Metalloproteinases. Biosens. Bioelectron. 2020, 169, 112647, DOI: 10.1016/j.bios.2020.112647Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVymsL3L&md5=6f3871dede2c49bc1420d4099c66ee22Construction of a sensitive protease sensor with DNA-peptide conjugates for single-molecule detection of multiple matrix metalloproteinasesLi, Yueying; Liu, Wen; Xu, Qinfeng; Hu, Juan; Zhang, Chun-yangBiosensors & Bioelectronics (2020), 169 (), 112647CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Protease expression is closely linked to various pathol. phenomena, and their accurate quantification is essential to clin. diagnosis and cancer therapy. Herein, we demonstrate for the first time the construction of a sensitive protease sensor by integrating protease-sensitive cleavage with nicking enzyme-assisted signal amplification (NESA) for single-mol. detection of multiple matrix metalloproteinases (MMPs). This protease sensor involves two DNA-peptide conjugates which contain both specific protease cleavage sites and trigger DNAs and two report DNAs which are modified with a fluorophore (Cy3 or Cy5) and a quencher (BHQ2). In the presence of specific MMPs, MMPs-mediated cleavage reactions lead to the release of specific trigger DNAs from the corresponding DNA-peptide conjugates. After the magnetic sepn., the resultant trigger DNAs may hybridize with the corresponding report DNAs to initiate the cyclic NESA reaction, releasing large amts. of Cy3/Cy5 fluorescent mols. which can be simply quantified by using total internal reflection fluorescence-based single-mol. detection. Taking advantage of the high specificity of proteolytic cleavage, the high amplification efficiency of cyclic NESA, and the high sensitivity of single-mol. detection, this protease sensor can simultaneously detect multiple MMPs with a detection limit of 3.33 pM for MMP-2 and 1.71 pM for MMP-7, superior to the target peptide-based methods. Moreover, this protease sensor can be applied for the measurement of MMP-2 and MMP-7 in cancer cells and the screening of protease inhibitors, holding great promise in clinic diagnosis and drug discovery.
- 25Luo, X.; Zhao, J.; Xie, X.; Liu, F.; Zeng, P.; Lei, C.; Nie, Z. Proteolysis-Responsive Rolling Circle Transcription Assay Enabling Femtomolar Sensitivity Detection of a Target Protease Biomarker. Anal. Chem. 2020, 92 (24), 16314– 16321, DOI: 10.1021/acs.analchem.0c04427Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVCnsr7M&md5=0901af0a864bfc37ecd696e8aa07f153Proteolysis-Responsive Rolling Circle Transcription Assay Enabling Femtomolar Sensitivity Detection of a Target Protease BiomarkerLuo, Xingyu; Zhao, Jiali; Xie, Xuan; Liu, Fang; Zeng, Pan; Lei, Chunyang; Nie, ZhouAnalytical Chemistry (Washington, DC, United States) (2020), 92 (24), 16314-16321CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Proteases play crucial roles in the malignant progression of tumor and thus have been regarded as biomarkers for many cancers. Although protease assays such as immunoassays and fluorogenic substrate probes have been developed, it remains challenging for them to give consideration to both sensitivity and accuracy. Here, we describe a proteolysis-responsive rolling circle transcription assay (PRCTA) for the ultrasensitive and accurate detection of protease activities by the rational integration of a protease-responsive RNA polymerase and rolling circle transcription. Taking cancer biomarker matrix metalloproteinase-2 (MMP-2) as the model, the PRCTA, which can transduce and amplify each proteolysis event catalyzed by MMP-2 into the output of multiple tandem fluorescent RNAs by in vitro transcription, is constructed for the sensitive anal. of MMP-2 activities. Such a rational integration greatly enhances the signal gain in PRCTA, and it enables the limit of detection of MMP-2 as low as 3 fM. The feasibility of PRCTA has been validated by the sensitive anal. of cellular MMP-2 activities of different cell lines with good accuracy, and the readout can be readily visualized by a fluorescence imaging system. Therefore, PRCTA has achieved the detection of target protease biomarkers with femtomolar sensitivity, exhibiting promising potential in biomedicine research and cancer diagnosis.
- 26Bui, H.; Brown, C. W.; Buckhout-White, S.; Díaz, S. A.; Stewart, M. H.; Susumu, K.; Oh, E.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors. Small 2019, 15 (14), 1805384, DOI: 10.1002/smll.201805384Google ScholarThere is no corresponding record for this reference.
- 27Yang, M.; Shi, K.; Liu, F.; Kang, W.; Lei, C.; Nie, Z. Coupling of Proteolysis-Triggered Transcription and CRISPR-Cas12a for Ultrasensitive Protease Detection. Sci. China Chem. 2021, 64 (2), 330– 336, DOI: 10.1007/s11426-020-9863-yGoogle ScholarThere is no corresponding record for this reference.
- 28Liu, F.; Yang, M.; Song, W.; Luo, X.; Tang, R.; Duan, Z.; Kang, W.; Xie, S.; Liu, Q.; Lei, C.; Huang, Y.; Nie, Z.; Yao, S. Target-Activated Transcription for the Amplified Sensing of Protease Biomarkers. Chem. Sci. 2020, 11 (11), 2993– 2998, DOI: 10.1039/C9SC04692EGoogle ScholarThere is no corresponding record for this reference.
- 29Kaminski, M. M.; Abudayyeh, O. O.; Gootenberg, J. S.; Zhang, F.; Collins, J. J. CRISPR-Based Diagnostics. Nat. Biomed. Eng. 2021, 5 (7), 643– 656, DOI: 10.1038/s41551-021-00760-7Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1ClsLrP&md5=5d3bc70679efc08e1629274086e57d6fCRISPR-based diagnosticsKaminski, Michael M.; Abudayyeh, Omar O.; Gootenberg, Jonathan S.; Zhang, Feng; Collins, James J.Nature Biomedical Engineering (2021), 5 (7), 643-656CODEN: NBEAB3; ISSN:2157-846X. (Nature Portfolio)Abstr.: The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiol. surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of mol. targets relevant to human health.
- 30Li, S. Y.; Cheng, Q. X.; Li, X. Y.; Zhang, Z. L.; Gao, S.; Cao, R. B.; Zhao, G. P.; Wang, J.; Wang, J. M. CRISPR-Cas12a-Assisted Nucleic Acid Detection. Cell Discovery 2018, 4 (1), 20, DOI: 10.1038/s41421-018-0028-zGoogle Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjnvVOksQ%253D%253D&md5=7b902affcc4e07d3ccf5efbe10a8453cCRISPR-Cas12a-assisted nucleic acid detectionLi Shi-Yuan; Zhao Guo-Ping; Wang Jin; Cheng Qiu-Xiang; Li Xiao-Yan; Wang Jing-Man; Zhang Zi-Long; Gao Song; Cao Rui-Bing; Zhao Guo-PingCell discovery (2018), 4 (), 20 ISSN:2056-5968.There is no expanded citation for this reference.
- 31Chen, J. S.; Ma, E.; Harrington, L. B.; Da Costa, M.; Tian, X.; Palefsky, J. M.; Doudna, J. A. CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity. Science 2018, 360, 436– 439, DOI: 10.1126/science.aar6245Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFyrsr8%253D&md5=63ed6929e10f1ec40154e1b18aeb9b82CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activityChen, Janice S.; Ma, Enbo; Harrington, Lucas B.; Da Costa, Maria; Tian, Xinran; Palefsky, Joel M.; Doudna, Jennifer A.Science (Washington, DC, United States) (2018), 360 (6387), 436-439CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA mols. We find that target-activated, nonspecific single-stranded DNase (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for mol. diagnostics.
- 32Broughton, J. P.; Deng, X.; Yu, G.; Fasching, C. L.; Servellita, V.; Singh, J.; Miao, X.; Streithorst, J. A.; Granados, A.; Sotomayor-Gonzalez, A.; Zorn, K.; Gopez, A.; Hsu, E.; Gu, W.; Miller, S.; Pan, C. Y.; Guevara, H.; Wadford, D. A.; Chen, J. S.; Chiu, C. Y. CRISPR–Cas12-Based Detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38 (7), 870– 874, DOI: 10.1038/s41587-020-0513-4Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntlejt7w%253D&md5=a1ccd699e2945474307f349518da37e5CRISPR-Cas12-based detection of SARS-CoV-2Broughton, James P.; Deng, Xianding; Yu, Guixia; Fasching, Clare L.; Servellita, Venice; Singh, Jasmeet; Miao, Xin; Streithorst, Jessica A.; Granados, Andrea; Sotomayor-Gonzalez, Alicia; Zorn, Kelsey; Gopez, Allan; Hsu, Elaine; Gu, Wei; Miller, Steve; Pan, Chao-Yang; Guevara, Hugo; Wadford, Debra A.; Chen, Janice S.; Chiu, Charles Y.Nature Biotechnology (2020), 38 (7), 870-874CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Abstr.: An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in Dec. 2019. COVID-19, the disease assocd. with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA exts. We validated our method using contrived ref. samples and clin. samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% pos. predictive agreement and 100% neg. predictive agreement.
- 33Rossetti, M.; Merlo, R.; Bagheri, N.; Moscone, D.; Valenti, A.; Saha, A.; Arantes, P. R.; Ippodrino, R.; Ricci, F.; Treglia, I.; Delibato, E.; Van Der Oost, J.; Palermo, G.; Perugino, G.; Porchetta, A. Enhancement of CRISPR/Cas12a Trans-Cleavage Activity Using Hairpin DNA Reporters. Nucleic Acids Res. 2022, 50 (14), 8377– 8391, DOI: 10.1093/nar/gkac578Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXpslCmtQ%253D%253D&md5=c4fe97010a2c84096d32e92cb66ff2b9Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reportersRossetti, Marianna; Merlo, Rosa; Bagheri, Neda; Moscone, Danila; Valenti, Anna; Saha, Aakash; Arantes, Pablo R.; Ippodrino, Rudy; Ricci, Francesco; Treglia, Ida; Delibato, Elisabetta; van der Oost, John; Palermo, Giulia; Perugino, Giuseppe; Porchetta, AlessandroNucleic Acids Research (2022), 50 (14), 8377-8391CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Mol. Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clin. relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.
- 34Li, J.; Yang, S.; Zuo, C.; Dai, L.; Guo, Y.; Xie, G. Applying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow Concentrations. ACS Sens. 2020, 5 (4), 970– 977, DOI: 10.1021/acssensors.9b02305Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkslWmtrY%253D&md5=4284c2b7b80cbd32ce94d5ef647aeecfApplying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow ConcentrationsLi, Junjie; Yang, Shuangshuang; Zuo, Chen; Dai, Ling; Guo, Yongcan; Xie, GuomingACS Sensors (2020), 5 (4), 970-977CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Efficient signal amplification is essential to construct ultrasensitive biosensors for biol. relevant species with abundant concomitant interferences. Here, the authors apply LbaCas12a as a signal amplifier to develop a versatile CRISPR-Cas12a platform to detect a wide range of analytes in ultralow concns. The platform relies on the indiscriminate single-stranded DNase activity of LbaCas12a, which recognizes single-stranded DNA intermediates generated by non-DNA targets down to femtomolar concns. and subsequently enhances the fluorescence signal output. With the help of functional nucleotides (DNAzyme and aptamer), ultrasensitive bioassays for Pb2+ and Acinetobacter baumannii have been designed with a limit of detection down to ~ 0.053 nM and ~ 3 CFU/mL, resp. It also allows simultaneous detection of four microRNAs (miRNAs) at a picomolar concn. without significant interferences by other counterparts, suggesting the potential of multiplexed miRNA expression profiles anal. in high throughput. Given the versatility and generality of the CRISPR-Cas12a platform, the authors expect the current work to advance the application of CRISPR-Cas-based platforms in bioanal. and provide new insights into ultrasensitive biosensor design.
- 35Xiong, Y.; Zhang, J.; Yang, Z.; Mou, Q.; Ma, Y.; Xiong, Y.; Lu, Y. Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. J. Am. Chem. Soc. 2020, 142 (1), 207– 213, DOI: 10.1021/jacs.9b09211Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlWhsrfF&md5=b680d3f70ac97b228f58a234a8691af8Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid TargetsXiong, Ying; Zhang, Jingjing; Yang, Zhenglin; Mou, Quanbing; Ma, Yuan; Xiong, Yonghua; Lu, YiJournal of the American Chemical Society (2020), 142 (1), 207-213CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Beyond its extraordinary genome editing ability, the CRISPR-Cas system has opened a new era of biosensing applications due to its high base resoln. and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic acid targets, we herein re-port CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) mols. such as aptamers and DNAzymes that are selective for small org. mol. and metal ion detections. The sensor is based on the Cas12a dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA) and its single stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA mols. that can lock a DNA activator for Cas12a-crRNA, preventing the ssD-NA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by com. available portable fluorimeters. Using this method, ATP and Na+ have been detected quant. under ambient temp. (25°C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system significantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanal. and biomedical applications.
- 36Hao, L.; Zhao, R. T.; Welch, N. L.; Tan, E. K. W.; Zhong, Q.; Harzallah, N. S.; Ngambenjawong, C.; Ko, H.; Fleming, H. E.; Sabeti, P. C.; Bhatia, S. N. CRISPR-Cas-Amplified Urinary Biomarkers for Multiplexed and Portable Cancer Diagnostics. Nat. Nanotechnol. 2023, 18 (7), 798– 807, DOI: 10.1038/s41565-023-01372-9Google ScholarThere is no corresponding record for this reference.
- 37Kang, W.; Xiao, F.; Zhu, X.; Ling, X.; Xie, S.; Li, R.; Yu, P.; Cao, L.; Lei, C.; Qiu, Y.; Liu, T.; Nie, Z. Engineering Anti-CRISPR Proteins to Create CRISPR-Cas Protein Switches for Activatable Genome Editing and Viral Protease Detection. Angew. Chem., Int. Ed. 2024, 63 (16), e202400599, DOI: 10.1002/anie.202400599Google ScholarThere is no corresponding record for this reference.
- 38Pandit, S.; Duchow, M.; Chao, W.; Capasso, A.; Samanta, D. DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing. Angew. Chem., Int. Ed. 2024, 63, e202310964, DOI: 10.1002/anie.202310964Google ScholarThere is no corresponding record for this reference.
- 39Dutta, A.; Li, J.; Lu, H.; Akech, J.; Pratap, J.; Wang, T.; Zerlanko, B. J.; Gerald, T. J. F.; Jiang, Z.; Birbe, R.; Wixted, J.; Violette, S. M.; Stein, J. L.; Stein, G. S.; Lian, J. B.; Languino, L. R. Integrin Avb6 Promotes an Osteolytic Program in Cancer Cells by Upregulating MMP2. Cancer Res. 2014, 74 (5), 1598– 1608, DOI: 10.1158/0008-5472.CAN-13-1796Google ScholarThere is no corresponding record for this reference.
- 40Tuszynski, G.; Jhon, A. The Role of Matrix Metalloproteinases in Tumor Angiogenesis and Tumor Metastasis. Pathol. Oncol. Res. 2001, 7, 14– 23, DOI: 10.1007/BF03032599Google ScholarThere is no corresponding record for this reference.
