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Nuclease Hydrolysis Does Not Drive the Rapid Signaling Decay of DNA Aptamer-Based Electrochemical Sensors in Biological Fluids

  • Alexander Shaver
    Alexander Shaver
    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United States
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    Orcidhttp://orcid.org/0000-0002-5478-5291
  • , 
  • Nandini Kundu
    Nandini Kundu
    Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
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  • Brian E. Young
    Brian E. Young
    Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
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  • Philip A. Vieira
    Philip A. Vieira
    Department of Psychology, California State University Dominguez Hills, Carson, California 90747, United States
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    Orcidhttp://orcid.org/0000-0003-0792-2836
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  • Jonathan T. Sczepanski
    Jonathan T. Sczepanski
    Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
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    Orcidhttp://orcid.org/0000-0002-9275-2597
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  • Netzahualcóyotl Arroyo-Currás*
    Netzahualcóyotl Arroyo-Currás
    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United States
    Department of Chemical and Biomolecular Engineering and Institute for NanoBioTechnology, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States
    *Email: [email protected]. Phone: 443-287-4798.
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    Orcidhttp://orcid.org/0000-0002-2740-6276
Cite this: Langmuir 2021, 37, 17, 5213–5221
Publication Date (Web):April 20, 2021
https://doi.org/10.1021/acs.langmuir.1c00166

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Electrochemical aptamer-based (E-AB) sensors are a technology capable of real-time monitoring of drug concentrations directly in the body. These sensors achieve their selectivity from surface-attached aptamers, which alter their conformation upon target binding, thereby causing a change in electron transfer kinetics between aptamer-bound redox reporters and the electrode surface. Because, in theory, aptamers can be selected for nearly any target of interest, E-AB sensors have far-reaching potential for diagnostic and biomedical applications. However, a remaining critical weakness in the platform lies in the time-dependent, spontaneous degradation of the bioelectronic interface. This progressive degradation─seen in part as a continuous drop in faradaic current from aptamer-attached redox reporters─limits the in vivo operational life of E-AB sensors to less than 12 h, prohibiting their long-term application for continuous molecular monitoring in humans. In this work, we study the effects of nuclease action on the signaling lifetime of E-AB sensors, to determine whether the progressive signal loss is caused by hydrolysis of DNA aptamers and thus the loss of signaling moieties from the sensor surface. We continuously interrogate sensors deployed in several undiluted biological fluids at 37 °C and inject nuclease to reach physiologically relevant concentrations. By employing both naturally occurring d-DNA and the nuclease-resistant enantiomer l-DNA, we determine that within the current lifespan of state-of-the-art E-AB sensors, nuclease hydrolysis is not the dominant cause of sensor signal loss under the conditions we tested. Instead, signal loss is driven primarily by the loss of monolayer elements─both blocking alkanethiol and aptamer monolayers─from the electrode surface. While use of l-DNA aptamers may extend the E-AB operational life in the long term, the critical issue of passive monolayer loss must be addressed before those effects can be seen.

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Introduction

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Synthetic nucleic acids are increasingly being used in biosensors, drug delivery systems, and other biotechnologies due to their ease of synthesis, wide range of available modifications, and cost effectiveness. However, these molecules are susceptible to catalytic hydrolysis by naturally occurring nucleases when deployed in biological environments, such as blood or cytosol, sometimes being fully degraded within minutes of exposure to such environments. (1) Additionally, nucleic acids are known to interact non-specifically with proteins and other biomolecules, (2) which can further hinder their usefulness when selective interactions are needed. These unwanted interactions, coupled with nuclease-driven degradation, can severely limit their lifetime when used in biotechnological applications that rely on the in vivo administration and functionality of synthetically produced nucleic acids.
Electrochemical aptamer-based (E-AB) sensors are an example platform that relies on the affinity and dynamics of synthetic oligonucleotides in biological fluids. (3,4) E-AB sensors consist of electrode-bound, redox reporter-modified nucleic acid aptamers co-deposited with alkanethiol monolayers onto gold electrodes via self-assembly (Figure 1A). (5) In the presence of their specific target, the aptamers undergo binding-induced conformational changes that alter the electron transfer efficiency between the redox reporters and the electrode surface, an event easily detected electrochemically. (6) E-AB sensors already support the continuous, real-time measurement of specific molecular targets within the body of living animals. (4) However, their in vivo operation is limited to less than 12 h due, in large part, to progressive signal degradation of the sensor interface. Specifically, in prior work, we (7) and others (8) have demonstrated that E-AB signal degradation is driven by the progressive desorption of monolayer components, including aptamers, from the electrode surface. In an effort to determine if nuclease-driven hydrolysis of aptamers also contributes to progressive E-AB signal decay, here, we study the effects of enantiomeric aptamer modifications on E-AB target binding and nuclease resistance.

Figure 1

Figure 1. Anatomy of E-AB sensors. (A) We fabricated sensors using aptamers of either naturally occurring d-DNA or the nuclease-resistant enantiomer l-DNA. These aptamers are modified on the 5′ end with an alkanethiol linker for self-assembly onto a gold electrode surface and on the 3′ end with a redox reporter for electrochemical interrogation. Both d- and l-DNA-functionalized sensors maintain similar signaling when interrogated by cyclic voltammetry (CV) (B) and square wave voltammetry (C). CV was measured in S1 buffer (5 mM HEPES, 50 mM NaCl, and 100 μΜ ZnCl2, pH ∼ 5.4) at 0.1 V/s. Square wave voltammetry was recorded in S1 buffer with a frequency of 80 Hz and an amplitude of 25 mV. Shaded areas represent the standard deviation calculated from four independent electrodes.

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Nucleic acid modifications such as peptide nucleic acids, locked nucleic acids, and phosphorothioate linkages are known to confer nuclease resistance to oligonucleotides. (9−12) However, the application of these and other oligonucleotide modifications in biological environments is limited by their potential toxicity and/or immunogenicity. (13−15) Additionally, such modifications can greatly alter the target binding and conformational dynamics of nucleic acids, negatively affecting their performance in biosensor technologies. (16) As a viable alternative, enantiomeric modification of nucleic acids is known to confer complete nuclease resistance while simultaneously maintaining the expected conformational dynamics, target affinity, and low immunogenicity. (17) While naturally occurring d-DNA can degrade within minutes in certain biological environments, chirally inverted l-DNA has been shown to be stable for up to 72 h within the same environments. (18−20) With this knowledge in mind, here, we fabricated E-AB sensors using either d- or l-DNA aptamers (Figure 1A) to determine the effects of enantiomeric modification on sensor operation and lifetime.