- 41Xie, T. X.; Wei, D.; Liu, M.; Gao, A. C.; Ali-Osman, F.; Sawaya, R.; Huang, S. Stat3 Activation Regulates the Expression of Matrix Metalloproteinase-2 and Tumor Invasion and Metastasis. Oncogene 2004, 23 (20), 3550– 3560, DOI: 10.1038/sj.onc.1207383Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjsV2ntLs%253D&md5=00c8970048472afee61306cd7d99f7dcStat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasisXie, Tong-Xin; Wei, Daoyan; Liu, Mingguang; Gao, Allen C.; Ali-Osman, Francis; Sawaya, Raymond; Huang, SuyunOncogene (2004), 23 (20), 3550-3560CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)The expression of matrix metalloproteinase-2 (MMP-2) has been linked with tumor invasion, angiogenesis, and metastasis. However, the mol. basis for MMP-2 overexpression in tumor cells remains unclear. In this study, by using K-1735 melanoma system, we demonstrated that highly metastatic C4, M2, and X21 tumor cells express elevated MMP-2 mRNA and enzymic activity, whereas poorly metastatic C10, C19, and C23 tumor cells express much lower levels. Moreover, a concomitant elevated Stat3 activity has been detected in these metastatic tumor cells that overexpress MMP-2. Transfection of constitutively activated Stat3 into poorly metastatic C23 tumor cells directly activated the MMP-2 promoter, whereas the expression of a dominant-neg. Stat3 in highly metastatic C4 tumor cells inhibited the MMP-2 promoter. A high-affinity Stat3-binding element was identified in the MMP-2 promoter and Stat3 protein bound directly to the MMP-2 promoter. Blockade of activated Stat3 through expression of a dominant-neg. Stat3 significantly suppressed MMP-2 expression in the metastatic tumor cells. Therefore, overexpression of MMP-2 in the metastatic melanoma cells can be attributed to elevated Stat3 activity, and Stat3 upregulates the transcription of MMP-2 through direct interaction with the MMP-2 promoter. Furthermore, blockade of activated Stat3 in highly metastatic C4 cells significantly suppressed the invasiveness of the tumor cells, inhibited tumor growth, and prevented metastasis in nude mice. Collectively, these studies suggest that Stat3 signaling directly regulates MMP-2 expression, tumor invasion, and metastasis, and that Stat3 activation might be a crucial event in the development of metastasis.
- 42Jezierska, A.; Motyl, T. Matrix Metalloproteinase-2 Involvement in Breast Cancer Progression: A Mini-Review. Med. Sci. Monit. 2009, 15 (2), RA32– RA40Google ScholarThere is no corresponding record for this reference.
- 43Von Maltzahn, G.; Harris, T. J.; Park, J. H.; Min, D. H.; Schmidt, A. J.; Sailor, M. J.; Bhatia, S. N. Nanoparticle Self-Assembly Gated by Logical Proteolytic Triggers. J. Am. Chem. Soc. 2007, 129 (19), 6064– 6065, DOI: 10.1021/ja070461lGoogle Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXksVSmsL4%253D&md5=6499978934c91f44f94e82bf0ecfa0f8Nanoparticle Self-Assembly Gated by Logical Proteolytic TriggersVon Maltzahn, Geoffrey; Harris, Todd J.; Park, Ji-Ho; Min, Dal-Hee; Schmidt, Alexander J.; Sailor, Michael J.; Bhatia, Sangeeta N.Journal of the American Chemical Society (2007), 129 (19), 6064-6065CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The emergent electromagnetic properties of nanoparticle self-assemblies are being harnessed to build new medical and biochem. assays with unprecedented sensitivity. While current self-assembly assays have displayed superior sensitivity for single mol. targets, the development of systems with the capacity to process multiple inputs may more effectively decipher complex disease signatures such as cancer. Herein, the authors present the design and synthesis of nanoparticles that perform Boolean logic operations using two proteolytic inputs assocd. with unique aspects of tumorigenesis (MMP2 and MMP7). Using dynamic light scattering, fluorescence, and MRI, the authors show that logical AND and OR functions can control the self-assembly of disperse superparamagnetic nanoparticles and enable remote, NMR detection of nanoparticle computation. In the future, by increasing the complexity of assembly triggers, nanoparticles may be tailored to sense a diversity of disease inputs in vitro and potentially in vivo.
- 44Jensen, K. K.; Ørum, H.; Nielsen, P. E.; Nordén, B. Kinetics for Hybridization of Peptide Nucleic Acids (PNA) with DNA and RNA Studied with the BIAcore Technique. Biochemistry 1997, 36 (16), 5072– 5077, DOI: 10.1021/bi9627525Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXit1Krsbc%253D&md5=9c3549eb1c2e26bdfcd2d6080cfd4156Kinetics for Hybridization of Peptide Nucleic Acids (PNA) with DNA and RNA Studied with the BIAcore TechniqueJensen, Kristine Kilsaa; Oerum, Henrik; Nielsen, Peter E.; Norden, BengtBiochemistry (1997), 36 (16), 5072-5077CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) contg. all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate consts. for assocn. (ka) and dissocn. (kd) of the duplex formation as well as the thermal stability (melting temp., Tm) of the duplexes have been detd. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences contg. single mismatches at various positions in the center resulted in increased dissocn. and decreased assocn. rate consts. Tm values for PNA•RNA duplexes are on av. 4° higher than for PNA•DNA duplexes and follow quant. the same variation with mismatches as do the PNA•DNA duplexes. Also a faster ka and a slower kd are found for PNA•RNA duplexes compared to the PNA•DNA duplexes. An overall fair correlation between Tm, ka, and kd is found for a series of PNA•DNA and PNA•RNA duplexes although the detn. of ka seemed to be prone to artifacts of the method and was not considered capable of providing abs. values representing the assocn. rate const. in bulk soln.
- 45Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson–Crick Hydrogen-Bonding Rules. Nature 1993, 365 (6446), 566– 568, DOI: 10.1038/365566a0Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXntFOksQ%253D%253D&md5=349ab1f3479e8e763f8555922e80fd12PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rulesEgholm, Michael; Buchardt, Ole; Christensen, Leif; Behrens, Carsten; Freier, Susan M.; Driver, David A.; Berg, Rolf H.; Kim, Seog K.; Norden, Bengt; Nielsen, Peter E.Nature (London, United Kingdom) (1993), 365 (6446), 566-8CODEN: NATUAS; ISSN:0028-0836.Recently, the authors designed a DNA analog, PNA (peptide nucleic acid), in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. Here, it is reported that PNA contg. all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.
- 46Davis, D. L.; Price, E. K.; Aderibigbe, S. O.; Larkin, M. X. H.; Barlow, E. D.; Chen, R.; Ford, L. C.; Gray, Z. T.; Gren, S. H.; Jin, Y.; Keddington, K. S.; Kent, A. D.; Kim, D.; Lewis, A.; Marrouche, R. S.; O’Dair, M. K.; Powell, D. R.; Scadden, M. H. C.; Session, C. B.; Tao, J.; Trieu, J.; Whiteford, K. N.; Yuan, Z.; Yun, G.; Zhu, J.; Heemstra, J. M. Effect of Buffer Conditions and Organic Cosolvents on the Rate of Strain-Promoted Azide-Alkyne Cycloaddition. J. Org. Chem. 2016, 81 (15), 6816– 6819, DOI: 10.1021/acs.joc.6b01112Google ScholarThere is no corresponding record for this reference.
- 47Liu, L.; Chu, H.; Yang, J.; Sun, Y.; Ma, P.; Song, D. Construction of a Magnetic-Fluorescent-Plasmonic Nanosensor for the Determination of MMP-2 Activity Based on SERS-Fluorescence Dual-Mode Signals. Biosens. Bioelectron. 2022, 212, 114389, DOI: 10.1016/j.bios.2022.114389Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhtl2hsrzN&md5=5ffa297f077667125924e50ae8e50cc6Construction of a magnetic-fluorescent-plasmonic nanosensor for the determination of MMP-2 activity based on SERS-fluorescence dual-mode signalsLiu, Lin; Chu, Hongyu; Yang, Jukun; Sun, Ying; Ma, Pinyi; Song, DaqianBiosensors & Bioelectronics (2022), 212 (), 114389CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Matrix metalloproteinase 2 (MMP-2) is a crucial biomarker of tumor growth, invasion and metastasis. In the present study, a core-satellite magnetic-fluorescent-plasmonic nanosensor (FMNS@Au) was constructed through biol. self-assembly to generate localized SERS "hot spots" and an efficient FRET system for the sensitive detn. of MMP-2 activity in a SERS-fluorescence dual-mode assay. In this hybrid nanosensor, a biotin-labeled peptide contg. a specific MMP-2 substrate (PLGVR) was employed as a bridge for the assembly of gold nanoparticles (AuNPs) and avidin functionalized fluorescent-magnetic nanospheres (FMNS). The modified RB on FMNS served as a Raman reporter and a donor of FRET, while the AuNPs assembled on FMNS acted as SERS substrates and acceptors of FRET. In the presence of MMP-2, the SERS "hot spot" effect was weakened and the FRET system was disrupted through enzymic cleavage of PLGVR, resulting in a redn. of SERS signal and the recovery of fluorescence emission. Importantly, this combination of SERS and fluorescence assay methods in the dual-mode nanosensor broadened the detection range for MMP-2 to 1-200 ng mL-1, with a limit of detection of 0.35 ng mL-1 and a limit of quantitation of 1.17 ng mL-1. In addn., our novel nanosensor affords semi-quant. sensing of MMP-2 by naked-eye observation and accurate detection of MMP-2 through dual-mode anal. The practicality of FMNS@Au was validated by detn. of MMP-2 activity in cell secretions and human serum samples. The designed FMNS@Au nanosensor holds great potential for clin. diagnosis of protease-related diseases.
- 48Yin, L.; Sun, H.; Zhang, H.; He, L.; Qiu, L.; Lin, J.; Xia, H.; Zhang, Y.; Ji, S.; Shi, H.; Gao, M. Quantitatively Visualizing Tumor-Related Protease Activity in Vivo Using a Ratiometric Photoacoustic Probe. J. Am. Chem. Soc. 2019, 141 (7), 3265– 3273, DOI: 10.1021/jacs.8b13628Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFKjtbg%253D&md5=92d3f71f014dcad961e1fd9cd621f656Quantitatively Visualizing Tumor-Related Protease Activity in Vivo Using a Ratiometric Photoacoustic ProbeYin, Ling; Sun, Hao; Zhang, Hao; He, Lei; Qiu, Ling; Lin, Jianguo; Xia, Huawei; Zhang, Yuqi; Ji, Shunjun; Shi, Haibin; Gao, MingyuanJournal of the American Chemical Society (2019), 141 (7), 3265-3273CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The abnormal expression of tumor-related proteases plays a crit. role in cancer invasion, progression, and metastasis. Therefore, it is considerably meaningful to noninvasively assess the proteases' activity in vivo for both tumor diagnosis and therapeutic evaluation. Herein, the authors report an activatable probe constructed with a near-IR dye (Cy5.5) and a quencher (QSY21) covalently linked through a peptide substrate of matrix metalloproteinases-2 (MMP-2) that was chosen as a model for tumor-assocd. proteases. Upon cleavage with activated MMP-2, this probe emitted an MMP-2-concn.-dependent fluorescence. Quite unexpectedly, owing to the variation in the aggregation state of both the dye and its quencher as a consequence of the cleavage, the responsive probe presented a dramatic MMP-2-concn.-dependent absorption at around 680 nm, while that at around 730 nm was MMP-2 concn. independent. These features allowed detection of MMP-2 activity via both fluorescence and photoacoustic (PA) imaging in vitro, resp. Moreover, taking the PA signal at 730 nm as an internal ref., the PA signal at 680 nm allowed quant. detection of MMP-2 expression in breast cancer in vivo. The authors thus envision that the current approach would offer a useful tool for studying the malignant impacts of versatile tumor-assocd. proteases in vivo.
- 49Nagase, H.; Ogata, Y.; Suzuki, K.; Enghild, J. J.; Salvesen, G. Substrate Specificities and Activation Mechanisms of Matrix Metalloproteinases. Biochem. Soc. Trans. 1991, 19, 715– 718, DOI: 10.1042/bst0190715Google ScholarThere is no corresponding record for this reference.
- 50Kwong, G. A.; Von Maltzahn, G.; Murugappan, G.; Abudayyeh, O.; Mo, S.; Papayannopoulos, I. A.; Sverdlov, D. Y.; Liu, S. B.; Warren, A. D.; Popov, Y.; Schuppan, D.; Bhatia, S. N. Mass-Encoded Synthetic Biomarkers for Multiplexed Urinary Monitoring of Disease. Nat. Biotechnol. 2013, 31 (1), 63– 70, DOI: 10.1038/nbt.2464Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVektbbM&md5=63625c88394c0cc19c012549eac5ab40Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of diseaseKwong, Gabriel A.; von Maltzahn, Geoffrey; Murugappan, Gayathree; Abudayyeh, Omar; Mo, Steven; Papayannopoulos, Ioannis A.; Sverdlov, Deanna Y.; Liu, Susan B.; Warren, Andrew D.; Popov, Yury; Schuppan, Detlef; Bhatia, Sangeeta N.Nature Biotechnology (2013), 31 (1), 63-70CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Biomarkers are becoming increasingly important in the clin. management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous mols. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiol. for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resoln. without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clin. used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to addnl. pathophysiol. processes and point-of-care diagnostics.
- 51Schuerle, S.; Dudani, J. S.; Christiansen, M. G.; Anikeeva, P.; Bhatia, S. N. Magnetically Actuated Protease Sensors for in Vivo Tumor Profiling. Nano Lett. 2016, 16 (10), 6303– 6310, DOI: 10.1021/acs.nanolett.6b02670Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVykurbO&md5=f268ff179a44dcd0271663ca9a867c43Magnetically Actuated Protease Sensors for in Vivo Tumor ProfilingSchuerle, Simone; Dudani, Jaideep S.; Christiansen, Michael G.; Anikeeva, Polina; Bhatia, Sangeeta N.Nano Letters (2016), 16 (10), 6303-6310CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Targeted cancer therapies require a precise detn. of the underlying biol. processes driving tumorigenesis within the complex tumor microenvironment. Therefore, new diagnostic tools that capture the mol. activity at the disease site in vivo are needed to better understand tumor behavior and ultimately maximize therapeutic responses. Matrix metalloproteinases (MMPs) drive multiple aspects of tumorigenesis, and their activity can be monitored using engineered peptide substrates as protease-specific probes. To identify tumor specific activity profiles, local sampling of the tumor microenvironment is necessary, such as through remote control of probes, which are only activated at the tumor site. Alternating magnetic fields (AMFs) provide an attractive option to remotely apply local triggering signals, as they penetrate deep into the body and are not likely to interfere with biol. processes due to weak magnetic properties of tissue. Here, the authors report the design and evaluation of a protease-activity nanosensor that can be remotely activated at the site of disease via an AMF at 515 kHz and 15 kA/m. The authors' nanosensor was comprised of thermosensitive liposomes contg. functionalized protease substrates that were unveiled at the target site by remotely triggered heat dissipation of coencapsulated magnetic nanoparticles (MNPs). This nanosensor was combined with a unique detection assay to quantify the amt. of cleaved substrates in the urine. The authors applied this spatiotemporally controlled system to det. tumor protease activity in vivo and identified differences in substrate cleavage profiles between two mouse models of human colorectal cancer.
- 52Xie, J.; Zhang, F.; Aronova, M.; Zhu, L.; Lin, X.; Quan, Q.; Liu, G.; Zhang, G.; Choi, K. Y.; Kim, K.; Sun, X.; Lee, S.; Sun, S.; Leapman, R.; Chen, X. Manipulating the Power of an Additional Phase: A Flower-like Au-Fe 3O4 Optical Nanosensor for Imaging Protease Expressions in Vivo. ACS Nano 2011, 5 (4), 3043– 3051, DOI: 10.1021/nn200161vGoogle Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjtVKhurc%253D&md5=e0986457030c62bf7438a6fe436c12aeManipulating the Power of an Additional Phase: A Flower-like Au-Fe3O4 Optical Nanosensor for Imaging Protease Expressions In vivoXie, Jin; Zhang, Fan; Aronova, Maria; Zhu, Lei; Lin, Xin; Quan, Qimeng; Liu, Gang; Zhang, Guofeng; Choi, Ki-Young; Kim, Kwangmeyung; Sun, Xiaolian; Lee, Seulki; Sun, Shouheng; Leapman, Richard; Chen, XiaoyuanACS Nano (2011), 5 (4), 3043-3051CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The authors and others have recently proposed the synthesis of composite nanoparticles that offer strongly enhanced functionality. Here the authors used a flower-shaped Au-Fe3O4 nanoparticle as a template to construct an optical probe contg. Cy5.5-GPLGVRG-TDOPA on the iron oxide surface and SH-PEG5000 on the gold surface that can be specifically activated by matrix metalloproteinases expressed in tumors. Gold nanoparticles have excellent quenching properties, but labile surface chem. in vivo; however, iron oxide nanoparticles afford robust surface chem., but are suboptimal as energy receptors. By a marriage of the two, the authors produced a unified structure with performance that is unachievable with the sep. components. The authors' results are a further demonstration that the architecture of nanoparticles can be modulated to tailor their function as mol. imaging/therapeutic agents.
- 53Ke, W.; Zha, Z.; Mukerabigwi, J. F.; Chen, W.; Wang, Y.; He, C.; Ge, Z. Matrix Metalloproteinase-Responsive Multifunctional Peptide-Linked Amphiphilic Block Copolymers for Intelligent Systemic Anticancer Drug Delivery. Bioconjugate Chem. 2017, 28 (8), 2190– 2198, DOI: 10.1021/acs.bioconjchem.7b00330Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVyhs7fO&md5=7962c23f31c9c7424c2b84e1afc85654Matrix Metalloproteinase-Responsive Multifunctional Peptide-Linked Amphiphilic Block Copolymers for Intelligent Systemic Anticancer Drug DeliveryKe, Wendong; Zha, Zengshi; Mukerabigwi, Jean Felix; Chen, Weijian; Wang, Yuheng; He, Chuanxin; Ge, ZhishenBioconjugate Chemistry (2017), 28 (8), 2190-2198CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The amphiphilic block copolymer anticancer drug nanocarriers clin. used or in the progress of clin. trials frequently suffer from modest final therapeutic efficacy due to a lack of intelligent features. For example, the biodegradable amphiphilic block copolymer, poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PDLLA) has been approved for clin. applications as a paclitaxel (PTX) nanocarrier (Genexol-PM) due to the optimized pharmacokinetics and biodistribution; however, a lack of intelligent features limits the intracellular delivery in tumor tissue. To endow the mediocre polymer with smart properties via a safe and facile method, we introduced a matrix metalloproteinase (MMP)-responsive peptide GPLGVRGDG into the block copolymer via efficient click chem. and ring-opening polymn. to prep. PEG-GPLGVRGDG-PDLLA (P1). P1 was further self-assembled into micellar nanoparticles (NPs) to load PTX, which show MMP-2-triggered dePEGylation due to cleavage of the peptide linkage. Moreover, the residual VRGDG sequences are retained on the surface of the NPs after dePEGylation, which can serve as ligands to facilitate the cellular uptake. The cytotoxicity of PTX loaded in P1 NPs against 4T1 cells is significantly enhanced as compared with free PTX or PTX-loaded PEG-GPLGVRG-PDLLA (P2) and PEG-PDLLA (P3) NPs. In vivo studies confirmed that PTX-loaded P1 NPs show prolonged blood circulation, which are similar to P2 and P3 NPs but exhibit more-efficient accumulation in the tumor site. Ultimately, PTX-loaded P1 NPs display statistically significant improvement of antitumor activity against tumor-bearing mice via systemic administration. Therefore, the strategy by facile incorporation of a responsive peptide linkage between PEG and PDLLA is a promising approach to improving the therapeutic efficacy of anticancer-drug-loaded amphiphilic block copolymer micelles.
- 54Harris, T. J.; Von Maltzahn, G.; Derfus, A. M.; Ruoslahti, E.; Bhatia, S. N. Proteolytic Actuation of Nanoparticle Self-Assembly. Angew. Chem., Int. Ed. 2006, 45 (19), 3161– 3165, DOI: 10.1002/anie.200600259Google ScholarThere is no corresponding record for this reference.
- 55Li, S.-Y.; Cheng, Q.-X.; Liu, J.-K.; Nie, X.-Q.; Zhao, G.-P.; Wang, J. CRISPR-Cas12a Has Both Cis- and Trans-Cleavage Activities on Single-Stranded DNA. Cell Res. 2018, 28, 491– 493, DOI: 10.1038/s41422-018Google ScholarThere is no corresponding record for this reference.
- 56Wang, B.; Wang, R.; Wang, D.; Wu, J.; Li, J.; Wang, J.; Liu, H.; Wang, Y. Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection. Anal. Chem. 2019, 91 (19), 12156– 12161, DOI: 10.1021/acs.analchem.9b01526Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1Kks7zI&md5=fb7777d8ab197f4e9503fe3343335652Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid DetectionWang, Bei; Wang, Rui; Wang, Daqi; Wu, Jian; Li, Jixi; Wang, Jin; Liu, Huihui; Wang, YongmingAnalytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12156-12161CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A rapid and sensitive method is crucial for nucleic acid detection. Recently, RNA-guided CRISPR/Cas12a nuclease-based methods present great promise for nucleic acid detection. In the present methods, however, DNA amplification and subsequent Cas12a cleavage is sepd. and the whole process takes as long as 2 h. Most importantly, the uncapping operation increases the risk of aerosol contamination. In this study, we propose a CRISPR/Cas12a-based method named "Cas12aVDet" for rapid nucleic acid detection. By integrating recombinase polymerase amplification (RPA) with Cas12a cleavage in a single reaction system, the detection can be accomplished in 30 min and uncapping contamination can be avoided. The detection signal can be obsd. by the naked eye under blue light. This method could detect DNA at single mol. level and demonstrated 100% accuracy for mycoplasma contamination detection, presenting great potential for a variety of nucleic acid detection applications.
- 57Feng, W.; Newbigging, A. M.; Tao, J.; Cao, Y.; Peng, H.; Le, C.; Wu, J.; Pang, B.; Li, J.; Tyrrell, D. L.; Zhang, H.; Le, X. C. CRISPR Technology Incorporating Amplification Strategies: Molecular Assays for Nucleic Acids, Proteins, and Small Molecules. Chem. Sci. 2021, 12 (13), 4683– 4698, DOI: 10.1039/D0SC06973FGoogle Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsVSmu7g%253D&md5=c1957341af7adda64699b9d2f62a935fCRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small moleculesFeng, Wei; Newbigging, Ashley M.; Tao, Jeffrey; Cao, Yiren; Peng, Hanyong; Le, Connie; Wu, Jinjun; Pang, Bo; Li, Juan; Tyrrell, D. Lorne; Zhang, Hongquan; Le, X. ChrisChemical Science (2021), 12 (13), 4683-4698CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A review. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-assocd. (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technol. has also advanced mol. detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance mol. targets, improvements in anal. specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temp. facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and mol. translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small mols. Successful integrations of CRISPR technol. with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.
- 58(a) https://www.anaspec.com/en/catalog/sensolyte-520-mmp-2-assay-kit-fluorimetric-1-kit∼9d6616da-4170-4e04-93d7-9bdcd084a456.Google ScholarThere is no corresponding record for this reference.(b) https://bpsbioscience.com/fluorogenic-mmp2-assay-kit-79918.Google ScholarThere is no corresponding record for this reference.
- 59Hananya, N.; Press, O.; Das, A.; Scomparin, A.; Satchi-Fainaro, R.; Sagi, I.; Shabat, D. Persistent Chemiluminescent Glow of Phenoxy-Dioxetane Luminophore Enables Unique CRET-Based Detection of Proteases. Chem.─Eur. J. 2019, 25 (64), 14679– 14687, DOI: 10.1002/chem.201903489Google ScholarThere is no corresponding record for this reference.
- 60den Hamer, A.; Den Dierickx, P.; Arts, R.; De Vries, J. S. P. M.; Brunsveld, L.; Merkx, M. Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity. ACS Sens. 2017, 2 (6), 729– 734, DOI: 10.1021/acssensors.7b00239Google ScholarThere is no corresponding record for this reference.
- 61Jian, M.; Su, M.; Gao, J.; Wang, Z. Peptide Microarray-Based Fluorescence Assay for Quantitatively Monitoring the Tumor-Associated Matrix Metalloproteinase-2 Activity. Sens. Actuators B Chem. 2020, 304. 127320 DOI: 10.1016/j.snb.2019.127320 .Google ScholarThere is no corresponding record for this reference.
- 62Chan, Y. C.; Chen, C. W.; Chan, M. H.; Chang, Y. C.; Chang, W. M.; Chi, L. H.; Yu, H. M.; Lin, Y. F.; Tsai, D. P.; Liu, R. S.; Hsiao, M. MMP2-Sensing up-Conversion Nanoparticle for Fluorescence Biosensing in Head and Neck Cancer Cells. Biosens. Bioelectron. 2016, 80, 131– 139, DOI: 10.1016/j.bios.2016.01.049Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKktb4%253D&md5=83a84b6cd7a5291a5ee274fe0f1d46f8MMP2-sensing up-conversion nanoparticle for fluorescencebiosensing in head and neck cancer cellsChan, Yung-Chieh; Chen, Chieh-Wei; Chan, Ming-Hsien; Chang, Yu-Chan; Chang, Wei-Min; Chi, Li-Hsing; Yu, Hui-Ming; Lin, Yuan-Feng; Tsai, Din Ping; Liu, Ru-Shi; Hsiao, MichaelBiosensors & Bioelectronics (2016), 80 (), 131-139CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Upconversion nanoparticles (UCNPs) have extensive biol.-applications because of their bio-compatibility, tunable optical properties and their ability to be excited by IR radiation. Matrix metalloproteinases (MMPs) play important roles in extracellular matrix remodelling; they are usually found to significantly increase during cancer progression, and these increases may lead to poor patient survival. In this study, we produced a biosensor that can be recognized by MMP2 and then be unravelled by the attached quencher to emit visible light. We used 3.5-nm gold nanoparticles as a quencher that absorbed emission from UCNPs at a wavelength of 540 nm. The biosensor consists of an upconversion nanoparticle, MMP2-recognized polypeptides and quenchers. To improve the biocompatibility and modify the UCNPs with a polypeptide, they were coated with a silica shell and further conjugated with MMP-recognizing polypeptides. The polypeptide has two ends of featuring carboxylic and thiol groups that react with UCNPs and AuNPs, and the resulting nanoparticles were referred to as UCNP@p-Au. According to the in vitro cell viability anal., UCNP@p-Au exhibited little toxicity and biocompatibility in head and neck cancer cells. Cellular uptake studies showed that the MMP-based biosensor was activated by 980-nm irradn. to emit green light. This MMP-based biosensor may serve as sensitive and specific mol. fluorescent probe in biol.-applications.
- 63https://www.merckmillipore.com/IT/it/product/InnoZyme-Gelatinase-MMP-2-MMP-9-Activity-Assay-kit-Fluorogenic,EMD_BIO-CBA003?ReferrerURL=https%3A%2F%2Fwww.google.com%2F.Google ScholarThere is no corresponding record for this reference.
- 64https://www.anaspec.com/en/catalog/sensolyte-520-mmp-2-assay-kit-fluorimetric-1-kit∼9d6616da-4170-4e04-93d7-9bdcd084a456.Google ScholarThere is no corresponding record for this reference.
- 65Bruch, R.; Urban, G. A.; Dincer, C. CRISPR/Cas Powered Multiplexed Biosensing. Trends Biotechnol. 2019, 37 (8), 791– 792, DOI: 10.1016/j.tibtech.2019.04.005Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXosFWjt7s%253D&md5=4dbc879ce82707bbf31181da3536aaadCRISPR/Cas Powered Multiplexed BiosensingBruch, Richard; Urban, Gerald A.; Dincer, CanTrends in Biotechnology (2019), 37 (8), 791-792CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)A review. Multiplexed CRISPR/Cas biosensing offers various possibilities for diagnosing different diseases by analyzing many analytes from one single specimen. However, in this relatively new field, nearly no multiplexing approaches exist, as many challenges need to be addressed. We discuss the reasons behind it and possible strategies to push the multiplexing level.
- 66Phan, Q. A.; Truong, L. B.; Medina-Cruz, D.; Dincer, C.; Mostafavi, E. CRISPR/Cas-Powered Nanobiosensors for Diagnostics. Biosens. Bioelectron. 2022, 197, 113732, DOI: 10.1016/j.bios.2021.113732Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVanurfM&md5=c93df123f5f64e524b944a4aa50ff1d4CRISPR/Cas-powered nanobiosensors for diagnosticsPhan, Quynh Anh; Truong, Linh B.; Medina-Cruz, David; Dincer, Can; Mostafavi, EbrahimBiosensors & Bioelectronics (2022), 197 (), 113732CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)A review. CRISPR diagnostics (CRISPR-Dx) offer a wide range of enhancements compared to traditional nanobiosensors by taking advantage of the excellent trans-cleavage activity of the CRISPR/Cas systems. However, the single-stranded DNA/RNA reporters of the current CRISPR-Dx suffer from poor stability and limited sensitivity, which make their application in complex biol. environments difficult. In comparison, nanomaterials, esp. metal nanoparticles, exhibits robust stability and desirable optical and electrocatalytical properties, which make them ideal as reporter mols. Therefore, biosensing research is moving towards the use of the trans-cleavage activity of CRISPR/Cas effectors on metal nanoparticles and apply the new phenomenon to develop novel nanobiosensors to target various targets such as viral infections, genetic mutations and tumor biomarkers, by using different sensing methods, including, but not limited to fluorescence, luminescence resonance, colorimetric and electrochem. signal readout. In this review, we explore some of the most recent advances in the field of CRISPR-powered nanotechnol. biosensors. Demonstrating high accuracy, sensitivity, selectivity and versatility, nanobiosensors along with CRISPR/Cas technol. offer tremendous potential for next-generation diagnostics of multiple targets, esp. at the point of care and without any target amplification.
- 67Boulware, K. T.; Daugherty, P. S. Protease Specificity Determination by Using Cellular Libraries of Peptide Substrates (CLiPS). Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (20), 7583– 7588, DOI: 10.1073/pnas.0511108103Google ScholarThere is no corresponding record for this reference.
- 68Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.; Ellman, J. A.; Craik, C. S. Rapid and General Profiling of Protease Specificity by Using Combinatorial Fluorogenic Substrate Libraries. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (14), 7754– 7759, DOI: 10.1073/pnas.140132697Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXkvFCnt7Y%253D&md5=669beba3367acd1911a2a39660117a78Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate librariesHarris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (14), 7754-7759CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A method is presented for the prepn. and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient prodn. of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approx. 3-fold-increased quantum yield of ACC over AMC permits redn. in enzyme and substrate concns. As a consequence, a greater no. of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Sol. positional protease substrate libraries of 137,180 and 6859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, resp., were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.
- 69Bertucci, A.; Porchetta, A.; Grosso, E.; Del Patiço, A.; Idili, A.; Ricci, F. Protein-Controlled Actuation of Dynamic Nucleic Acid Networks by Using Synthetic DNA Translators. Angew. Chem., Int. Ed. 2020, 59, 20577– 20581, DOI: 10.1002/anie.202008553Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslGqt7jP&md5=88b58d6f0b1cbcdc4bc74038962f1704Protein-Controlled Actuation of Dynamic Nucleic Acid Networks by Using Synthetic DNA TranslatorsBertucci, Alessandro; Porchetta, Alessandro; Del Grosso, Erica; Patino, Tania; Idili, Andrea; Ricci, FrancescoAngewandte Chemie, International Edition (2020), 59 (46), 20577-20581CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Integrating dynamic DNA nanotechnol. with protein-controlled actuation will expand the authors' ability to process mol. information. The authors have developed a strategy to actuate strand displacement reactions using DNA-binding proteins by engineering synthetic DNA translators that convert specific protein-binding events into trigger inputs through a programmed conformational change. The authors have constructed synthetic DNA networks responsive to two different DNA-binding proteins, TATA-binding protein and Myc-Max, and demonstrated multi-input activation of strand displacement reactions. The authors achieved protein-controlled regulation of a synthetic RNA and of an enzyme through artificial DNA-based communication, showing the potential of the authors' mol. system in performing further programmable tasks.