Results and Discussion

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l-DNA Sensors Maintain Achiral Target Affinity

The signaling performance of E-AB sensors is highly sensitive to the structure of their bioelectronic interface. (21,22) As such, any alteration to the chemistries of the blocking monolayer, aptamers, or redox reporters must be evaluated for its effects on sensor selectivity, affinity, and signal gain. Specifically, structural changes to the aptamers such as enantiomeric reversal must be evaluated for effects on target binding. To evaluate these effects, we used the cocaine-binding aptamer (Table 1) as a model system, (23) synthesized in both the d- and l-DNA forms. Notably, this aptamer has been shown to lack specificity, binding to many structurally different targets including chiral and achiral molecules. (24) This characteristic is important because chirality plays a critical role in DNA–small molecule interactions. For example, prior studies have demonstrated that selection of a d-DNA aptamer for a chiral molecule allows the l-DNA form of the aptamer to bind equally to the enantiomer of the aforesaid chiral molecule, a property referred to as reciprocal chiral specificity. (1,25) Relatedly, achiral targets have the ability to bind both d- and l-forms of an aptamer selected against them. (26) As such, we hypothesized that this phenomenon would translate to the surface of E-AB sensors.
Table 1. DNA Sequences Employed in This Work
targetsequenceref.
Cocaine5′- GGC GAC AAG GAA AAT CCT TCA ACG AAG GTG GGT GGC C -3′ (19)
N/A5′- GGA TCG AAC TGG TAC GCC -3′ 
To test our hypothesis, we first examined the achiral target procaine (Figure S1), which binds to the cocaine-binding aptamer. We chemically synthesized both d- and l-DNA versions of this aptamer (d-coc and l-coc, respectively) using commercially available d- and l-nucleoside phosphoramidites. The 3′ end of each aptamer was functionalized with a thiol and the 5′ end was functionalized with the redox reporter methylene blue (MB). We then fabricated sensors by incubating gold electrodes overnight in an aqueous solution containing 1 mM 6-mercaptohexanol (MCH) and 500 nM (either) d-coc or l-coc. Evaluating the redox response of these sensors by either CV or square wave voltammetry (SWV), we see no significant difference between the baseline voltammograms obtained from either d-coc or l-coc sensors (Figure 1B,C) under identical deposition conditions and technique parameters. To then determine the optimal frequencies for E-AB interrogation using SWV, we built “quasi-reversible maximum” maps as initially described by Lovrić and colleagues (Figures 2A and S2) (27,28) and adapted to E-AB interrogation by Dauphin-Ducharme and Plaxco. (29) Briefly, we interrogated both d-coc and l-coc sensors across a range of square wave frequencies in both the absence and presence of a saturating amount of procaine and identified two maxima, one corresponding to the unbound state and the other to the bound state of the aptamer. We selected the frequencies at which these maxima are observed, 5 and 250 Hz, for further SWV interrogation of our E-AB sensors.

Figure 2

Figure 2. Sensors employing d- or l-coc aptamers respond equivalently to achiral target additions. (A) We demonstrate this equivalence by, first, highlighting the fact that signal ON and OFF frequencies remain the same between sensor types. (B) By challenging both d-coc and l-coc-functionalized sensors with increasing concentrations of the achiral target procaine, we determine that the enantiomeric inversion of aptamers does not alter either signal gain (magnitude of current change at saturation, GainD-coc = 910% vs GainL-coc = 890%) or apparent target affinity (KD.d-DNA = 10.6 ± 0.6 mM vs KD.l-DNA = 10.3 ± 0.6 mM). These consistencies remain when sensors are challenged with targets other than procaine. Specifically, both d-coc and l-coc sensors respond similarly to the achiral target 6-hydroxyquinoline (C, Gaind-coc = 260% vs Gainl-coc = 300%; KD.d-DNA = 2.37 ± 0.10 mM vs KD.l-DNA = 2.29 ± 0.10 mM) and the chiral target quinine (D, Gaind-coc = 390% vs Gainl-coc = 360%; KD.d-DNA = 523 ± 40 μM vs KD.l-DNA = 686 ± 32 μM). The latter still binds to l-coc sensors because quinine contains an achiral quinoline backbone moiety. All SWV experiments were recorded from 0 to −0.5 V versus Ag/AgCl with an amplitude of 25 mV. Shaded areas in all panels represent the standard deviation calculated from eight independent sensors.

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The chirality of the nucleic acid aptamers (d vs l) is inconsequential for binding achiral targets and chiral targets with achiral moieties involved in aptamer binding. Accordingly, binding of achiral procaine to either d-coc or l-coc sensors is not significantly different (Figure 2B). We demonstrate this by building calibration curves for each sensor type, in which we challenge them with increasing amounts of targets while serially interrogating at 5 and 250 Hz. Procaine has been previously identified as a 1:1 minor groove binder and an electron donor to DNA, with strongest interactions with adenine and thymine, which represent 50% of the cocaine aptamer sequence used in this work. (30) Moreover, it is speculated that the structural flexibility of procaine allows it to reorient itself and bind to either DNA enantiomer, with no significant difference in affinity. To further demonstrate the idea that achiral structures can interact with both d- and l-DNA-functionalized sensors equally, we also built calibration curves for the chiral molecule quinine and its achiral backbone analogue 6-hydroxyquinoline. Johnson and colleagues demonstrated that the cocaine aptamer binds to the achiral quinoline ring, commonly shared between these two targets. (23,24) Interrogating E-AB sensors at each target’s respective maxima (Figure S2), again, we observed no significant difference between the dissociation constants for d-coc and l-coc (Figure 2C,D). These results demonstrate that although quinine is a chiral molecule, the strongest interaction of the cocaine aptamer is with the quinoline ring. Therefore, E-AB sensors employing aptamers that are optimized in the d-DNA form against achiral targets or achiral target moieties can be directly translated to the l-DNA enantiomer without affinity losses.

E-AB Sensors Functionalized with l-DNA Resist Nuclease-Driven Hydrolysis

Motivated by the retained analytical performance of l-coc sensors with achiral targets, we investigated how sensitive these sensors were to the presence of nucleases in buffer. As a model enzyme, we used S1 nuclease, which is known to have strong activity against single-stranded DNA. (31) For these experiments, we used unstructured oligonucleotides (Table 1) to ensure that the secondary structure did not affect nuclease activity. Of note, when comparing the cocaine aptamer and the unstructured oligonucleotides, we saw no difference in their sensitivity to nuclease-driven signal decay (Figure S3). We prepared sensors via overnight incubation in an aqueous solution of 500 nM DNA and 1 mM 1-hexanethiol (HxSH). In prior work, (7) we demonstrated that HxSH monolayers remain on the surface of E-AB sensors for periods of days under continuous electrochemical interrogation, whereas benchmark MCH monolayers significantly desorb from sensor surfaces starting at 24 h. Additionally, HxSH monolayers allow greater resolution of faradaic peaks related to the redox conversion of DNA-attached MB. Thus, here, we employed HxSH to decouple signal loss caused by short-chain monolayer desorption from that of receptor desorption or nuclease-driven hydrolysis of receptors (all processes cause a drop in E-AB signaling).
l-DNA sensors resist nuclease-dependent degradation across four orders of magnitude of nuclease concentration. To demonstrate this resistance, we simultaneously interrogated electrodes functionalized with either d-DNA or l-DNA constructs within the same electrochemical cell (Figure 3). We note that 0 h in all graphs represents the time immediately after sensor fabrication, when electrodes are first placed into the cell without any prior electrochemical pre-treatment. We interrogated these sensors in S1 buffer (5 mM HEPES, 50 mM NaCl, and 100 μM ZnCl2, pH ∼ 5.4) by CV every 25 s for 4 h and extracted the peak current related to the oxidation of DNA-attached redox reporters (leucomethylene blue to MB) over time. We then normalized the peak current at every point to the initial peak current for each sensor, giving us a readout of the remaining concentration of intact DNA constructs on the surface, while removing noise due to variability in sensor-to-sensor fabrication. Additionally, for these experiments, the electrochemical cell was held at a constant temperature of 37 °C using a temperature-controlled bath to ensure maximum nuclease activity and more accurately reflect an in vivo environment. After determining a baseline of sensor drift (Figure 3, left of vertical lines), we then injected S1 nuclease directly into the cell in an amount sufficient to reach the final intended concentration in U/mL. To most accurately represent the concentration of nuclease found in the human body, where we ultimately want to deploy our E-AB sensors, we initially tested three orders of magnitude in nuclease concentration, as this entire range has been reported in the previous literature. (32,33) Following additions of 0.1–10 U/mL S1 nuclease, d-DNA sensors reveal an increased rate of degradation, as indicated by the increased slopes after the injection of nuclease (Figure 3A, vertical line). In contrast, the slow drift of l-DNA sensors remains constant and unaffected by nuclease additions, save for a transient increase in signal loss upon addition of 10 U/mL, indicating that the l-DNA constructs are resistant to hydrolysis across the entire physiological range of nuclease concentrations (Figure 3B). To further demonstrate this point of nuclease resistance, we also injected a large, non-physiological excess of S1 nuclease, 500 U/mL, after which we see near-complete signal disappearance in d-DNA functionalized sensors within 1 h, while l-DNA-functionalized sensors again remain resistant to hydrolysis by the added nuclease (Figure 3C). Note that the slightly decreased current seen with l-DNA upon the addition of nuclease (relative to the absence of nuclease) is most likely due to a combination of low nuclease activity on l-DNA and non-specific binding of protein onto the electrode surface. This conclusion is supported by a control experiment where we injected 10 U/mL inactivated nuclease (Figure 3D) and saw equivalent signal loss, albeit less than that seen in Figure 3B, with both d-DNA and l-DNA sensors.