- 70Bucci, J.; Irmisch, P.; Del Grosso, E.; Seidel, R.; Ricci, F. Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions. J. Am. Chem. Soc. 2022, 144 (43), 19791– 19798, DOI: 10.1021/jacs.2c06599Google ScholarThere is no corresponding record for this reference.
- 71Xiang, Z.; Zhao, J.; Yi, D.; Di, Z.; Li, L. Peptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular Imaging. Angew. Chem., Int. Ed. 2021, 60 (42), 22659– 22663, DOI: 10.1002/anie.202106639Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVCntb7L&md5=6645b9f7993c9da3ea3a7fda50caf4fePeptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular ImagingXiang, Zhichu; Zhao, Jian; Yi, Deyu; Di, Zhenghan; Li, LeleAngewandte Chemie, International Edition (2021), 60 (42), 22659-22663CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Protease-triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnol. Herein, we report the design of a protease-activatable aptamer system that can perform mol. sensing and imaging in a tumor-specific manner. The system is constructed by locking the structure-switching activity of an aptamer using a rationally designed PNA-peptide-PNA triblock copolymer. Highly selective protease-mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell-selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.
- 72Watson, E. E.; Angerani, S.; Sabale, P. M.; Winssinger, N. Biosupramolecular Systems: Integrating Cues into Responses. J. Am. Chem. Soc. 2021, 143 (12), 4467– 4482, DOI: 10.1021/jacs.0c12970Google Scholar72https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjsVKhu7g%253D&md5=d03a2c63507bd7266e8d000044a65633Biosupramolecular Systems: Integrating Cues into ResponsesWatson, Emma E.; Angerani, Simona; Sabale, Pramod M.; Winssinger, NicolasJournal of the American Chemical Society (2021), 143 (12), 4467-4482CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A review. Life is orchestrated by biomols. interacting in complex networks of biol. circuitry with emerging function. Progress in different areas of chem. has made the design of systems that can recapitulate elements of such circuitry possible. Herein we review prominent examples of networks, the methodologies available to translate an input into various outputs, and speculate on potential applications and directions for the field. The programmability of nucleic acid hybridization has inspired applications beyond its function in heredity. At the circuitry level, DNA provides a powerful platform to design dynamic systems that respond to nucleic acid input sequences with output sequences through diverse logic gates, enabling the design of ever more complex circuitry. In order to interface with more diverse biomol. inputs and yield outputs other than oligonucleotide sequences, an array of nucleic acid conjugates have been reported that can engage proteins as their input and yield a turn-on of enzymic activity, a bioactive small mol., or morphol. changes in nanoobjects. While the programmability of DNA makes it an obvious starting point to design circuits, other biosupramol. interactions have also been demonstrated, and harnessing progress in protein design is bound to deliver further integration of macromols. in artificial circuits.
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Abstract
Figure 1
Figure 1. Schematic illustration of the proposed MMP2 activity-based detection method, leveraging a synthetic protein-to-DNA input exchange. A peptide-PNA chemical translator is used to convert MMP2 enzymatic activity into an input for CRISPR-Cas12a-based signal amplification.
Figure 2
Figure 2. Design of the peptide-PNA chemical translator and HPLC-HRMS analysis of protease cleavage products. (a) Illustration of the molecular design of the chemical translator, reporting the amino acid sequence of the peptide unit and the nucleobase sequence of the PNA unit. An azide group is conjugated to the 5′-end of the probe to enable click chemistry for anchoring to solid supports. A fluorescent version of the translator was obtained by conjugating 5(6)-carboxy-tetramethylrhodamine (TAMRA) to additional lysine residue present at the 3′-end. (b) Schematic representation of the MMP2-mediated enzymatic cleavage of the chemical translator (red circle-line probe), highlighting the cleavage site and the formation of two reaction products, a small peptide fragment (blue circles) and a larger fragment containing the PNA unit (orange circles + orange line). (c) HPLC-ESI-HRMS chromatogram of the chemical translator after incubation with MMP2 (24 h, 37 °C, [MMP2]:[translator] 1:10). The three main peaks correspond to the PNA-containing fragment (rt 13.25 min, m/z = 528.7370 [M+6H]6+, 634.2828 [M+5H]5+, 792.6016 [M+4H]4+, 1056.4669 [M+3H]3+ orange box), to unreacted molecular translator present in large excess in the reaction mixture (rt 16.42 min, m/z = 620.9489 [M+6H]6+, 744.9370 [M+5H]5+, 930.6689 [M+4H]4+ red box), and to the peptide fragment (rt 21.45 min, m/z = 571.2845 [M + H]+, 593.2655 [M + Na]+ blue box).
Figure 3
Figure 3. Design of the sensing platform and characterization of the activity-based sensing mechanism. (a) Multistep functionalization scheme of the magnetic beads with the chemical translator. Bifunctional DBCO-NHS allows for conversion of the amine groups initially present on the bead surface into DBCO functionalities. The peptide-PNA translator was then grafted onto these beads through an SPAAC reaction between the azide moiety on the 5′-end of the peptide unit of the translator and DBCO. (b) Zeta potential measurements during the various functionalization steps. After functionalization with DBCO, the zeta potential of a dispersion of the magnetic beads in PBS buffer shifts from 33 ± 1 mV to −4 ± 1 mV. Conjugation with the chemical translator increases the zeta potential to 11 ± 1 mV (n = 3, mean + SD). (c) Illustration of the MMP2 cleavage assay using magnetic beads functionalized with a fluorophore-tagged chemical translator. When the sensing platform is incubated with MMP2, proteolytic cleavage followed by magnetic separation of the beads results in a supernatant enriched in the PNA-containing cleavage product. (d) Normalized fluorescence intensity obtained for the cleavage assay conducted using different MMP2 incubation times ([MMP2] 100 nM, magnetic beads 1 mg/mL, n = 3, mean + SD). (e) Normalized fluorescence intensity obtained with MMP2 (100 nM) and other nonspecific MMPs (100 nM) (n = 3, mean + SD). (f) Fluorescence intensity values obtained for the assay conducted using different concentrations of MMP2 in the range of 0.03–10 nM (cleavage time 2h), from which it was possible to calculate an LOD of 104 pM (n = 3, mean ± SD), calculated as the concentration of MMP2 leading to a signal change equal to 3 times the standard deviation of the background (absence of the target).
Figure 4
Figure 4. CRISPR-Cas12a-based amplification of MMP2-derived nucleic acid output. (a) Pictorial illustration of the proposed MMP2 activity-based assay using CRISPR-Cas12a-based signal amplification. The peptide-PNA translator on the magnetic bead surface is hybridized with an ssDNA sequence serving as the trigger input for target-induced CRISPR-Cas12 trans-cleavage nuclease activity. Following incubation with MMP2 (2 h) and magnetic separation of the beads, the nucleic acid-containing cleavage product enriches the supernatant. This is incubated with a preformed crRNA/Cas12a complex (20 nM) in the presence of an excess of ssDNA hairpin reporter (100 nM). (b) Fluorescence kinetic profiles of CRISPR-Cas12a trans-cleavage activity (i.e., Cas12a-mediated degradation of the DNA hairpin reporters) upon interaction with MMP2-derived ssDNA activator in the MMP2 concentration range 1–300 pM. (c) Calibration curve obtained through a linear fit of signal gain % values in the 3–100 pM MMP2 concentration range. The curve is described by the following equation: y = (−452.1 ± 48.7) + (174.6 ± 11.4)x, R2 = 0.96. (d) Signal gain % values, calculated at the 2 h end point, obtained for CRISPR-Cas12a-based (red bars) and unamplified fluorescence assays (blue bars) for different concentrations of MMP2 (n = 3, mean ± SD).
Figure 5
Figure 5. Analysis of MMP2 in undiluted cell culture medium. (a) Calibration curve obtained through a linear fit of fluorescence intensity values in the 30–300 pM MMP2 concentration range using spiked samples in undiluted DMEM cell culture medium. The curve is described by the following equation: y = (−1,660,085 ± 353,763) + (849,889 ± 71,517)x, R2 = 0.93. (b) Recovery values for different concentrations of MMP2 in undiluted DMEM cell culture medium when using the developed CRISPR-Cas assay or a commercially available standard ELISA (n = 3, mean ± SD).
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- 1Soleimany, A. P.; Bhatia, S. N. Activity-Based Diagnostics: An Emerging Paradigm for Disease Detection and Monitoring. Trends Mol. Med. 2020, 26 (5), 450– 468, DOI: 10.1016/j.molmed.2020.01.0131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXks1Kmtr8%253D&md5=4f24850c46de76ccf09462a92c89f1ffActivity-Based Diagnostics: An Emerging Paradigm for Disease Detection and MonitoringSoleimany, Ava P.; Bhatia, Sangeeta N.Trends in Molecular Medicine (2020), 26 (5), 450-468CODEN: TMMRCY; ISSN:1471-4914. (Elsevier Ltd.)A review. Diagnostics to accurately detect disease and monitor therapeutic response are essential for effective clin. management. Bioengineering, chem. biol., mol. biol., and computer science tools are converging to guide the design of diagnostics that leverage enzymic activity to measure or produce biomarkers of disease. Advances in the development of these 'activity-based diagnostics' (ABDx) and their application in infectious and noncommunicable diseases. We highlight efforts towards both mol. probes that respond to disease-specific catalytic activity to produce a diagnostic readout, as well as diagnostics that use enzymes as an engineered component of their sense-and-respond cascade. These technologies exemplify how integrating techniques from multiple disciplines with preclin. validation has enabled ABDx that may realize the goals of precision medicine.
- 2Muir, R. K.; Guerra, M.; Bogyo, M. M. Activity-Based Diagnostics: Recent Advances in the Development of Probes for Use with Diverse Detection Modalities. ACS Chem. Biol. 2022, 17 (2), 281– 291, DOI: 10.1021/acschembio.1c007532https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XptlShtA%253D%253D&md5=1b7e018e69b93bb5c13e176adcdb54a5Activity-Based Diagnostics: Recent Advances in the Development of Probes for Use with Diverse Detection ModalitiesMuir, Ryan K.; Guerra, Matteo; Bogyo, Matthew M.ACS Chemical Biology (2022), 17 (2), 281-291CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Abnormal enzyme expression and activity is a hallmark of many diseases. Activity-based diagnostics are a class of chem. probes that aim to leverage this dysregulated metabolic signature to produce a detectable signal specific to diseased tissue. In this review, we highlight recent methodologies employed in activity-based diagnostics that provide exquisite signal sensitivity and specificity in complex biol. systems for multiple disease states. We divide these examples based upon their unique signal readout modalities and highlight those that have advanced into clin. trials.
- 3Kandell, R. M.; Kudryashev, J. A.; Kwon, E. J. Targeting the Extracellular Matrix in Traumatic Brain Injury Increases Signal Generation from an Activity-Based Nanosensor. ACS Nano 2021, 15 (12), 20504– 20516, DOI: 10.1021/acsnano.1c090643https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXis1KgurjJ&md5=e7acd15bb2d6be4834880779b89b5066Targeting the Extracellular Matrix in Traumatic Brain Injury Increases Signal Generation from an Activity-Based NanosensorKandell, Rebecca M.; Kudryashev, Julia A.; Kwon, Ester J.ACS Nano (2021), 15 (12), 20504-20516CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Traumatic brain injury (TBI) is a crit. public health concern and major contributor to death and long-term disability. After the initial trauma, a sustained secondary injury involving a complex continuum of pathophysiol. unfolds, ultimately leading to the destruction of nervous tissue. One disease hallmark of TBI is ectopic protease activity, which can mediate cell death, extracellular matrix breakdown, and inflammation. We previously engineered a fluorogenic activity-based nanosensor for TBI (TBI-ABN) that passively accumulates in the injured brain across the disrupted vasculature and generates fluorescent signal in response to calpain-1 cleavage, thus enabling in situ visualization of TBI-assocd. calpain-1 protease activity. In this work, we hypothesized that actively targeting the extracellular matrix (ECM) of the injured brain would improve nanosensor accumulation in the injured brain beyond passive delivery alone and lead to increased nanosensor activation. We evaluated several peptides that bind exposed/enriched ECM constituents in the brain and discovered that nanomaterials modified with peptides that target hyaluronic acid (HA) displayed widespread distribution across the injury lesion, in particular colocalizing with perilesional and hippocampal neurons. Modifying TBI-ABN with HA-targeting peptide led to increases in activation in a ligand-valency-dependent manner, up to 6.6-fold in the injured cortex compared to a nontargeted nanosensor. This robust nanosensor activation enabled 3D visualization of injury-specific protease activity in a cleared and intact brain. In our work, we establish that targeting brain ECM with peptide ligands can be leveraged to improve the distribution and function of a bioresponsive imaging nanomaterial.
- 4Kudryashev, J. A.; Madias, M. I.; Kandell, R. M.; Lin, Q. X.; Kwon, E. J. An Activity-Based Nanosensor for Minimally-Invasive Measurement of Protease Activity in Traumatic Brain Injury. Adv. Funct. Mater. 2023, 33 (28), 2300218, DOI: 10.1002/adfm.202300218There is no corresponding record for this reference.
- 5Nagano, N.; Ichihashi, Y.; Komatsu, T.; Matsuzaki, H.; Hata, K.; Watanabe, T.; Misawa, Y.; Suzuki, M.; Sakamoto, S.; Kagami, Y.; Kashiro, A.; Takeuchi, K.; Kanemitsu, Y.; Ochiai, H.; Watanabe, R.; Honda, K.; Urano, Y. Development of Fluorogenic Substrates for Colorectal Tumor-Related Neuropeptidases for Activity-Based Diagnosis. Chem. Sci. 2023, 14 (17), 4495– 4499, DOI: 10.1039/D2SC07029DThere is no corresponding record for this reference.
- 6Sanman, L. E.; Bogyo, M. Activity-Based Profiling of Proteases. Annu. Rev. Biochem. 2014, 83, 249– 273, DOI: 10.1146/annurev-biochem-060713-0353526https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFOhtrjN&md5=1348a1c09d9506aa29cda49b376b0dc9Activity-based profiling of proteasesSanman, Laura E.; Bogyo, MatthewAnnual Review of Biochemistry (2014), 83 (), 249-273CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)A review. Proteolytic enzymes are key signaling mols. in both normal physiol. processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease mRNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochem. methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymol., discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.
- 7Zmudzinski, M.; Malon, O.; Poręba, M.; Drąg, M. Imaging of Proteases Using Activity-Based Probes. Curr. Opin. Chem. Biol. 2023, 74, 102299, DOI: 10.1016/j.cbpa.2023.102299There is no corresponding record for this reference.
- 8Zhang, H.; Wu, M.; Ta, H. T.; Xu, Z. P.; Zhang, R. Recent Development and Applications of Sensors for the Detection of Matrix Metalloproteinases. Adv. Mater. Technol. 2023, 8, 2201786, DOI: 10.1002/admt.202201786There is no corresponding record for this reference.
- 9Ćwilichowska, N.; Świderska, K. W.; Dobrzyń, A.; Drąg, M.; Poręba, M. Diagnostic and Therapeutic Potential of Protease Inhibition. Mol. Aspects Med. 2022, 88, 101144, DOI: 10.1016/j.mam.2022.101144There is no corresponding record for this reference.
- 10Dudani, J. S.; Warren, A. D.; Bhatia, S. N. Harnessing Protease Activity to Improve Cancer Care. Annu. Rev. Cancer Biol. 2018, 2, 353– 376, DOI: 10.1146/annurev-cancerbio-030617-050549There is no corresponding record for this reference.
- 11Egeblad, M.; Werb, Z. New Functions for the Matrix Metalloproteinases in Cancer Progression. Nat. Rev. Cancer 2002, 2 (3), 161– 174, DOI: 10.1038/nrc74511https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xis1KktL8%253D&md5=56dd558362155cb25c9adbea4d478aa8New functions for the matrix metalloproteinases in cancer progressionEgeblad, Mikala; Werb, ZenaNature Reviews Cancer (2002), 2 (3), 161-174CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. Matrix metalloproteinases (MMPs) have long been assocd. with cancer-cell invasion and metastasis. This provided the rationale for clin. trials of MMP inhibitors, unfortunately with disappointing results. We now know, however, that the MMPs have functions other than promotion of invasion, have substrates other than components of the extracellular matrix, and that they function before invasion in the development of cancer. With this knowledge in hand, can we rethink the use of MMP inhibitors in the clinic.
- 12Quintero-Fabián, S.; Arreola, R.; Becerril-Villanueva, E.; Torres-Romero, J. C.; Arana-Argáez, V.; Lara-Riegos, J.; Ramírez-Camacho, M. A.; Alvarez-Sánchez, M. E. Role of Matrix Metalloproteinases in Angiogenesis and Cancer. Front. Oncol. 2019, 9, 1370, DOI: 10.3389/fonc.2019.0137012https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MbosFWkuw%253D%253D&md5=5315e512fdf249018b0bd8541c5eec7dRole of Matrix Metalloproteinases in Angiogenesis and CancerQuintero-Fabian Saray; Arreola Rodrigo; Becerril-Villanueva Enrique; Torres-Romero Julio Cesar; Lara-Riegos Julio; Arana-Argaez Victor; Ramirez-Camacho Mario Alberto; Alvarez-Sanchez Maria ElizbethFrontiers in oncology (2019), 9 (), 1370 ISSN:2234-943X.During angiogenesis, new vessels emerge from existing endothelial lined vessels to promote the degradation of the vascular basement membrane and remodel the extracellular matrix (ECM), followed by endothelial cell migration, and proliferation and the new generation of matrix components. Matrix metalloproteinases (MMPs) participate in the disruption, tumor neovascularization, and subsequent metastasis while tissue inhibitors of metalloproteinases (TIMPs) downregulate the activity of these MMPs. Then, the angiogenic response can be directly or indirectly mediated by MMPs through the modulation of the balance between pro- and anti-angiogenic factors. This review analyzes recent knowledge on MMPs and their participation in angiogenesis.
- 13Gobin, E.; Bagwell, K.; Wagner, J.; Mysona, D.; Sandirasegarane, S.; Smith, N.; Bai, S.; Sharma, A.; Schleifer, R.; She, J. X. A Pan-Cancer Perspective of Matrix Metalloproteases (MMP) Gene Expression Profile and Their Diagnostic/Prognostic Potential. BMC Cancer 2019, 19 (1), 1– 10, DOI: 10.1186/s12885-019-5768-0There is no corresponding record for this reference.
- 14Fields, G. B. Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases. In Matrix Metalloproteinase Protocols; Clark, I. M.; Young, D. A.; Rowan, A. D., Eds.; Humana Press, 2010; Vol. 622, pp 393– 433.There is no corresponding record for this reference.
- 15Pham, W.; Choi, Y.; Weissleder, R.; Tung, C. H. Developing a Peptide-Based near-Infrared Molecular Probe for Protease Sensing. Bioconjugate Chem. 2004, 15 (6), 1403– 1407, DOI: 10.1021/bc049924s15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnslGqsLs%253D&md5=9be677530b817fe8c7134f9dacc047ffDeveloping a peptide-based near-infrared molecular probe for protease sensingPham, Wellington; Choi, Yongdoo; Weissleder, Ralph; Tung, Ching-HsuanBioconjugate Chemistry (2004), 15 (6), 1403-1407CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Recently near-IR (NIR) mol. probes have become important reporter mols. for a no. of types of in vivo biomedical imaging. A peptide-based NIR fluorescence probe consisting of a NIR fluorescence emitter (Cy5.5), a NIR fluorescence absorber (NIRQ820), and a protease selective peptide sequence was designed to sense protease activity. Using a MMP-7 model, we showed that NIRQ820 efficiently absorbs the emission energy of Cy5.5 resulting in a low initial signal. Upon reacting with its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a Kcat/Km of 100 000 M-1 s-1. The described synthetic strategy should have wide application for other NIR probe prepns.
- 16Ryu, J. H.; Lee, A.; Lee, S.; Ahn, C. H.; Park, J. W.; Leary, J. F.; Park, S.; Kim, K.; Kwon, I. C.; Youn, I. C.; Choi, K. One-Step” Detection of Matrix Metalloproteinase Activity Using a Fluorogenic Peptide Probe-Immobilized Diagnostic Kit. Bioconjugate Chem. 2010, 21 (7), 1378– 1384, DOI: 10.1021/bc100008b16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvV2kurc%253D&md5=779eb471d5afec751a2fb5beed6e2b0c"One-Step" Detection of Matrix Metalloproteinase Activity Using a Fluorogenic Peptide Probe-Immobilized Diagnostic KitRyu, Ju Hee; Lee, Aeju; Lee, Seulki; Ahn, Cheol-Hee; Park, Jong Woong; Leary, James F.; Park, Sangjin; Kim, Kwangmeyung; Kwon, Ick Chan; Youn, In-Chan; Choi, KuiwonBioconjugate Chemistry (2010), 21 (7), 1378-1384CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Matrix metalloproteinases (MMPs) have been shown to be abundant in pathol. conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biol. samples provides important clin. information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96-well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-IR (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concns. of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concn. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Addnl., the MMP-D-KIT was used to detect MMP activity in biol. samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biol. samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.
- 17Li, X.; Deng, D.; Xue, J.; Qu, L.; Achilefu, S.; Gu, Y. Quantum Dots Based Molecular Beacons for in Vitro and in Vivo Detection of MMP-2 on Tumor. Biosens. Bioelectron. 2014, 61, 512– 518, DOI: 10.1016/j.bios.2014.05.03517https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtFSgs77F&md5=b72dd49e0341a5a20eda8bcf4e6aa2cbQuantum dots based molecular beacons for in vitro and in vivo detection of MMP-2 on tumorLi, Xin; Deng, Dawei; Xue, Jianpeng; Qu, Lingzhi; Achilefu, Samuel; Gu, YueqingBiosensors & Bioelectronics (2014), 61 (), 512-518CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Matrix metalloproteinase-2 (MMP-2) is a protease related to tumor invasion and metastasis. It is heavily secreted by malignant tumor cells, allowing the protease to serve as an imaging biomarker of cancer. In this study, a novel sensing system based on fluorescence resonance energy transfer (FRET) from quantum dot (QD, the donor) to org. dye (the acceptor) was constructed for the in vitro and in vivo detection of matrix metalloproteinases-2 via a MMP-2-specific peptide substrate (GPLGVRGKGG). Specifically, 535 nm-emitting CdTe QD were bound to Rhodamine B (RB) through the peptide for in vitro detection of MMP-2, while 720 nm-emitting CdTeS QDs was linked to near IR dye ICG-Der-02 (MPA) by the peptide for measurement in vivo. When these probes were exposed to MMP-2, the selective cleavage of the peptide resulted in the recovery of fluorescence from QDs. By using the produced 540QD-peptide-RB and 720QD-peptide-MPA probes, we successfully examd. MMP-2 in live cells and tumor on nude mouse, resp. Due to the tunable fluorescence of Qds, this nanosensor can be fine-tuned for a wide range of applications such as the detection of different biomarkers and early diagnosis of disease.
- 18Feng, D.; Zhang, Y.; Feng, T.; Shi, W.; Li, X.; Ma, H. A Graphene Oxide-Peptide Fluorescence Sensor Tailor-Made for Simple and Sensitive Detection of Matrix Metalloproteinase 2. Chem. Commun. 2011, 47 (38), 10680– 10682, DOI: 10.1039/c1cc13975d18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFyqtLfP&md5=481c624299ce90d5e041ff7518844af7A graphene oxide-peptide fluorescence sensor tailor-made for simple and sensitive detection of matrix metalloproteinase 2Feng, Duan; Zhang, Yangyang; Feng, Tingting; Shi, Wen; Li, Xiaohua; Ma, HuiminChemical Communications (Cambridge, United Kingdom) (2011), 47 (38), 10680-10682CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A graphene oxide-peptide based fluorescence sensor has been developed for matrix metalloproteinase 2 (MMP2), and its applicability has been demonstrated by monitoring the concn. of MMP2 secreted by HeLa cells, revealing that HeLa cells with a d. of 5.48 × 105 cells per mL can produce 22 nM in cell culture media in 24 h.
- 19Welser, K.; Adsley, R.; Moore, B. M.; Chan, W. C.; Aylott, J. W. Protease Sensing with Nanoparticle Based Platforms. Analyst 2011, 136 (1), 29– 41, DOI: 10.1039/C0AN00429DThere is no corresponding record for this reference.
- 20Jin, Z.; Dridi, N.; Palui, G.; Palomo, V.; Jokerst, J. V.; Dawson, P. E.; Sang, Q. X. A.; Mattoussi, H. Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14. Anal. Chem. 2023, 95 (5), 2713– 2722, DOI: 10.1021/acs.analchem.2c03400There is no corresponding record for this reference.
- 21Lei, Z.; Zhang, H.; Wang, Y.; Meng, X.; Wang, Z. Peptide Microarray-Based Metal Enhanced Fluorescence Assay for Multiple Profiling of Matrix Metalloproteinases Activities. Anal. Chem. 2017, 89 (12), 6749– 6757, DOI: 10.1021/acs.analchem.7b01037There is no corresponding record for this reference.
- 22Biela, A.; Watkinson, M.; Meier, U. C.; Baker, D.; Giovannoni, G.; Becer, C. R.; Krause, S. Disposable MMP-9 Sensor Based on the Degradation of Peptide Cross-Linked Hydrogel Films Using Electrochemical Impedance Spectroscopy. Biosens. Bioelectron. 2015, 68, 660– 667, DOI: 10.1016/j.bios.2015.01.06022https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsleqsL8%253D&md5=e896e54c77078ddcae50c289e108555aDisposable MMP-9 sensor based on the degradation of peptide crosslinked hydrogel films using electrochemical impedance spectroscopyBiela, Anna; Watkinson, Michael; Meier, Ute C.; Baker, David; Giovannoni, Gavin; Becer, C. Remzi; Krause, SteffiBiosensors & Bioelectronics (2015), 68 (), 660-667CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Matrix metalloproteinase-9 (MMP-9) plays an important role in both physiol. and pathol. processes. This enzyme is a peripheral biomarker of neuroinflammation in multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system. Presently, expensive magnetic resonance imaging (MRI) studies are used to monitor subclin. disease activity in MS. An alternative to costly MRI scans could be the detection of MMP-9, using a low-cost, disposable sensor system for MMP-9 suitable for home-monitoring of inflammation. This would allow an early prediction of the failure of anti-inflammatory therapies and more timely clin. intervention to limit neuronal damage and prevent disability. Here, the authors present the development of a disposable sensor for fast and straightforward detection of MMP-9. Biosensors were produced by coating electrodes with oxidized dextran and subsequent crosslinking with peptides contg. specific cleavage sites for MMP-9. Exposure of the films to the enzyme resulted in the degrdn. of the films, which was monitored using impedance measurements. The sensor response was rapid, a significant impedance change was usually obsd. within 5 min after the addn. of MMP-9. Sensors showed a negligible response to matrix metalloproteinase-2 (MMP-2), a protease which may interfere with MMP-9 detection. The peptide sequence with the highest sensitivity and selectivity Leu-Gly-Arg-Met-Gly-Leu-Pro-Gly-Lys was selected to construct calibration curves. MMP-9 was successfully detected in a clin. relevant range from 50 to 400 ng/mL. Two different processes of hydrogel degrdn. were obsd. on electrode surfaces with different roughness, and both appeared suitable to monitor MMP-9 activity. The sensor materials are generic and can be easily adopted to respond to other proteases by selecting peptide crosslinkers with suitable cleavage sites.
- 23Kou, B. B.; Chai, Y. Q.; Yuan, Y. L.; Yuan, R. PtNPs as Scaffolds to Regulate Interenzyme Distance for Construction of Efficient Enzyme Cascade Amplification for Ultrasensitive Electrochemical Detection of MMP-2. Anal. Chem. 2017, 89 (17), 9383– 9387, DOI: 10.1021/acs.analchem.7b02210There is no corresponding record for this reference.
- 24Li, Y.; Liu, W.; Xu, Q.; Hu, J.; Zhang, C.-y. Construction of a Sensitive Protease Sensor with DNA-Peptide Conjugates for Single-Molecule Detection of Multiple Matrix Metalloproteinases. Biosens. Bioelectron. 2020, 169, 112647, DOI: 10.1016/j.bios.2020.11264724https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVymsL3L&md5=6f3871dede2c49bc1420d4099c66ee22Construction of a sensitive protease sensor with DNA-peptide conjugates for single-molecule detection of multiple matrix metalloproteinasesLi, Yueying; Liu, Wen; Xu, Qinfeng; Hu, Juan; Zhang, Chun-yangBiosensors & Bioelectronics (2020), 169 (), 112647CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Protease expression is closely linked to various pathol. phenomena, and their accurate quantification is essential to clin. diagnosis and cancer therapy. Herein, we demonstrate for the first time the construction of a sensitive protease sensor by integrating protease-sensitive cleavage with nicking enzyme-assisted signal amplification (NESA) for single-mol. detection of multiple matrix metalloproteinases (MMPs). This protease sensor involves two DNA-peptide conjugates which contain both specific protease cleavage sites and trigger DNAs and two report DNAs which are modified with a fluorophore (Cy3 or Cy5) and a quencher (BHQ2). In the presence of specific MMPs, MMPs-mediated cleavage reactions lead to the release of specific trigger DNAs from the corresponding DNA-peptide conjugates. After the magnetic sepn., the resultant trigger DNAs may hybridize with the corresponding report DNAs to initiate the cyclic NESA reaction, releasing large amts. of Cy3/Cy5 fluorescent mols. which can be simply quantified by using total internal reflection fluorescence-based single-mol. detection. Taking advantage of the high specificity of proteolytic cleavage, the high amplification efficiency of cyclic NESA, and the high sensitivity of single-mol. detection, this protease sensor can simultaneously detect multiple MMPs with a detection limit of 3.33 pM for MMP-2 and 1.71 pM for MMP-7, superior to the target peptide-based methods. Moreover, this protease sensor can be applied for the measurement of MMP-2 and MMP-7 in cancer cells and the screening of protease inhibitors, holding great promise in clinic diagnosis and drug discovery.
- 25Luo, X.; Zhao, J.; Xie, X.; Liu, F.; Zeng, P.; Lei, C.; Nie, Z. Proteolysis-Responsive Rolling Circle Transcription Assay Enabling Femtomolar Sensitivity Detection of a Target Protease Biomarker. Anal. Chem. 2020, 92 (24), 16314– 16321, DOI: 10.1021/acs.analchem.0c0442725https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVCnsr7M&md5=0901af0a864bfc37ecd696e8aa07f153Proteolysis-Responsive Rolling Circle Transcription Assay Enabling Femtomolar Sensitivity Detection of a Target Protease BiomarkerLuo, Xingyu; Zhao, Jiali; Xie, Xuan; Liu, Fang; Zeng, Pan; Lei, Chunyang; Nie, ZhouAnalytical Chemistry (Washington, DC, United States) (2020), 92 (24), 16314-16321CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Proteases play crucial roles in the malignant progression of tumor and thus have been regarded as biomarkers for many cancers. Although protease assays such as immunoassays and fluorogenic substrate probes have been developed, it remains challenging for them to give consideration to both sensitivity and accuracy. Here, we describe a proteolysis-responsive rolling circle transcription assay (PRCTA) for the ultrasensitive and accurate detection of protease activities by the rational integration of a protease-responsive RNA polymerase and rolling circle transcription. Taking cancer biomarker matrix metalloproteinase-2 (MMP-2) as the model, the PRCTA, which can transduce and amplify each proteolysis event catalyzed by MMP-2 into the output of multiple tandem fluorescent RNAs by in vitro transcription, is constructed for the sensitive anal. of MMP-2 activities. Such a rational integration greatly enhances the signal gain in PRCTA, and it enables the limit of detection of MMP-2 as low as 3 fM. The feasibility of PRCTA has been validated by the sensitive anal. of cellular MMP-2 activities of different cell lines with good accuracy, and the readout can be readily visualized by a fluorescence imaging system. Therefore, PRCTA has achieved the detection of target protease biomarkers with femtomolar sensitivity, exhibiting promising potential in biomedicine research and cancer diagnosis.