Figure 3

Figure 3. l-DNA-functionalized sensors resist nuclease-driven hydrolysis. To illustrate this effect, we serially measured cyclic voltammograms on electrodes functionalized with d-DNA and l-DNA oligonucleotides, 18 nt long, every 25 s for 4 h. By monitoring the peak current from the oxidation of DNA-attached leucomethylene blue reporters, (A) we observed changes in the d-DNA electrode signal driven by both progressive monolayer desorption (constant, initial negative drift in all panels) and nuclease-driven hydrolysis across the range of physiological concentrations. The solid lines indicate the time at which we injected S1 nuclease into the electrochemical cell. (B) l-DNA-functionalized electrodes, in contrast, presented only slight signal loss upon exposure to added S1 nuclease. (C) This remains true even with a 50× excess of S1 nuclease relative to physiological levels of nucleases in human circulation. (D) If we first inactivate the nuclease by heating to 70 °C in the presence of ethylenediaminetetraacetic acid (EDTA), no additional degradation is seen on either form of DNA. Peak currents were extracted from cyclic voltammograms measured at scanning rates of 0.1 V s–1. Shaded areas for all traces represent the standard deviation calculated from at least four electrodes. 0 h in all graphs represents the time immediately after sensor fabrication, when electrodes are first placed into the cell without any prior electrochemical pre-treatment.

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Rapid Signal Decay of State-of-the-Art Sensors in Biological Fluids is Not Driven by Nuclease Hydrolysis

The nuclease-dependent degradation of d-DNA-functionalized sensors seen in buffer does not translate to biological fluids. Because our ultimate goal for E-AB sensing is prolonged deployment in vivo, we repeated the previously described nuclease injection experiments in six different biological fluids: serum, saliva, urine, plasma, cerebrospinal fluid (CSF), and whole blood (Figure 4). For these experiments, we used both E-AB sensors functionalized with the benchmark MCH monolayer and sensors with a HxSH monolayer. Of note, when using MCH monolayers in S1 buffer, we are unable to see nuclease-dependent degradation of the sensor signal (Figure S4). We suggest that this could be due to either or both of the following effects: (1) the polarity of MCH groups could slow down or prevent non-specific binding of nucleases to the sensor surface relative to hydrophobic HxSH monolayers, decreasing the extent of aptamer hydrolysis, or (2) the enhanced signal-to-noise ratio we see with HxSH monolayers (Figure S5) could enable better visualization of aptamer hydrolysis by nucleases. In our measurements, we used both d-coc and l-coc to elucidate the stability between enantiomers. We again interrogated these sensors at 37 °C by CV, extracted the peak current related to the oxidation of DNA-attached leucomethylene blue over time, and normalized values to the original peak current for each sensor. In these experiments, we injected 250 U/mL S1 nuclease after determining the baseline signal drift. While this concentration of nuclease is much higher than the physiological range, we used a large excess to allow clear distinction of nuclease-driven signal decay versus the drift caused by monolayer desorption, as seen in Figure 3C. Notably, the baseline drift seen in biological fluids is accelerated compared to that in buffer, seen as biexponential decay versus linear, respectively. This difference is expected because in biological fluids, signal decay occurs through both the loss of monolayer materials and adsorption of proteins on the sensor surface, whereas decay in buffer occurs only through the former mechanism.

Figure 4

Figure 4. E-AB signal decay in biological fluids. By interrogating d-coc and l-coc sensors with either MCH or HxSH monolayers in (A) unfiltered human serum, (B) saliva, (C) urine, (D) plasma, (E) CSF, or (F) whole blood, we demonstrate that signal decay is not driven by nuclease hydrolysis. While some difference in signal loss can be seen for sensors with HxSH monolayers before injection of excess nuclease (left of vertical lines), no significant difference is seen between d-coc and l-coc sensors with MCH monolayers in any of the biological fluids, either before or after injection of excess nuclease. This lack of nuclease-dependent signal decay likely indicates that the operational life of E-AB sensors is not limited by nuclease action but rather by monolayer desorption. Peak currents were extracted from cyclic voltammograms measured at scanning rates of 0.1 V s–1. Shaded areas for all traces represent the standard deviation calculated from at least four electrodes.

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Across both enantiomers of the aptamer, both monolayer chemistries, and all six biological fluids, there is no significant drop in the E-AB signal upon the injection of excess nuclease (Figure 4). Specifically, addition of S1 nuclease (vertical lines in all graphs) to unfiltered human serum (Figure 4A), saliva (Figure 4B), urine (Figure 4C) plasma (Figure 4D), CSF (Figure 4E), or whole blood (Figure 4F) did not cause the expected slope change seen with d-DNA/HxSH-functionalized sensors in S1 buffer (Figure 3). We speculate that this effect is due to two reasons. On one hand, native d-DNA has been reported to tolerate longer exposure to serum and other extracellular fluids than to intracellular environments, either because of low nuclease activity or low enzyme concentrations. (34) On the other hand, sensors functionalized with hydrophobic HxSH monolayers may undergo rapid fouling via non-specific protein adsorption in each of the biological fluids considered, forming films that preclude the approach of added nuclease to the sensor interface and subsequent cleavage of aptamers. However, because nucleases inherent to each fluid also contribute to the sensor-fouling process, a significant acceleration of signal decay is still observed for sensors containing d-DNA relative to those functionalized with l-DNA. This point is illustrated by the steeper slope of d-DNA traces compared to l-DNA traces before nuclease injection in each of the HxSH graphs (Figure 4, right columns, left of vertical lines). We presume that this effect is maintained in sensors functionalized with MCH monolayers, except that the total current magnitude in these is much lower (Figure S5), resulting in larger noise and standard deviations that overlap between sensor types (not significantly different).
The fact that this signal decay is related to the disappearance of the leucomethylene blue oxidation peak indicates one or more of the following degradative mechanisms. (A) MB could be cleaved from the end of the DNA, causing signal decay from loss of the reporter alone. While possible in small amounts, previous studies (35,36) have demonstrated that the weakest bonds in thiol-on-gold SAMs are the S–Au and Au–Au bonds, so cleavage there is more likely than within the linker between the DNA and MB. (B) MB could be degrading, thereby reducing the signal without loss of surface materials. This possibility is an area of active research in our laboratory. However, this mechanism would likely not portray the increase in CV capacitance we see over time. As such, MB degradation can only account for part of the signal decay. (C) The aptamer monolayer is desorbing from the electrode surface. This mechanism accounts for both the disappearance of the oxidation peak and the accompanying increase in CV capacitance. Based on the data presented, this desorption occurs in faster time scales than the signal decay caused by nuclease-driven aptamer hydrolysis. Thus, our results suggest that state-of-the-art E-AB sensors are not limited in operational life by the action of nucleases at a time scale of hours. On the contrary, the main issue concerning the lifetime of E-AB sensors is that of monolayer loss of both aptamers and short-chain alkanethiols.