- 26Bui, H.; Brown, C. W.; Buckhout-White, S.; Díaz, S. A.; Stewart, M. H.; Susumu, K.; Oh, E.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors. Small 2019, 15 (14), 1805384, DOI: 10.1002/smll.201805384There is no corresponding record for this reference.
- 27Yang, M.; Shi, K.; Liu, F.; Kang, W.; Lei, C.; Nie, Z. Coupling of Proteolysis-Triggered Transcription and CRISPR-Cas12a for Ultrasensitive Protease Detection. Sci. China Chem. 2021, 64 (2), 330– 336, DOI: 10.1007/s11426-020-9863-yThere is no corresponding record for this reference.
- 28Liu, F.; Yang, M.; Song, W.; Luo, X.; Tang, R.; Duan, Z.; Kang, W.; Xie, S.; Liu, Q.; Lei, C.; Huang, Y.; Nie, Z.; Yao, S. Target-Activated Transcription for the Amplified Sensing of Protease Biomarkers. Chem. Sci. 2020, 11 (11), 2993– 2998, DOI: 10.1039/C9SC04692EThere is no corresponding record for this reference.
- 29Kaminski, M. M.; Abudayyeh, O. O.; Gootenberg, J. S.; Zhang, F.; Collins, J. J. CRISPR-Based Diagnostics. Nat. Biomed. Eng. 2021, 5 (7), 643– 656, DOI: 10.1038/s41551-021-00760-729https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1ClsLrP&md5=5d3bc70679efc08e1629274086e57d6fCRISPR-based diagnosticsKaminski, Michael M.; Abudayyeh, Omar O.; Gootenberg, Jonathan S.; Zhang, Feng; Collins, James J.Nature Biomedical Engineering (2021), 5 (7), 643-656CODEN: NBEAB3; ISSN:2157-846X. (Nature Portfolio)Abstr.: The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiol. surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of mol. targets relevant to human health.
- 30Li, S. Y.; Cheng, Q. X.; Li, X. Y.; Zhang, Z. L.; Gao, S.; Cao, R. B.; Zhao, G. P.; Wang, J.; Wang, J. M. CRISPR-Cas12a-Assisted Nucleic Acid Detection. Cell Discovery 2018, 4 (1), 20, DOI: 10.1038/s41421-018-0028-z30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjnvVOksQ%253D%253D&md5=7b902affcc4e07d3ccf5efbe10a8453cCRISPR-Cas12a-assisted nucleic acid detectionLi Shi-Yuan; Zhao Guo-Ping; Wang Jin; Cheng Qiu-Xiang; Li Xiao-Yan; Wang Jing-Man; Zhang Zi-Long; Gao Song; Cao Rui-Bing; Zhao Guo-PingCell discovery (2018), 4 (), 20 ISSN:2056-5968.There is no expanded citation for this reference.
- 31Chen, J. S.; Ma, E.; Harrington, L. B.; Da Costa, M.; Tian, X.; Palefsky, J. M.; Doudna, J. A. CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity. Science 2018, 360, 436– 439, DOI: 10.1126/science.aar624531https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFyrsr8%253D&md5=63ed6929e10f1ec40154e1b18aeb9b82CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activityChen, Janice S.; Ma, Enbo; Harrington, Lucas B.; Da Costa, Maria; Tian, Xinran; Palefsky, Joel M.; Doudna, Jennifer A.Science (Washington, DC, United States) (2018), 360 (6387), 436-439CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA mols. We find that target-activated, nonspecific single-stranded DNase (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for mol. diagnostics.
- 32Broughton, J. P.; Deng, X.; Yu, G.; Fasching, C. L.; Servellita, V.; Singh, J.; Miao, X.; Streithorst, J. A.; Granados, A.; Sotomayor-Gonzalez, A.; Zorn, K.; Gopez, A.; Hsu, E.; Gu, W.; Miller, S.; Pan, C. Y.; Guevara, H.; Wadford, D. A.; Chen, J. S.; Chiu, C. Y. CRISPR–Cas12-Based Detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38 (7), 870– 874, DOI: 10.1038/s41587-020-0513-432https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntlejt7w%253D&md5=a1ccd699e2945474307f349518da37e5CRISPR-Cas12-based detection of SARS-CoV-2Broughton, James P.; Deng, Xianding; Yu, Guixia; Fasching, Clare L.; Servellita, Venice; Singh, Jasmeet; Miao, Xin; Streithorst, Jessica A.; Granados, Andrea; Sotomayor-Gonzalez, Alicia; Zorn, Kelsey; Gopez, Allan; Hsu, Elaine; Gu, Wei; Miller, Steve; Pan, Chao-Yang; Guevara, Hugo; Wadford, Debra A.; Chen, Janice S.; Chiu, Charles Y.Nature Biotechnology (2020), 38 (7), 870-874CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Abstr.: An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in Dec. 2019. COVID-19, the disease assocd. with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA exts. We validated our method using contrived ref. samples and clin. samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% pos. predictive agreement and 100% neg. predictive agreement.
- 33Rossetti, M.; Merlo, R.; Bagheri, N.; Moscone, D.; Valenti, A.; Saha, A.; Arantes, P. R.; Ippodrino, R.; Ricci, F.; Treglia, I.; Delibato, E.; Van Der Oost, J.; Palermo, G.; Perugino, G.; Porchetta, A. Enhancement of CRISPR/Cas12a Trans-Cleavage Activity Using Hairpin DNA Reporters. Nucleic Acids Res. 2022, 50 (14), 8377– 8391, DOI: 10.1093/nar/gkac57833https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXpslCmtQ%253D%253D&md5=c4fe97010a2c84096d32e92cb66ff2b9Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reportersRossetti, Marianna; Merlo, Rosa; Bagheri, Neda; Moscone, Danila; Valenti, Anna; Saha, Aakash; Arantes, Pablo R.; Ippodrino, Rudy; Ricci, Francesco; Treglia, Ida; Delibato, Elisabetta; van der Oost, John; Palermo, Giulia; Perugino, Giuseppe; Porchetta, AlessandroNucleic Acids Research (2022), 50 (14), 8377-8391CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Mol. Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clin. relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.
- 34Li, J.; Yang, S.; Zuo, C.; Dai, L.; Guo, Y.; Xie, G. Applying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow Concentrations. ACS Sens. 2020, 5 (4), 970– 977, DOI: 10.1021/acssensors.9b0230534https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkslWmtrY%253D&md5=4284c2b7b80cbd32ce94d5ef647aeecfApplying CRISPR-Cas12a as a Signal Amplifier to Construct Biosensors for Non-DNA Targets in Ultralow ConcentrationsLi, Junjie; Yang, Shuangshuang; Zuo, Chen; Dai, Ling; Guo, Yongcan; Xie, GuomingACS Sensors (2020), 5 (4), 970-977CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)Efficient signal amplification is essential to construct ultrasensitive biosensors for biol. relevant species with abundant concomitant interferences. Here, the authors apply LbaCas12a as a signal amplifier to develop a versatile CRISPR-Cas12a platform to detect a wide range of analytes in ultralow concns. The platform relies on the indiscriminate single-stranded DNase activity of LbaCas12a, which recognizes single-stranded DNA intermediates generated by non-DNA targets down to femtomolar concns. and subsequently enhances the fluorescence signal output. With the help of functional nucleotides (DNAzyme and aptamer), ultrasensitive bioassays for Pb2+ and Acinetobacter baumannii have been designed with a limit of detection down to ~ 0.053 nM and ~ 3 CFU/mL, resp. It also allows simultaneous detection of four microRNAs (miRNAs) at a picomolar concn. without significant interferences by other counterparts, suggesting the potential of multiplexed miRNA expression profiles anal. in high throughput. Given the versatility and generality of the CRISPR-Cas12a platform, the authors expect the current work to advance the application of CRISPR-Cas-based platforms in bioanal. and provide new insights into ultrasensitive biosensor design.
- 35Xiong, Y.; Zhang, J.; Yang, Z.; Mou, Q.; Ma, Y.; Xiong, Y.; Lu, Y. Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. J. Am. Chem. Soc. 2020, 142 (1), 207– 213, DOI: 10.1021/jacs.9b0921135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlWhsrfF&md5=b680d3f70ac97b228f58a234a8691af8Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid TargetsXiong, Ying; Zhang, Jingjing; Yang, Zhenglin; Mou, Quanbing; Ma, Yuan; Xiong, Yonghua; Lu, YiJournal of the American Chemical Society (2020), 142 (1), 207-213CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Beyond its extraordinary genome editing ability, the CRISPR-Cas system has opened a new era of biosensing applications due to its high base resoln. and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic acid targets, we herein re-port CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) mols. such as aptamers and DNAzymes that are selective for small org. mol. and metal ion detections. The sensor is based on the Cas12a dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA) and its single stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA mols. that can lock a DNA activator for Cas12a-crRNA, preventing the ssD-NA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by com. available portable fluorimeters. Using this method, ATP and Na+ have been detected quant. under ambient temp. (25°C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system significantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanal. and biomedical applications.
- 36Hao, L.; Zhao, R. T.; Welch, N. L.; Tan, E. K. W.; Zhong, Q.; Harzallah, N. S.; Ngambenjawong, C.; Ko, H.; Fleming, H. E.; Sabeti, P. C.; Bhatia, S. N. CRISPR-Cas-Amplified Urinary Biomarkers for Multiplexed and Portable Cancer Diagnostics. Nat. Nanotechnol. 2023, 18 (7), 798– 807, DOI: 10.1038/s41565-023-01372-9There is no corresponding record for this reference.
- 37Kang, W.; Xiao, F.; Zhu, X.; Ling, X.; Xie, S.; Li, R.; Yu, P.; Cao, L.; Lei, C.; Qiu, Y.; Liu, T.; Nie, Z. Engineering Anti-CRISPR Proteins to Create CRISPR-Cas Protein Switches for Activatable Genome Editing and Viral Protease Detection. Angew. Chem., Int. Ed. 2024, 63 (16), e202400599, DOI: 10.1002/anie.202400599There is no corresponding record for this reference.
- 38Pandit, S.; Duchow, M.; Chao, W.; Capasso, A.; Samanta, D. DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing. Angew. Chem., Int. Ed. 2024, 63, e202310964, DOI: 10.1002/anie.202310964There is no corresponding record for this reference.
- 39Dutta, A.; Li, J.; Lu, H.; Akech, J.; Pratap, J.; Wang, T.; Zerlanko, B. J.; Gerald, T. J. F.; Jiang, Z.; Birbe, R.; Wixted, J.; Violette, S. M.; Stein, J. L.; Stein, G. S.; Lian, J. B.; Languino, L. R. Integrin Avb6 Promotes an Osteolytic Program in Cancer Cells by Upregulating MMP2. Cancer Res. 2014, 74 (5), 1598– 1608, DOI: 10.1158/0008-5472.CAN-13-1796There is no corresponding record for this reference.
- 40Tuszynski, G.; Jhon, A. The Role of Matrix Metalloproteinases in Tumor Angiogenesis and Tumor Metastasis. Pathol. Oncol. Res. 2001, 7, 14– 23, DOI: 10.1007/BF03032599There is no corresponding record for this reference.
- 41Xie, T. X.; Wei, D.; Liu, M.; Gao, A. C.; Ali-Osman, F.; Sawaya, R.; Huang, S. Stat3 Activation Regulates the Expression of Matrix Metalloproteinase-2 and Tumor Invasion and Metastasis. Oncogene 2004, 23 (20), 3550– 3560, DOI: 10.1038/sj.onc.120738341https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjsV2ntLs%253D&md5=00c8970048472afee61306cd7d99f7dcStat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasisXie, Tong-Xin; Wei, Daoyan; Liu, Mingguang; Gao, Allen C.; Ali-Osman, Francis; Sawaya, Raymond; Huang, SuyunOncogene (2004), 23 (20), 3550-3560CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)The expression of matrix metalloproteinase-2 (MMP-2) has been linked with tumor invasion, angiogenesis, and metastasis. However, the mol. basis for MMP-2 overexpression in tumor cells remains unclear. In this study, by using K-1735 melanoma system, we demonstrated that highly metastatic C4, M2, and X21 tumor cells express elevated MMP-2 mRNA and enzymic activity, whereas poorly metastatic C10, C19, and C23 tumor cells express much lower levels. Moreover, a concomitant elevated Stat3 activity has been detected in these metastatic tumor cells that overexpress MMP-2. Transfection of constitutively activated Stat3 into poorly metastatic C23 tumor cells directly activated the MMP-2 promoter, whereas the expression of a dominant-neg. Stat3 in highly metastatic C4 tumor cells inhibited the MMP-2 promoter. A high-affinity Stat3-binding element was identified in the MMP-2 promoter and Stat3 protein bound directly to the MMP-2 promoter. Blockade of activated Stat3 through expression of a dominant-neg. Stat3 significantly suppressed MMP-2 expression in the metastatic tumor cells. Therefore, overexpression of MMP-2 in the metastatic melanoma cells can be attributed to elevated Stat3 activity, and Stat3 upregulates the transcription of MMP-2 through direct interaction with the MMP-2 promoter. Furthermore, blockade of activated Stat3 in highly metastatic C4 cells significantly suppressed the invasiveness of the tumor cells, inhibited tumor growth, and prevented metastasis in nude mice. Collectively, these studies suggest that Stat3 signaling directly regulates MMP-2 expression, tumor invasion, and metastasis, and that Stat3 activation might be a crucial event in the development of metastasis.
- 42Jezierska, A.; Motyl, T. Matrix Metalloproteinase-2 Involvement in Breast Cancer Progression: A Mini-Review. Med. Sci. Monit. 2009, 15 (2), RA32– RA40There is no corresponding record for this reference.
- 43Von Maltzahn, G.; Harris, T. J.; Park, J. H.; Min, D. H.; Schmidt, A. J.; Sailor, M. J.; Bhatia, S. N. Nanoparticle Self-Assembly Gated by Logical Proteolytic Triggers. J. Am. Chem. Soc. 2007, 129 (19), 6064– 6065, DOI: 10.1021/ja070461l43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXksVSmsL4%253D&md5=6499978934c91f44f94e82bf0ecfa0f8Nanoparticle Self-Assembly Gated by Logical Proteolytic TriggersVon Maltzahn, Geoffrey; Harris, Todd J.; Park, Ji-Ho; Min, Dal-Hee; Schmidt, Alexander J.; Sailor, Michael J.; Bhatia, Sangeeta N.Journal of the American Chemical Society (2007), 129 (19), 6064-6065CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The emergent electromagnetic properties of nanoparticle self-assemblies are being harnessed to build new medical and biochem. assays with unprecedented sensitivity. While current self-assembly assays have displayed superior sensitivity for single mol. targets, the development of systems with the capacity to process multiple inputs may more effectively decipher complex disease signatures such as cancer. Herein, the authors present the design and synthesis of nanoparticles that perform Boolean logic operations using two proteolytic inputs assocd. with unique aspects of tumorigenesis (MMP2 and MMP7). Using dynamic light scattering, fluorescence, and MRI, the authors show that logical AND and OR functions can control the self-assembly of disperse superparamagnetic nanoparticles and enable remote, NMR detection of nanoparticle computation. In the future, by increasing the complexity of assembly triggers, nanoparticles may be tailored to sense a diversity of disease inputs in vitro and potentially in vivo.
- 44Jensen, K. K.; Ørum, H.; Nielsen, P. E.; Nordén, B. Kinetics for Hybridization of Peptide Nucleic Acids (PNA) with DNA and RNA Studied with the BIAcore Technique. Biochemistry 1997, 36 (16), 5072– 5077, DOI: 10.1021/bi962752544https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXit1Krsbc%253D&md5=9c3549eb1c2e26bdfcd2d6080cfd4156Kinetics for Hybridization of Peptide Nucleic Acids (PNA) with DNA and RNA Studied with the BIAcore TechniqueJensen, Kristine Kilsaa; Oerum, Henrik; Nielsen, Peter E.; Norden, BengtBiochemistry (1997), 36 (16), 5072-5077CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) contg. all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate consts. for assocn. (ka) and dissocn. (kd) of the duplex formation as well as the thermal stability (melting temp., Tm) of the duplexes have been detd. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences contg. single mismatches at various positions in the center resulted in increased dissocn. and decreased assocn. rate consts. Tm values for PNA•RNA duplexes are on av. 4° higher than for PNA•DNA duplexes and follow quant. the same variation with mismatches as do the PNA•DNA duplexes. Also a faster ka and a slower kd are found for PNA•RNA duplexes compared to the PNA•DNA duplexes. An overall fair correlation between Tm, ka, and kd is found for a series of PNA•DNA and PNA•RNA duplexes although the detn. of ka seemed to be prone to artifacts of the method and was not considered capable of providing abs. values representing the assocn. rate const. in bulk soln.
- 45Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson–Crick Hydrogen-Bonding Rules. Nature 1993, 365 (6446), 566– 568, DOI: 10.1038/365566a045https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXntFOksQ%253D%253D&md5=349ab1f3479e8e763f8555922e80fd12PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rulesEgholm, Michael; Buchardt, Ole; Christensen, Leif; Behrens, Carsten; Freier, Susan M.; Driver, David A.; Berg, Rolf H.; Kim, Seog K.; Norden, Bengt; Nielsen, Peter E.Nature (London, United Kingdom) (1993), 365 (6446), 566-8CODEN: NATUAS; ISSN:0028-0836.Recently, the authors designed a DNA analog, PNA (peptide nucleic acid), in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. Here, it is reported that PNA contg. all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.
- 46Davis, D. L.; Price, E. K.; Aderibigbe, S. O.; Larkin, M. X. H.; Barlow, E. D.; Chen, R.; Ford, L. C.; Gray, Z. T.; Gren, S. H.; Jin, Y.; Keddington, K. S.; Kent, A. D.; Kim, D.; Lewis, A.; Marrouche, R. S.; O’Dair, M. K.; Powell, D. R.; Scadden, M. H. C.; Session, C. B.; Tao, J.; Trieu, J.; Whiteford, K. N.; Yuan, Z.; Yun, G.; Zhu, J.; Heemstra, J. M. Effect of Buffer Conditions and Organic Cosolvents on the Rate of Strain-Promoted Azide-Alkyne Cycloaddition. J. Org. Chem. 2016, 81 (15), 6816– 6819, DOI: 10.1021/acs.joc.6b01112There is no corresponding record for this reference.
- 47Liu, L.; Chu, H.; Yang, J.; Sun, Y.; Ma, P.; Song, D. Construction of a Magnetic-Fluorescent-Plasmonic Nanosensor for the Determination of MMP-2 Activity Based on SERS-Fluorescence Dual-Mode Signals. Biosens. Bioelectron. 2022, 212, 114389, DOI: 10.1016/j.bios.2022.11438947https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhtl2hsrzN&md5=5ffa297f077667125924e50ae8e50cc6Construction of a magnetic-fluorescent-plasmonic nanosensor for the determination of MMP-2 activity based on SERS-fluorescence dual-mode signalsLiu, Lin; Chu, Hongyu; Yang, Jukun; Sun, Ying; Ma, Pinyi; Song, DaqianBiosensors & Bioelectronics (2022), 212 (), 114389CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Matrix metalloproteinase 2 (MMP-2) is a crucial biomarker of tumor growth, invasion and metastasis. In the present study, a core-satellite magnetic-fluorescent-plasmonic nanosensor (FMNS@Au) was constructed through biol. self-assembly to generate localized SERS "hot spots" and an efficient FRET system for the sensitive detn. of MMP-2 activity in a SERS-fluorescence dual-mode assay. In this hybrid nanosensor, a biotin-labeled peptide contg. a specific MMP-2 substrate (PLGVR) was employed as a bridge for the assembly of gold nanoparticles (AuNPs) and avidin functionalized fluorescent-magnetic nanospheres (FMNS). The modified RB on FMNS served as a Raman reporter and a donor of FRET, while the AuNPs assembled on FMNS acted as SERS substrates and acceptors of FRET. In the presence of MMP-2, the SERS "hot spot" effect was weakened and the FRET system was disrupted through enzymic cleavage of PLGVR, resulting in a redn. of SERS signal and the recovery of fluorescence emission. Importantly, this combination of SERS and fluorescence assay methods in the dual-mode nanosensor broadened the detection range for MMP-2 to 1-200 ng mL-1, with a limit of detection of 0.35 ng mL-1 and a limit of quantitation of 1.17 ng mL-1. In addn., our novel nanosensor affords semi-quant. sensing of MMP-2 by naked-eye observation and accurate detection of MMP-2 through dual-mode anal. The practicality of FMNS@Au was validated by detn. of MMP-2 activity in cell secretions and human serum samples. The designed FMNS@Au nanosensor holds great potential for clin. diagnosis of protease-related diseases.
- 48Yin, L.; Sun, H.; Zhang, H.; He, L.; Qiu, L.; Lin, J.; Xia, H.; Zhang, Y.; Ji, S.; Shi, H.; Gao, M. Quantitatively Visualizing Tumor-Related Protease Activity in Vivo Using a Ratiometric Photoacoustic Probe. J. Am. Chem. Soc. 2019, 141 (7), 3265– 3273, DOI: 10.1021/jacs.8b1362848https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFKjtbg%253D&md5=92d3f71f014dcad961e1fd9cd621f656Quantitatively Visualizing Tumor-Related Protease Activity in Vivo Using a Ratiometric Photoacoustic ProbeYin, Ling; Sun, Hao; Zhang, Hao; He, Lei; Qiu, Ling; Lin, Jianguo; Xia, Huawei; Zhang, Yuqi; Ji, Shunjun; Shi, Haibin; Gao, MingyuanJournal of the American Chemical Society (2019), 141 (7), 3265-3273CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The abnormal expression of tumor-related proteases plays a crit. role in cancer invasion, progression, and metastasis. Therefore, it is considerably meaningful to noninvasively assess the proteases' activity in vivo for both tumor diagnosis and therapeutic evaluation. Herein, the authors report an activatable probe constructed with a near-IR dye (Cy5.5) and a quencher (QSY21) covalently linked through a peptide substrate of matrix metalloproteinases-2 (MMP-2) that was chosen as a model for tumor-assocd. proteases. Upon cleavage with activated MMP-2, this probe emitted an MMP-2-concn.-dependent fluorescence. Quite unexpectedly, owing to the variation in the aggregation state of both the dye and its quencher as a consequence of the cleavage, the responsive probe presented a dramatic MMP-2-concn.-dependent absorption at around 680 nm, while that at around 730 nm was MMP-2 concn. independent. These features allowed detection of MMP-2 activity via both fluorescence and photoacoustic (PA) imaging in vitro, resp. Moreover, taking the PA signal at 730 nm as an internal ref., the PA signal at 680 nm allowed quant. detection of MMP-2 expression in breast cancer in vivo. The authors thus envision that the current approach would offer a useful tool for studying the malignant impacts of versatile tumor-assocd. proteases in vivo.
- 49Nagase, H.; Ogata, Y.; Suzuki, K.; Enghild, J. J.; Salvesen, G. Substrate Specificities and Activation Mechanisms of Matrix Metalloproteinases. Biochem. Soc. Trans. 1991, 19, 715– 718, DOI: 10.1042/bst0190715There is no corresponding record for this reference.
- 50Kwong, G. A.; Von Maltzahn, G.; Murugappan, G.; Abudayyeh, O.; Mo, S.; Papayannopoulos, I. A.; Sverdlov, D. Y.; Liu, S. B.; Warren, A. D.; Popov, Y.; Schuppan, D.; Bhatia, S. N. Mass-Encoded Synthetic Biomarkers for Multiplexed Urinary Monitoring of Disease. Nat. Biotechnol. 2013, 31 (1), 63– 70, DOI: 10.1038/nbt.246450https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVektbbM&md5=63625c88394c0cc19c012549eac5ab40Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of diseaseKwong, Gabriel A.; von Maltzahn, Geoffrey; Murugappan, Gayathree; Abudayyeh, Omar; Mo, Steven; Papayannopoulos, Ioannis A.; Sverdlov, Deanna Y.; Liu, Susan B.; Warren, Andrew D.; Popov, Yury; Schuppan, Detlef; Bhatia, Sangeeta N.Nature Biotechnology (2013), 31 (1), 63-70CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Biomarkers are becoming increasingly important in the clin. management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous mols. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiol. for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resoln. without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clin. used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to addnl. pathophysiol. processes and point-of-care diagnostics.
- 51Schuerle, S.; Dudani, J. S.; Christiansen, M. G.; Anikeeva, P.; Bhatia, S. N. Magnetically Actuated Protease Sensors for in Vivo Tumor Profiling. Nano Lett. 2016, 16 (10), 6303– 6310, DOI: 10.1021/acs.nanolett.6b0267051https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVykurbO&md5=f268ff179a44dcd0271663ca9a867c43Magnetically Actuated Protease Sensors for in Vivo Tumor ProfilingSchuerle, Simone; Dudani, Jaideep S.; Christiansen, Michael G.; Anikeeva, Polina; Bhatia, Sangeeta N.Nano Letters (2016), 16 (10), 6303-6310CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Targeted cancer therapies require a precise detn. of the underlying biol. processes driving tumorigenesis within the complex tumor microenvironment. Therefore, new diagnostic tools that capture the mol. activity at the disease site in vivo are needed to better understand tumor behavior and ultimately maximize therapeutic responses. Matrix metalloproteinases (MMPs) drive multiple aspects of tumorigenesis, and their activity can be monitored using engineered peptide substrates as protease-specific probes. To identify tumor specific activity profiles, local sampling of the tumor microenvironment is necessary, such as through remote control of probes, which are only activated at the tumor site. Alternating magnetic fields (AMFs) provide an attractive option to remotely apply local triggering signals, as they penetrate deep into the body and are not likely to interfere with biol. processes due to weak magnetic properties of tissue. Here, the authors report the design and evaluation of a protease-activity nanosensor that can be remotely activated at the site of disease via an AMF at 515 kHz and 15 kA/m. The authors' nanosensor was comprised of thermosensitive liposomes contg. functionalized protease substrates that were unveiled at the target site by remotely triggered heat dissipation of coencapsulated magnetic nanoparticles (MNPs). This nanosensor was combined with a unique detection assay to quantify the amt. of cleaved substrates in the urine. The authors applied this spatiotemporally controlled system to det. tumor protease activity in vivo and identified differences in substrate cleavage profiles between two mouse models of human colorectal cancer.
- 52Xie, J.; Zhang, F.; Aronova, M.; Zhu, L.; Lin, X.; Quan, Q.; Liu, G.; Zhang, G.; Choi, K. Y.; Kim, K.; Sun, X.; Lee, S.; Sun, S.; Leapman, R.; Chen, X. Manipulating the Power of an Additional Phase: A Flower-like Au-Fe 3O4 Optical Nanosensor for Imaging Protease Expressions in Vivo. ACS Nano 2011, 5 (4), 3043– 3051, DOI: 10.1021/nn200161v52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjtVKhurc%253D&md5=e0986457030c62bf7438a6fe436c12aeManipulating the Power of an Additional Phase: A Flower-like Au-Fe3O4 Optical Nanosensor for Imaging Protease Expressions In vivoXie, Jin; Zhang, Fan; Aronova, Maria; Zhu, Lei; Lin, Xin; Quan, Qimeng; Liu, Gang; Zhang, Guofeng; Choi, Ki-Young; Kim, Kwangmeyung; Sun, Xiaolian; Lee, Seulki; Sun, Shouheng; Leapman, Richard; Chen, XiaoyuanACS Nano (2011), 5 (4), 3043-3051CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The authors and others have recently proposed the synthesis of composite nanoparticles that offer strongly enhanced functionality. Here the authors used a flower-shaped Au-Fe3O4 nanoparticle as a template to construct an optical probe contg. Cy5.5-GPLGVRG-TDOPA on the iron oxide surface and SH-PEG5000 on the gold surface that can be specifically activated by matrix metalloproteinases expressed in tumors. Gold nanoparticles have excellent quenching properties, but labile surface chem. in vivo; however, iron oxide nanoparticles afford robust surface chem., but are suboptimal as energy receptors. By a marriage of the two, the authors produced a unified structure with performance that is unachievable with the sep. components. The authors' results are a further demonstration that the architecture of nanoparticles can be modulated to tailor their function as mol. imaging/therapeutic agents.
- 53Ke, W.; Zha, Z.; Mukerabigwi, J. F.; Chen, W.; Wang, Y.; He, C.; Ge, Z. Matrix Metalloproteinase-Responsive Multifunctional Peptide-Linked Amphiphilic Block Copolymers for Intelligent Systemic Anticancer Drug Delivery. Bioconjugate Chem. 2017, 28 (8), 2190– 2198, DOI: 10.1021/acs.bioconjchem.7b0033053https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVyhs7fO&md5=7962c23f31c9c7424c2b84e1afc85654Matrix Metalloproteinase-Responsive Multifunctional Peptide-Linked Amphiphilic Block Copolymers for Intelligent Systemic Anticancer Drug DeliveryKe, Wendong; Zha, Zengshi; Mukerabigwi, Jean Felix; Chen, Weijian; Wang, Yuheng; He, Chuanxin; Ge, ZhishenBioconjugate Chemistry (2017), 28 (8), 2190-2198CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The amphiphilic block copolymer anticancer drug nanocarriers clin. used or in the progress of clin. trials frequently suffer from modest final therapeutic efficacy due to a lack of intelligent features. For example, the biodegradable amphiphilic block copolymer, poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PDLLA) has been approved for clin. applications as a paclitaxel (PTX) nanocarrier (Genexol-PM) due to the optimized pharmacokinetics and biodistribution; however, a lack of intelligent features limits the intracellular delivery in tumor tissue. To endow the mediocre polymer with smart properties via a safe and facile method, we introduced a matrix metalloproteinase (MMP)-responsive peptide GPLGVRGDG into the block copolymer via efficient click chem. and ring-opening polymn. to prep. PEG-GPLGVRGDG-PDLLA (P1). P1 was further self-assembled into micellar nanoparticles (NPs) to load PTX, which show MMP-2-triggered dePEGylation due to cleavage of the peptide linkage. Moreover, the residual VRGDG sequences are retained on the surface of the NPs after dePEGylation, which can serve as ligands to facilitate the cellular uptake. The cytotoxicity of PTX loaded in P1 NPs against 4T1 cells is significantly enhanced as compared with free PTX or PTX-loaded PEG-GPLGVRG-PDLLA (P2) and PEG-PDLLA (P3) NPs. In vivo studies confirmed that PTX-loaded P1 NPs show prolonged blood circulation, which are similar to P2 and P3 NPs but exhibit more-efficient accumulation in the tumor site. Ultimately, PTX-loaded P1 NPs display statistically significant improvement of antitumor activity against tumor-bearing mice via systemic administration. Therefore, the strategy by facile incorporation of a responsive peptide linkage between PEG and PDLLA is a promising approach to improving the therapeutic efficacy of anticancer-drug-loaded amphiphilic block copolymer micelles.