Conclusions

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We have studied the effects of enantiomeric inversion of aptamers on the signaling and operational lifetime of E-AB sensors. Our results indicate two major points. First, direct conversion of d-DNA aptamers to the enantiomeric l-DNA form does not affect E-AB sensor signaling where an achiral target is concerned. While multiple groups have demonstrated before that aptamer affinity is not altered upon enantiomeric inversion, (1,25,37) it has never been shown with respect to E-AB sensors, where conformational dynamics and response to electrochemical perturbations, in addition to aptamer affinity, play a critical role in signaling. (29,38) As such, we have demonstrated that the concept of enantiomeric inversion can be translated to this up-and-coming technology. Additionally, our results suggest that even if an aptamer is selected to bind a chiral target, if the main binding motif is an achiral portion of that target, the aptamer can still be converted to the l-DNA form without significant losses in target affinity.
The second major point is that of the nuclease-dependent sensor signal loss. When using a HxSH monolayer─which we have previously shown to resist desorption from the electrode surface and thus allow distinction of alternate causes of signal loss (7)─our results indicate that d-DNA-functionalized sensors do experience some nuclease-dependent signal loss in the physiological range of nuclease concentrations related to human circulation. When switched to l-DNA, the sensors are resistant to nuclease-dependent signal loss in buffer, even at concentrations much higher than those found in human circulation. Additionally, sensors with HxSH monolayers reveal a slight difference in signal loss between d-DNA and l-DNA when deployed in various biological fluids. However, this effect is not seen when using sensors with a MCH monolayer, the most commonly used and state-of-the-art monolayer chemistry. Across buffer and six different biological fluids, our results indicate no difference in signal loss between d-DNA- and l-DNA-functionalized sensors when using MCH monolayers. We attribute this result to a combination of the low signal-to-noise measurements obtained when using the MCH monolayer and the rapid desorption of the aptamer monolayer from the electrode surface. Likely, the desorption of aptamers is causing signal loss at a much faster rate than nuclease-driven hydrolysis of aptamers.
The results of this work reveal a critical deficiency in the technology of E-AB sensors. While a main goal of the technology is prolonged sensing in biological fluids, past studies have shown that sensors can last no more than 12 h in such media, (39) greatly limiting their widespread application. This work now demonstrates that the limited operational life is, under the conditions we tested, not due to hydrolysis of surface-attached aptamers by the nucleases in biological fluids. On the contrary, desorption of the self-assembled monolayers appears to be the limiting factor in sensor lifetime. While short-chain monolayer desorption can be limited with highly hydrophobic monolayer chemistries, (7) such chemistries prevent sensor use in biological fluids due to rapid fouling of the surface. (40,41) As such, if the operational lifetime of E-AB sensors in biological fluids is to be extended beyond the current limit, it appears that the monolayer chemistry itself must be changed. Perhaps then, with a more stable monolayer, E-AB sensor lifetime will be limited by nuclease action, at which time use of l-DNA aptamers could be a viable solution.

Materials and Methods

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Chemicals and Materials

Oligonucleotide synthesis reagents, d-nucleoside phosphoramidites, 3′-PT-Amino-Modifier C6 CPG, Thiol-Modifier C6 S–S phosphoramidite, MB NHS ester, and Glen-Pak DNA purification cartridges were purchased from Glen Research (Sterling, VA). l-nucleoside phosphoramidites were purchased from ChemGenes (Wilmington, MA). Acrylamide/bisacrylamide solution for denaturing polyacrylamide gel electrophoresis (PAGE) was purchased from BioRad Laboratories (Hercules, CA). Deprotection reagents were purchased from Sigma-Aldrich (St. Louis, MO).
6-mercapto-1-hexanol (MCH), HxSH, and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS, 11.9 mM HPO32–; 137 mM NaCl; 2.7 mM KCl; pH = 7.4) trace-metal grade sulfuric acid (H2SO4), sodium hydroxide (NaOH), EDTA, and sodium chloride (NaCl) were purchased from Fisher Scientific (Waltham, MA). Zinc chloride was purchased from Alfa Aesar (Ward Hill, MA). HEPES was purchased from J.T. Baker (Phillipsburg, NJ). Biological fluids were purchased from BioreclamationIVT (Washington, D.C.). S1 nuclease was purchased from ThermoScientific (Lithuania). We prepared all aqueous solutions using deionized water from a Milli-Q Direct purification system, with a resistivity of 18 MΩ. S1 buffer (pH ∼ 5.4) contained 5 mM HEPES, 50 mM NaCl, and 100 μM ZnCl2. Gold working electrodes (PN: 002314, diameter: 1.6 mm) and coiled platinum wire counter electrodes (PN: 012961) were obtained from ALS Inc. (Tokyo, Japan). Silver/silver chloride reference electrodes (PN: CHI111) were purchased from CH Instruments (Austin, TX). For polishing electrodes, 1200/P2500 silicon carbide grinding paper (PN: 36-08-1200) was bought from Buehler (Lake Bluff, IL); cloth pads (PN: MF-1040) and alumina slurry (PN: CF-1050) were purchased from BASi (West Lafayette, IN).

Oligonucleotide Synthesis and Purification

Both d-DNA and l-DNA versions of the cocaine aptamer (Table 1) were prepared by solid-phase synthesis on an Expedite 8909 DNA/RNA synthesizer. A 3′-amine modification was installed by initiating each synthesis on 3′-PT-amino-modifier CPGs, followed by coupling of either d-nucleoside phosphoramidites or l-nucleoside phosphoramidites according to the manufacturer’s recommended protocols. The 5′-thiol modification was introduced as a disulfide during synthesis by coupling the thiol-modifier C6 S-S phosphoramidite to the 5′-terminus of each oligonucleotide as recommended by the manufacturer. The terminal 4,4′-dimethoxytrityl (DMT) group was retained for downstream purification. Following deprotection of the oligonucleotides by AMA (ammonium hydroxide/40% aqueous methylamine 1:1 v/v), the oligonucleotides were desalted using a Glen-Pak DNA purification cartridge and concentrated by ethanol precipitation. In order to attach the MB reporter, the crude oligonucleotide pellet was dissolved in 500 μL of 0.1 M bicarbonate buffer (pH 9) to which a solution containing ∼2 mg of MB NHS ester in 12 μL of DMSO was added. The coupling reaction was allowed to proceed overnight at room temperature and was quenched by ethanol precipitation. The modified oligonucleotides were purified by 20% denaturing PAGE (19:1 acrylamide/bisacrylamide), and the products were excised from the gel and eluted overnight at room temperature in a buffer consisting of 200 mM NaCl, 10 mM EDTA, and 10 mM Tris (pH 7.6). The solution was filtered to remove gel fragments, and the oligonucleotides were desalted by ethanol precipitation. The obtained pellet was resuspended in water, and the concentration was determined by measuring the absorbance at 260 nm using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The purity and identity of all synthetic oligonucleotides were confirmed by mass spectrometric analysis (Novatia LLC, Newton, PA) (Figures S6 and S7).
The oligonucleotide modifications employed in this work had the following structures:
Hexanethiol modification at the 5′ terminus:
MB modification at the 3′ terminus:
To prepare the DNA solutions, we first incubated 1 μL of 100 μM thiolated MB-modified DNA with 2 μL of 5 mM TCEP to reduce the disulfide bond. We then diluted the DNA with freshly prepared 1 mM thiol solution to a final concentration of 500 nM, as determined via molecular absorbance measurements employing an Implen NP80 NanoPhotometer.

Electrode and Nuclease Preparation

Gold electrodes were polished for ∼2 min on 1200/P2500 silicon carbide grinding paper and subsequently for ∼4 min on a cloth pad with alumina slurry. After rinsing with water to remove polishing debris, they were then electrochemically cleaned in 0.5 M NaOH and 0.5 M H2SO4 following a previously reported protocol. (5) Briefly, (1) in 0.5 M NaOH, we scanned from −1 to −1.8 V versus Ag/AgCl, 400 times at a scan rate of 1 V/s and (2) in 0.5 M H2SO4, we scanned from 0.4 to −0.5 to 1.75 back to 0.4 V versus Ag/AgCl, 300 times at a scan rate of 1 V/s. Once electrodes were clean, we rinsed them with water and placed them immediately into 1 mM alkanethiol solutions with 500 nM DNA and then left them to incubate overnight. Post incubation, we rinsed the electrodes with water and placed them into a custom, water-jacketed electrochemical cell containing 1× PBS, S1 buffer, or biological fluid. The cell was kept at a constant temperature of either 25 or 37 °C by running temperature-controlled water through the cell jacket using a Huber Microprocessor Control MPC water recirculation bath. For control nuclease experiments, S1 nuclease was inactivated by incubation in S1 buffer with 5 mM EDTA at 70 °C for 10 min, according to the product information manual’s instructions for inhibition.

Electrochemical Measurements

A CH Instruments electrochemical analyzer (CHI 1040C, Austin, TX) multichannel potentiostat and associated software were used for all CV and SWV measurements. We used a three-electrode cell configuration consisting of gold disk working, coiled platinum wire counter, and Ag/AgCl (saturated KCl) reference electrodes. CV measurements were recorded at a scan rate of 100 mV/s in one of the two potential windows: (1) −0.5 to 0.2 V vs Ag/AgCl in PBS or biological fluids and (2) −0.3 to 0.2 V versus Ag/AgCl in S1 buffer. Current was sampled every millisecond. SWV measurements were performed from 0 to −0.5 V versus Ag/AgCl with a square wave amplitude of 25 mV, step size of 1 mV, and various frequencies. Temperature of the electrochemical cell was held constant using a Huber Microprocessor Control MPC water recirculation bath (Huber, Cary, NC).

Data Analysis

We employed a previously reported, open-source Python script called SACMES (42) for the batch processing of our electrochemical measurements. SACMES allows us to extract specific capacitive and faradaic currents from our electrochemical measurements, including peak currents, in real time. We determined dissociation constants for each sensor architecture using the specific signal ON frequency data, producing the best fit to the Hill equation. We employed non-linear regression function Igor Pro v8 graphing software to determine KD values.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.1c00166.

  • Aptamer secondary structure and target chemical structures, quasi-reversible maximum maps for hydroxyquinoline and quinine, deconvoluted mass spectrum of the synthesiszed l-cocaine aptamer, and deconvoluted mass spectrum of the synthesiszed d-cocaine aptamer (PDF)

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Author Information

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  • Corresponding Author
    • Netzahualcóyotl Arroyo-Currás - Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United States;  Department of Chemical and Biomolecular Engineering and Institute for NanoBioTechnology, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States;  Orcidhttp://orcid.org/0000-0002-2740-6276;  Email: [email protected]
  • Authors
    • Alexander Shaver - Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21202, United States;  Orcidhttp://orcid.org/0000-0002-5478-5291
    • Nandini Kundu - Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
    • Brian E. Young - Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
    • Philip A. Vieira - Department of Psychology, California State University Dominguez Hills, Carson, California 90747, United States;  Orcidhttp://orcid.org/0000-0003-0792-2836
    • Jonathan T. Sczepanski - Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States;  Orcidhttp://orcid.org/0000-0002-9275-2597
  • Author Contributions

    A.S., N.A.-C., and J.T.S. developed the idea. N.K. and B.E.Y. synthesized and purified DNA constructs. A.S. and P.A.V. fabricated E-AB sensors. A.S. performed all electrochemical experiments. All authors participated in the writing and editing of this manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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A.S. and N.A.-C. thank the Johns Hopkins University School of Medicine for providing the funds used to perform the research reported in this work. N.K., B.E.Y., and J.T.S. were supported by the National Institute of General Medical Sciences (R35GM124974) and the Welch Foundation (A1909). P.A.V. was also supported by the National Institute of General Medical Sciences (SC2GM127268). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References