- 54Harris, T. J.; Von Maltzahn, G.; Derfus, A. M.; Ruoslahti, E.; Bhatia, S. N. Proteolytic Actuation of Nanoparticle Self-Assembly. Angew. Chem., Int. Ed. 2006, 45 (19), 3161– 3165, DOI: 10.1002/anie.200600259There is no corresponding record for this reference.
- 55Li, S.-Y.; Cheng, Q.-X.; Liu, J.-K.; Nie, X.-Q.; Zhao, G.-P.; Wang, J. CRISPR-Cas12a Has Both Cis- and Trans-Cleavage Activities on Single-Stranded DNA. Cell Res. 2018, 28, 491– 493, DOI: 10.1038/s41422-018There is no corresponding record for this reference.
- 56Wang, B.; Wang, R.; Wang, D.; Wu, J.; Li, J.; Wang, J.; Liu, H.; Wang, Y. Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection. Anal. Chem. 2019, 91 (19), 12156– 12161, DOI: 10.1021/acs.analchem.9b0152656https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1Kks7zI&md5=fb7777d8ab197f4e9503fe3343335652Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid DetectionWang, Bei; Wang, Rui; Wang, Daqi; Wu, Jian; Li, Jixi; Wang, Jin; Liu, Huihui; Wang, YongmingAnalytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12156-12161CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A rapid and sensitive method is crucial for nucleic acid detection. Recently, RNA-guided CRISPR/Cas12a nuclease-based methods present great promise for nucleic acid detection. In the present methods, however, DNA amplification and subsequent Cas12a cleavage is sepd. and the whole process takes as long as 2 h. Most importantly, the uncapping operation increases the risk of aerosol contamination. In this study, we propose a CRISPR/Cas12a-based method named "Cas12aVDet" for rapid nucleic acid detection. By integrating recombinase polymerase amplification (RPA) with Cas12a cleavage in a single reaction system, the detection can be accomplished in 30 min and uncapping contamination can be avoided. The detection signal can be obsd. by the naked eye under blue light. This method could detect DNA at single mol. level and demonstrated 100% accuracy for mycoplasma contamination detection, presenting great potential for a variety of nucleic acid detection applications.
- 57Feng, W.; Newbigging, A. M.; Tao, J.; Cao, Y.; Peng, H.; Le, C.; Wu, J.; Pang, B.; Li, J.; Tyrrell, D. L.; Zhang, H.; Le, X. C. CRISPR Technology Incorporating Amplification Strategies: Molecular Assays for Nucleic Acids, Proteins, and Small Molecules. Chem. Sci. 2021, 12 (13), 4683– 4698, DOI: 10.1039/D0SC06973F57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsVSmu7g%253D&md5=c1957341af7adda64699b9d2f62a935fCRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small moleculesFeng, Wei; Newbigging, Ashley M.; Tao, Jeffrey; Cao, Yiren; Peng, Hanyong; Le, Connie; Wu, Jinjun; Pang, Bo; Li, Juan; Tyrrell, D. Lorne; Zhang, Hongquan; Le, X. ChrisChemical Science (2021), 12 (13), 4683-4698CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A review. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-assocd. (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technol. has also advanced mol. detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance mol. targets, improvements in anal. specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temp. facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and mol. translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small mols. Successful integrations of CRISPR technol. with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.
- 58(a) https://www.anaspec.com/en/catalog/sensolyte-520-mmp-2-assay-kit-fluorimetric-1-kit∼9d6616da-4170-4e04-93d7-9bdcd084a456.There is no corresponding record for this reference.(b) https://bpsbioscience.com/fluorogenic-mmp2-assay-kit-79918.There is no corresponding record for this reference.
- 59Hananya, N.; Press, O.; Das, A.; Scomparin, A.; Satchi-Fainaro, R.; Sagi, I.; Shabat, D. Persistent Chemiluminescent Glow of Phenoxy-Dioxetane Luminophore Enables Unique CRET-Based Detection of Proteases. Chem.─Eur. J. 2019, 25 (64), 14679– 14687, DOI: 10.1002/chem.201903489There is no corresponding record for this reference.
- 60den Hamer, A.; Den Dierickx, P.; Arts, R.; De Vries, J. S. P. M.; Brunsveld, L.; Merkx, M. Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity. ACS Sens. 2017, 2 (6), 729– 734, DOI: 10.1021/acssensors.7b00239There is no corresponding record for this reference.
- 61Jian, M.; Su, M.; Gao, J.; Wang, Z. Peptide Microarray-Based Fluorescence Assay for Quantitatively Monitoring the Tumor-Associated Matrix Metalloproteinase-2 Activity. Sens. Actuators B Chem. 2020, 304. 127320 DOI: 10.1016/j.snb.2019.127320 .There is no corresponding record for this reference.
- 62Chan, Y. C.; Chen, C. W.; Chan, M. H.; Chang, Y. C.; Chang, W. M.; Chi, L. H.; Yu, H. M.; Lin, Y. F.; Tsai, D. P.; Liu, R. S.; Hsiao, M. MMP2-Sensing up-Conversion Nanoparticle for Fluorescence Biosensing in Head and Neck Cancer Cells. Biosens. Bioelectron. 2016, 80, 131– 139, DOI: 10.1016/j.bios.2016.01.04962https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKktb4%253D&md5=83a84b6cd7a5291a5ee274fe0f1d46f8MMP2-sensing up-conversion nanoparticle for fluorescencebiosensing in head and neck cancer cellsChan, Yung-Chieh; Chen, Chieh-Wei; Chan, Ming-Hsien; Chang, Yu-Chan; Chang, Wei-Min; Chi, Li-Hsing; Yu, Hui-Ming; Lin, Yuan-Feng; Tsai, Din Ping; Liu, Ru-Shi; Hsiao, MichaelBiosensors & Bioelectronics (2016), 80 (), 131-139CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Upconversion nanoparticles (UCNPs) have extensive biol.-applications because of their bio-compatibility, tunable optical properties and their ability to be excited by IR radiation. Matrix metalloproteinases (MMPs) play important roles in extracellular matrix remodelling; they are usually found to significantly increase during cancer progression, and these increases may lead to poor patient survival. In this study, we produced a biosensor that can be recognized by MMP2 and then be unravelled by the attached quencher to emit visible light. We used 3.5-nm gold nanoparticles as a quencher that absorbed emission from UCNPs at a wavelength of 540 nm. The biosensor consists of an upconversion nanoparticle, MMP2-recognized polypeptides and quenchers. To improve the biocompatibility and modify the UCNPs with a polypeptide, they were coated with a silica shell and further conjugated with MMP-recognizing polypeptides. The polypeptide has two ends of featuring carboxylic and thiol groups that react with UCNPs and AuNPs, and the resulting nanoparticles were referred to as UCNP@p-Au. According to the in vitro cell viability anal., UCNP@p-Au exhibited little toxicity and biocompatibility in head and neck cancer cells. Cellular uptake studies showed that the MMP-based biosensor was activated by 980-nm irradn. to emit green light. This MMP-based biosensor may serve as sensitive and specific mol. fluorescent probe in biol.-applications.
- 63https://www.merckmillipore.com/IT/it/product/InnoZyme-Gelatinase-MMP-2-MMP-9-Activity-Assay-kit-Fluorogenic,EMD_BIO-CBA003?ReferrerURL=https%3A%2F%2Fwww.google.com%2F.There is no corresponding record for this reference.
- 64https://www.anaspec.com/en/catalog/sensolyte-520-mmp-2-assay-kit-fluorimetric-1-kit∼9d6616da-4170-4e04-93d7-9bdcd084a456.There is no corresponding record for this reference.
- 65Bruch, R.; Urban, G. A.; Dincer, C. CRISPR/Cas Powered Multiplexed Biosensing. Trends Biotechnol. 2019, 37 (8), 791– 792, DOI: 10.1016/j.tibtech.2019.04.00565https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXosFWjt7s%253D&md5=4dbc879ce82707bbf31181da3536aaadCRISPR/Cas Powered Multiplexed BiosensingBruch, Richard; Urban, Gerald A.; Dincer, CanTrends in Biotechnology (2019), 37 (8), 791-792CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)A review. Multiplexed CRISPR/Cas biosensing offers various possibilities for diagnosing different diseases by analyzing many analytes from one single specimen. However, in this relatively new field, nearly no multiplexing approaches exist, as many challenges need to be addressed. We discuss the reasons behind it and possible strategies to push the multiplexing level.
- 66Phan, Q. A.; Truong, L. B.; Medina-Cruz, D.; Dincer, C.; Mostafavi, E. CRISPR/Cas-Powered Nanobiosensors for Diagnostics. Biosens. Bioelectron. 2022, 197, 113732, DOI: 10.1016/j.bios.2021.11373266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVanurfM&md5=c93df123f5f64e524b944a4aa50ff1d4CRISPR/Cas-powered nanobiosensors for diagnosticsPhan, Quynh Anh; Truong, Linh B.; Medina-Cruz, David; Dincer, Can; Mostafavi, EbrahimBiosensors & Bioelectronics (2022), 197 (), 113732CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)A review. CRISPR diagnostics (CRISPR-Dx) offer a wide range of enhancements compared to traditional nanobiosensors by taking advantage of the excellent trans-cleavage activity of the CRISPR/Cas systems. However, the single-stranded DNA/RNA reporters of the current CRISPR-Dx suffer from poor stability and limited sensitivity, which make their application in complex biol. environments difficult. In comparison, nanomaterials, esp. metal nanoparticles, exhibits robust stability and desirable optical and electrocatalytical properties, which make them ideal as reporter mols. Therefore, biosensing research is moving towards the use of the trans-cleavage activity of CRISPR/Cas effectors on metal nanoparticles and apply the new phenomenon to develop novel nanobiosensors to target various targets such as viral infections, genetic mutations and tumor biomarkers, by using different sensing methods, including, but not limited to fluorescence, luminescence resonance, colorimetric and electrochem. signal readout. In this review, we explore some of the most recent advances in the field of CRISPR-powered nanotechnol. biosensors. Demonstrating high accuracy, sensitivity, selectivity and versatility, nanobiosensors along with CRISPR/Cas technol. offer tremendous potential for next-generation diagnostics of multiple targets, esp. at the point of care and without any target amplification.
- 67Boulware, K. T.; Daugherty, P. S. Protease Specificity Determination by Using Cellular Libraries of Peptide Substrates (CLiPS). Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (20), 7583– 7588, DOI: 10.1073/pnas.0511108103There is no corresponding record for this reference.
- 68Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.; Ellman, J. A.; Craik, C. S. Rapid and General Profiling of Protease Specificity by Using Combinatorial Fluorogenic Substrate Libraries. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (14), 7754– 7759, DOI: 10.1073/pnas.14013269768https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXkvFCnt7Y%253D&md5=669beba3367acd1911a2a39660117a78Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate librariesHarris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (14), 7754-7759CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A method is presented for the prepn. and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient prodn. of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approx. 3-fold-increased quantum yield of ACC over AMC permits redn. in enzyme and substrate concns. As a consequence, a greater no. of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Sol. positional protease substrate libraries of 137,180 and 6859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, resp., were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.
- 69Bertucci, A.; Porchetta, A.; Grosso, E.; Del Patiço, A.; Idili, A.; Ricci, F. Protein-Controlled Actuation of Dynamic Nucleic Acid Networks by Using Synthetic DNA Translators. Angew. Chem., Int. Ed. 2020, 59, 20577– 20581, DOI: 10.1002/anie.20200855369https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslGqt7jP&md5=88b58d6f0b1cbcdc4bc74038962f1704Protein-Controlled Actuation of Dynamic Nucleic Acid Networks by Using Synthetic DNA TranslatorsBertucci, Alessandro; Porchetta, Alessandro; Del Grosso, Erica; Patino, Tania; Idili, Andrea; Ricci, FrancescoAngewandte Chemie, International Edition (2020), 59 (46), 20577-20581CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Integrating dynamic DNA nanotechnol. with protein-controlled actuation will expand the authors' ability to process mol. information. The authors have developed a strategy to actuate strand displacement reactions using DNA-binding proteins by engineering synthetic DNA translators that convert specific protein-binding events into trigger inputs through a programmed conformational change. The authors have constructed synthetic DNA networks responsive to two different DNA-binding proteins, TATA-binding protein and Myc-Max, and demonstrated multi-input activation of strand displacement reactions. The authors achieved protein-controlled regulation of a synthetic RNA and of an enzyme through artificial DNA-based communication, showing the potential of the authors' mol. system in performing further programmable tasks.
- 70Bucci, J.; Irmisch, P.; Del Grosso, E.; Seidel, R.; Ricci, F. Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions. J. Am. Chem. Soc. 2022, 144 (43), 19791– 19798, DOI: 10.1021/jacs.2c06599There is no corresponding record for this reference.
- 71Xiang, Z.; Zhao, J.; Yi, D.; Di, Z.; Li, L. Peptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular Imaging. Angew. Chem., Int. Ed. 2021, 60 (42), 22659– 22663, DOI: 10.1002/anie.20210663971https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitVCntb7L&md5=6645b9f7993c9da3ea3a7fda50caf4fePeptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular ImagingXiang, Zhichu; Zhao, Jian; Yi, Deyu; Di, Zhenghan; Li, LeleAngewandte Chemie, International Edition (2021), 60 (42), 22659-22663CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Protease-triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnol. Herein, we report the design of a protease-activatable aptamer system that can perform mol. sensing and imaging in a tumor-specific manner. The system is constructed by locking the structure-switching activity of an aptamer using a rationally designed PNA-peptide-PNA triblock copolymer. Highly selective protease-mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell-selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.
- 72Watson, E. E.; Angerani, S.; Sabale, P. M.; Winssinger, N. Biosupramolecular Systems: Integrating Cues into Responses. J. Am. Chem. Soc. 2021, 143 (12), 4467– 4482, DOI: 10.1021/jacs.0c1297072https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjsVKhu7g%253D&md5=d03a2c63507bd7266e8d000044a65633Biosupramolecular Systems: Integrating Cues into ResponsesWatson, Emma E.; Angerani, Simona; Sabale, Pramod M.; Winssinger, NicolasJournal of the American Chemical Society (2021), 143 (12), 4467-4482CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A review. Life is orchestrated by biomols. interacting in complex networks of biol. circuitry with emerging function. Progress in different areas of chem. has made the design of systems that can recapitulate elements of such circuitry possible. Herein we review prominent examples of networks, the methodologies available to translate an input into various outputs, and speculate on potential applications and directions for the field. The programmability of nucleic acid hybridization has inspired applications beyond its function in heredity. At the circuitry level, DNA provides a powerful platform to design dynamic systems that respond to nucleic acid input sequences with output sequences through diverse logic gates, enabling the design of ever more complex circuitry. In order to interface with more diverse biomol. inputs and yield outputs other than oligonucleotide sequences, an array of nucleic acid conjugates have been reported that can engage proteins as their input and yield a turn-on of enzymic activity, a bioactive small mol., or morphol. changes in nanoobjects. While the programmability of DNA makes it an obvious starting point to design circuits, other biosupramol. interactions have also been demonstrated, and harnessing progress in protein design is bound to deliver further integration of macromols. in artificial circuits.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c02622.
Materials, nucleic acid sequences, including those of the PNA-peptide translator, a description of the experimental conditions for SPAAC reaction; FT-IR spectra, fluorescence emission spectra, zeta potential measurements, chromatogram for the peptide-PNA conjugate characterization, two-photon microscopy image of functionalized magnetic beads, and experimental results from ELISA (PDF)
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