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    Seth, Punit P.; Jazayeri, Ali; Yu, Jeff; Allerson, Charles R.; Bhat, Balkrishen; Swayze, Eric E.
    Molecular Therapy--Nucleic Acids (2012), 1 (Oct.), E47/1-E47/8CODEN: MTAOC5; ISSN:2162-2531. (Nature Publishing Group)
    We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifiations targeting phosphatase and tensin homolog (PTEN) mRNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN mRNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen wts. suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ wts. Examn. of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clin. setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.
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  14. 14
    Swayze, E. E.; Siwkowski, A. M.; Wancewicz, E. V.; Migawa, M. T.; Wyrzykiewicz, T. K.; Hung, G.; Monia, B. P.; Bennett, C. F. Antisense Oligonucleotides Containing Locked Nucleic Acid Improve Potency but Cause Significant Hepatotoxicity in Animals. Nucleic Acids Res. 2007, 35, 687– 700,  DOI: 10.1093/nar/gkl1071
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    14
    Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals
    Swayze, Eric E.; Siwkowski, Andrew M.; Wancewicz, Edward V.; Migawa, Michael T.; Wyrzykiewicz, Tadeusz K.; Hung, Gene; Monia, Brett P.; Bennett, C. Frank
    Nucleic Acids Research (2007), 35 (2), 687-700CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    A series of antisense oligonucleotides (ASOs) contg. either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA contg. oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE contg. ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ wts. and body wts. This toxicity was evident for multiple sequences targeting three different biol. targets, as well as in mismatch control sequences having no known mRNA targets. Histopathol. evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was obsd. as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.
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  15. 15
    Shen, W.; De Hoyos, C. L.; Sun, H.; Vickers, T. A.; Liang, X.-h.; Crooke, S. T. Acute Hepatotoxicity of 2′ Fluoro-Modified 5–10–5 Gapmer Phosphorothioate Oligonucleotides in Mice Correlates with Intracellular Protein Binding and the Loss of DBHS Proteins. Nucleic Acids Res. 2018, 46, 2204– 2217,  DOI: 10.1093/nar/gky060
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    15
    Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins
    Shen, Wen; De Hoyos, Cheryl L.; Sun, Hong; Vickers, Timothy A.; Liang, Xue-Hai; Crooke, Stanley T.
    Nucleic Acids Research (2018), 46 (5), 2204-2217CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    We reported previously that a 2' fluoro-modified (2' F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5-10-5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2'-O-(2-methoxyethyl) (2' MOE) or 2',4'-constrained 2'-O-Et (cEt) did. Rapid degrdn. of these proteins and cytotoxicity were obsd. in cells treated with 2' F PS-ASO. Here, we report that 2' F gapmer PS-ASOs of different sequences caused redn. in levels of DBHS proteins and hepatotoxicity in mice. 2'F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were obsd. as early as 24 to 48 h after a single administration of the 2' F PS-ASO. The obsd. effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2' F nucleoside metabolites. Instead, we found that 2' F PS-ASO assocd. with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates pos. with the 2' F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2' F PS-ASO in vivo.
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  16. 16
    Lee, E. J.; Lim, H. K.; Cho, Y. S.; Hah, S. S. Peptide Nucleic Acids Are an Additional Class of Aptamers. RSC Adv. 2013, 3, 5828– 5831,  DOI: 10.1039/c3ra40553b
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    16
    Peptide nucleic acids are an additional class of aptamers
    Lee, Eun Jeong; Lim, Hyun Kyung; Cho, Yea Seul; Hah, Sang Soo
    RSC Advances (2013), 3 (17), 5828-5831CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    Peptide nucleic acids (PNAs) have been known for their similar or superior properties to DNA or RNA since 1991, but not regarded as a sep. class of aptamers. We report here an interesting addnl. property of PNAs revealed by graphene oxide (GO)-based assays, suggesting that nucleobases in certain aptamers play more important roles in strong and selective binding to the target mols.
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  17. 17
    Young, B. E.; Kundu, N.; Sczepanski, J. T. Mirror-Image Oligonucleotides: History and Emerging Applications. Chem.─Eur. J. 2019, 25, 7981– 7990,  DOI: 10.1002/chem.201900149
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    17
    Mirror-Image Oligonucleotides: History and Emerging Applications
    Young, Brian E.; Kundu, Nandini; Sczepanski, Jonathan T.
    Chemistry - A European Journal (2019), 25 (34), 7981-7990CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. As chiral mols., naturally occurring D-oligonucleotides have enantiomers, L-DNA and L-RNA, which are comprised of L-(deoxy)ribose sugars. These mirror-image oligonucleotides have the same phys. and chem. properties as that of their native D-counterparts, yet are highly orthogonal to the stereospecific environment of biol. Consequently, L-oligonucleotides are resistant to nuclease degrdn. and many of the off-target interactions that plague traditional D-oligonucleotide-based technologies; thus making them ideal for biomedical applications. Despite a flurry of interest during the early 1990s, the inability of D- and L-oligonucleotides to form contiguous Watson-Crick base pairs with each other has ultimately led to the perception that L-oligonucleotides have only limited utility. Recently, however, scientists have begun to uncover novel strategies to harness the bio-orthogonality of L-oligonucleotides, while overcoming (and even exploiting) their inability to Watson-Crick base pair with the natural polymer. Herein, a brief history of L-oligonucleotide research is presented and emerging L-oligonucleotide-based technologies, as well as their applications in research and therapy, are presented.
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  18. 18
    Cui, L.; Peng, R.; Fu, T.; Zhang, X.; Wu, C.; Chen, H.; Liang, H.; Yang, C. J.; Tan, W. Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems. Anal. Chem. 2016, 88, 1850– 1855,  DOI: 10.1021/acs.analchem.5b04170
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    18
    Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems
    Cui, Liang; Peng, Ruizi; Fu, Ting; Zhang, Xiaobing; Wu, Cuichen; Chen, Huapei; Liang, Hao; Yang, Chaoyong James; Tan, Weihong
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (3), 1850-1855CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    DNAzymes, an important type of metal ion-dependent functional nucleic acid, are widely applied in bioanal. and biomedicine. However, the use of DNAzymes in practical applications has been impeded by the intrinsic drawbacks of natural nucleic acids, such as interferences from nuclease digestion and protein binding, as well as undesired intermol. interactions with other nucleic acids. On the basis of reciprocal chiral substrate specificity, the enantiomer of D-DNAzyme, L-DNAzyme, could initiate catalytic cleavage activity with the same achiral metal ion as a cofactor. Meanwhile, by using the advantage of nonbiol. L-DNAzyme, which is not subject to the interferences of biol. matrixes, as recognition units, a facile and stable L-DNAzyme sensor was proposed for sensing metal ions in complex biol. samples and live cells.
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  19. 19
    Ke, G.; Wang, C.; Ge, Y.; Zheng, N.; Zhu, Z.; Yang, C. J. L-DNA Molecular Beacon: A Safe, Stable, and Accurate Intracellular Nano-Thermometer for Temperature Sensing in Living Cells. J. Am. Chem. Soc. 2012, 134, 18908– 18911,  DOI: 10.1021/ja3082439
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    19
    L-DNA Molecular Beacon: A Safe, Stable, and Accurate Intracellular Nano-thermometer for Temperature Sensing in Living Cells
    Ke, Guoliang; Wang, Chunming; Ge, Yun; Zheng, Nanfeng; Zhu, Zhi; Yang, Chaoyong James
    Journal of the American Chemical Society (2012), 134 (46), 18908-18911CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Noninvasive and accurate measurement of intracellular temp. is of great significance in biol. and medicine. This paper describes a safe, stable, and accurate intracellular nano-thermometer based on an L-DNA mol. beacon (L-MB), a dual-labeled hairpin oligonucleotide built from the optical isomer of naturally occurring D-DNA. Relying on the temp.-responsive hairpin structure and the FRET signaling mechanism of MBs, the fluorescence of L-MBs is quenched below the melting temp. and enhanced with increasing temp. Because of the excellent reversibility and tunable response range, L-MBs are very suitable for temp. sensing. More importantly, the non-natural L-DNA backbone prevents the L-MBs from binding to cellular nucleic acids and proteins as well as from being digested by nucleases inside the cells, thus ensuring excellent stability and accuracy of the nano-thermometer in a complex cellular environment. The L-MB nano-thermometer was used for the photothermal study of Pd nanosheets in living cells, establishing the nano-thermometer as a useful tool for intracellular temp. measurement.
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  20. 20
    Kim, K.-R.; Lee, T.; Kim, B.-S.; Ahn, D.-R. Utilizing the Bioorthogonal Base-Pairing System of l-DNA to Design Ideal DNA Nanocarriers for Enhanced Delivery of Nucleic Acid Cargos. Chem. Sci. 2014, 5, 1533– 1537,  DOI: 10.1039/c3sc52601a
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    20
    Utilizing the bioorthogonal base-pairing system of l-DNA to design ideal DNA nanocarriers for enhanced delivery of nucleic acid cargos
    Kim, Kyoung-Ran; Lee, Taemin; Kim, Byeong-Su; Ahn, Dae-Ro
    Chemical Science (2014), 5 (4), 1533-1537CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    DNA nanoconstructs are a potential drug carrier with high biocompatibility. They are promising particularly when therapeutic nucleic acids are the cargos to be delivered, since both the carrier and the cargo are nucleic acids which can be designed, synthesized and assembled in a seamless feature without using post-synthetic conjugation chem. However, the unwanted base-paring events between the cargo and the carrier may potentially disturb the desired structure of the cargo-loaded carrier. To address this concern, we propose a DNA nanocarrier composed of l-DNA strands having a bioorthogonal base-paring system. The study presented here provides useful properties of l-DNA as a backbone for the DNA nanocarrier and demonstrates superiority of l-DNA over the natural d-DNA backbone in the delivery of an anti-proliferative aptamer as well as in the construction of the cargo-carrier assembly.
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  21. 21
    Phares, N.; White, R. J.; Plaxco, K. W. Improving the Stability and Sensing of Electrochemical Biosensors by Employing Trithiol-Anchoring Groups in a Six-Carbon Self-Assembled Monolayer. Anal. Chem. 2009, 81, 1095– 1100,  DOI: 10.1021/ac8021983
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    21
    Improving the Stability and Sensing of Electrochemical Biosensors by Employing Trithiol-Anchoring Groups in a Six-Carbon Self-Assembled Monolayer
    Phares, Noelle; White, Ryan J.; Plaxco, Kevin W.
    Analytical Chemistry (Washington, DC, United States) (2009), 81 (3), 1095-1100CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Alkane thiol self-assembled monolayers (SAMs) have seen widespread utility in the fabrication of electrochem. biosensors. Their utility, however, reflects a potentially significant compromise. While shorter SAMs support efficient electron transfer, they pack poorly and are thus relatively unstable. Longer SAMs are more stable but suffer from less efficient electron transfer, thus degrading sensor performance. Here the authors use the electrochem. DNA (E-DNA) sensor platform to compare the signaling and stability of biosensors fabricated using a short, six-carbon monothiol with those employing either of two com. available trihexylthiol anchors (a flexible Letsinger type and a rigid adamantane type). The authors find that all three anchors support efficient electron transfer and E-DNA signaling, with the gain, specificity, and selectivity of all three being effectively indistinguishable. The stabilities of the three anchors, however, vary significantly. Sensors anchored with the flexible trithiol exhibit enhanced stability, retaining 75% of their original signal and maintaining excellent signaling properties after 50 days storage in buffer. Likewise these sensors exhibit excellent temp. stability and robustness to electrochem. interrogation. The stability of sensors fabricated using the rigid trithiol anchor, by comparison, are similar to those of the monothiol, with both exhibiting significant (>60%) loss of signal upon wet storage or thermocycling. Employing a flexible trithiol anchor in the fabrication of SAM-based electrochem. biosensors may provide a means of improving sensor robustness without sacrificing electron transfer efficiency or otherwise impeding sensor performance.
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  22. 22
    Ricci, F.; Zari, N.; Caprio, F.; Recine, S.; Amine, A.; Moscone, D.; Palleschi, G.; Plaxco, K. W. Surface Chemistry Effects on the Performance of an Electrochemical DNA Sensor. Bioelectrochemistry 2009, 76, 208– 213,  DOI: 10.1016/j.bioelechem.2009.03.007
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    22
    Surface chemistry effects on the performance of an electrochemical DNA sensor
    Ricci, Francesco; Zari, Nadia; Caprio, Felice; Recine, Simona; Amine, Aziz; Moscone, Danila; Palleschi, Giuseppe; Plaxco, Kevin W.
    Bioelectrochemistry (2009), 76 (1-2), 208-213CODEN: BIOEFK; ISSN:1567-5394. (Elsevier B.V.)
    E-DNA sensors are a reagentless, electrochem. oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. The authors have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. The authors find that, among the thiols tested, the pos. charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, resp., the neg. charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chem. can lead to significant improvements in the performance of this broad class of electrochem. DNA sensors.
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  23. 23
    Reinstein, O.; Yoo, M.; Han, C.; Palmo, T.; Beckham, S. A.; Wilce, M. C. J.; Johnson, P. E. Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an off-Target Ligand. Biochemistry 2013, 52, 8652– 8662,  DOI: 10.1021/bi4010039
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    23
    Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand
    Reinstein, Oren; Yoo, Mina; Han, Chris; Palmo, Tsering; Beckham, Simone A.; Wilce, Matthew C. J.; Johnson, Philip E.
    Biochemistry (2013), 52 (48), 8652-8662CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The cocaine-binding aptamer is unusual in that it tightly binds mols. other than the ligand it was selected for. Here, the authors study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titrn. calorimetry was used to quantify the quinine-binding affinity and thermodn. of a set of sequence variants of the cocaine-binding aptamer. The authors find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more neg. change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle x-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.
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  24. 24
    Slavkovic, S.; Altunisik, M.; Reinstein, O.; Johnson, P. E. Structure-Affinity Relationship of the Cocaine-Binding Aptamer with Quinine Derivatives. Bioorg. Med. Chem. 2015, 23, 2593– 2597,  DOI: 10.1016/j.bmc.2015.02.052
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    24
    Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives
    Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E.
    Bioorganic & Medicinal Chemistry (2015), 23 (10), 2593-2597CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    In addn. to binding its target mol., cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titrn. calorimetry-based binding expts. to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic arom. ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliph. portion of quinine, quinuclidine, does not show detectable binding. Compds. with three fused arom. rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alc. or an amino group all result in lower binding affinity. For all ligands that bind, assocn. is driven by a neg. enthalpy compensated by unfavorable binding entropy.
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  25. 25
    Dey, S.; Sczepanski, J. T. In Vitro Selection of L-DNA Aptamers That Bind a Structured d-RNA Molecule. Nucleic Acids Res. 2020, 48, 1669– 1680,  DOI: 10.1093/nar/gkz1236
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    25
    In vitro selection of L-DNA aptamers that bind a structured D-RNA molecule
    Dey, Sougata; Sczepanski, Jonathan T.
    Nucleic Acids Research (2020), 48 (4), 1669-1680CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    The development of structure-specific RNA binding reagents remains a central challenge in RNA biochem. and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve L-RNA aptamers that bind tightly to structured D-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of L-DNA, which has several practical advantages compared to L-RNA, remains unknown. Here, we report the discovery and characterization of the first L-DNA aptamers against a structured RNA mol., precursor microRNA-155, thereby establishing the capacity of DNA and RNA mols. of the opposite handedness to form tight and specific 'cross-chiral' interactions with each other. L-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of L-DNA to interact with native D-RNA via Watson- Crick base pairing. Furthermore, L-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of L-DNA aptamers are distinct from previously reported L-RNA aptamers against pre-miR-155, indicating that L-DNA and L-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that L-DNA may be pursued as an alternative to L-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.
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  26. 26
    Feng, X.-N.; Cui, Y.-X.; Zhang, J.; Tang, A.-N.; Mao, H.-B.; Kong, D.-M. Chiral Interaction Is a Decisive Factor to Replace d-DNA with l-DNA Aptamers. Anal. Chem. 2020, 92, 6470– 6477,  DOI: 10.1021/acs.analchem.9b05676
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    26
    Chiral Interaction Is a Decisive Factor To Replace D-DNA with L-DNA Aptamers
    Feng, Xue-Nan; Cui, Yun-Xi; Zhang, Jing; Tang, An-Na; Mao, Han-Bin; Kong, De-Ming
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (9), 6470-6477CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Nucleic acid aptamers have been widely used in various fields such as biosensing, DNA chip, and medical diagnosis. However, the high susceptibility of nucleic acids to ubiquitous nucleases reduces the biostability of aptamers and limits their applications in biol. contexts. Therefore, improving the biostability of aptamers becomes an urgent need. Herein, the authors present a simple strategy to resolve this problem by directly replacing the D-DNA-based aptamers with left-handed L-DNA. By testing several reported aptamers against resp. targets, the authors found that the proposed strategy stood up well for nonchiral small mol. targets (e.g., Hemin and cationic porphyrin) and chiral targets whose interactions with aptamers are chirality-independent (e.g., ATP). The authors also found that the L-DNA aptamers were indeed endowed with greatly improved biostability due to the extraordinary resistance of L-DNA to nuclease digestion. With respect to other small-mol. targets whose interactions with aptamers are chirality-dependent (e.g., kanamycin) and biomacromols. (e.g., tyrosine kinase-7), however, the proposed strategy was not entirely effective likely due to the participation of the DNA backbone chirality into the target recognition. In spite of this limitation, this strategy indeed paves an easy way to screen highly biostable aptamers important for the applications in many fields.
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  27. 27
    Lovrić, M.; Komorsky-Lovric, Š. Square-Wave Voltammetry of an Adsorbed Reactant. J. Electroanal. Chem. 1988, 248, 239– 253,  DOI: 10.1016/0022-0728(88)85089-7
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    27
    Square-wave voltammetry of an adsorbed reactant
    Lovric, Milivoj; Komorsky-Lovric, Sebojka
    Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1988), 248 (2), 239-53CODEN: JEIEBC; ISSN:0022-0728.
    Many electroanal. methods employ the adsorptive accumulation at the hanging Hg drop electrode combined with different stripping voltammetric techniques. One of the more sensitive techniques is square-wave voltammetry (SWV). The theory of SWV responses of adsorbed reactants is presented. The pronounced sensitivity of the SWV to quasi-reversible redox reactions from the adsorbed state is demonstrated. The relations between the properties of the SW response and the parameters of a charge transfer and of the excitation signal are discussed. The SW voltammograms of methylene blue are analyzed according to theor. predictions.
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    Komorsky-Lovrić, Š.; Lovrić, M. Measurements of Redox Kinetics of Adsorbed Azobenzene by “a Quasireversible Maximum” in Square-Wave Voltammetry. Electrochim. Acta 1995, 40, 1781– 1784,  DOI: 10.1016/0013-4686(95)00097-X
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    Measurements of redox kinetics of adsorbed azobenzene by "a quasireversible maximum" in square-wave voltammetry
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    The std. redox reaction rate consts. of azobenzene adsorbed to the mercury electrode are ks = 350 ± 25 s-1 in the acidic perchlorate medium (pH 2) and ks = 175 ± 20 s-1 in the acetate buffer pH 4.7. Under the influence of 0.2%, 0.5% and 1% acetonitrile in these electrolytes, the rate consts. are reduced to 265 ± 5 s-1, 160 ± 20 s-1 and 77 ± 10 s-1, at pH 2, and to 150 ± 5 s-1 and 56 ± 13 s-1 at pH 4.7.
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    Dauphin-Ducharme, P.; Plaxco, K. W. Maximizing the Signal Gain of Electrochemical-DNA Sensors. Anal. Chem. 2016, 88, 11654– 11662,  DOI: 10.1021/acs.analchem.6b03227
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    Analytical Chemistry (Washington, DC, United States) (2016), 88 (23), 11654-11662CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Electrochem. DNA (E-DNA) sensors have emerged as a promising class of biosensors capable of detecting a wide range of mol. analytes (nucleic acids, proteins, small mols., inorg. ions) without the need for exogenous reagents or wash steps. In these sensors a binding-induced conformational change in an electrode-bound "probe" (a target-binding nucleic acid or nucleic-acid-peptide chimera) alters the location of an attached redox reporter, leading to a change in electron transfer that is typically monitored using square-wave voltammetry. Because signaling in this class of sensors relies on binding-induced changes in electron transfer rate, the signal gain of such sensors (change in signal upon the addn. of satg. target) is dependent on the frequency of the square-wave potential pulse used to interrogate them, with the optimal square-wave frequency depending on the structure of the probe, the nature of the redox reporter, and other features of the sensor. Because it alters the driving force of the redox reaction and thus electron transfer kinetics, signal gain in this class of sensors is also strongly dependent on the amplitude of the square-wave potential pulse. Specifically, the authors show here that the simultaneous optimization of square-wave frequency and amplitude produces large (often more than two-fold) increases in the signal gain of a wide range of E-DNA-type sensors.
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    RSC Advances (2018), 8 (17), 9083-9093CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    Procaine is an anesthetic drug commonly administrated topically or i.v. for use in local anesthesia. Promisingly, some anticancer activities of procaine have also been reported. Therefore, the mechanism of interaction between anesthetic drug procaine with ct-DNA was detd. collectively by means of various spectroscopic and mol. docking methods. Minor groove 1 : 1 binding of procaine to the ct-DNA was evidenced from absorption spectroscopy, fluorescence quenching, DNA melting, competitive binding measurements with EB and DAPI dyes, viscosity and CD spectroscopy together with mol. docking simulations and DFT calcns. Mol. docking on five different B-DNA structures (taken from the Protein Data Bank) shows that procaine binds in the AT rich region of all five B-DNA structures. Thermodn. parameters, evaluated using van't Hoff's isotherm, shown that the interaction was feasible and the binding forces involved were hydrophobic as well as hydrogen bonding which were, further, confirmed by mol. docking. The frontier MOs (HOMO and LUMO) of procaine and DNA bases have been calcd. by DFT method and the chem. potential (μ), chem. hardness (η) and fraction no. of electrons (ΔN) from procaine to DNA bases were evaluated, which have shown that procaine acts as an electron donor to the DNA bases. Simultaneously, anticancer activities of procaine alone and in combination with doxorubicin were obsd. on the MCF-7 breast cancer cell line. The results showed that the combined treatment with both procaine and doxorubicin enhanced the cytotoxic and apoptotic inducing potential of doxorubicin.
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    Annals of the New York Academy of Sciences (2006), 1075 (), 191-196CODEN: ANYAA9; ISSN:0077-8923. (Blackwell Publishing, Inc.)
    The concn. of circulating DNA (cirDNA) and DNase activity in blood plasma of healthy donors and patients with colon or stomach cancer were analyzed. The concn. of DNA was measured using Hoechst 33258 fluorescent assay after the isolation by the glass-milk protocol. A 1-kbp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers was used as a substrate for DNase. DNase activity was estd. from the data of immunochem. detection of the nonhydrolyzed amplicon. The av. concn. of cirDNA in the plasma of healthy donors was low (34±34 ng/mL), and was accompanied with high DNase activity (0.356±0.410 U/mL). The increased concns. of cirDNA in blood plasma of patients with colon and stomach cancer were accompanied by a decrease in DNase activity below the detection level of the assay. The data obtained demonstrate that low DNase activity in blood plasma of cancer patients can cause an increase in the concn. of cirDNA.
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    Ershova, E.; Sergeeva, V.; Klimenko, M.; Avetisova, K.; Klimenko, P.; Kostyuk, E.; Veiko, N.; Veiko, R.; Izevskaya, V.; Kutsev, S. Circulating Cell-Free DNA Concentration and DNase I Activity of Peripheral Blood Plasma Change in Case of Pregnancy with Intrauterine Growth Restriction Compared to Normal Pregnancy. Biomed. Rep. 2017, 7, 319– 324,  DOI: 10.3892/br.2017.968
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    Circulating cell-free DNA concentration and DNase I activity of peripheral blood plasma change in case of pregnancy with intrauterine growth restriction compared to normal pregnancy
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    Biomedical Reports (2017), 7 (4), 319-324CODEN: BREIB7; ISSN:2049-9442. (Spandidos Publications Ltd.)
    The level of apoptosis is increased during pregnancy. Dying cells emit DNA that remains in blood circulation and is known as cell free DNA (cfDNA). The concn. of cfDNA can reflect the level of cell death. The present article is the result of studying cfDNA concn. and DNase I activity in the blood plasma of 40 non pregnant women (control), 40 healthy pregnant women (over 37 wk) and 40 pregnant women with a diagnosis of intrauterine growth restriction (IUGR). In order to explain the obtained results, a program modeling the change of cfDNA concn. under the influence of different internal and external factors was written. It was reported that, despite the fact that the level of cell death is increased, cfDNA concn. in blood can be decreased due to activation of cfDNA elimination system. A significant increase of DNase I activity has been reported in cases of IUGR. Increase in DNase I activity over a certain threshold indicates presence of pathol. processes in the organism. CfDNA circulating in blood cannot be a reliable marker of increased cell death during pregnancy. Thus, assessment of the level of cell death during pregnancy should be done by simultaneous anal. of cfDNA level and DNase I activity.
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    Nature Communications (2014), 5 (), 4348CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    The strength of the thiol-gold interactions provides the basis to fabricate robust self-assembled monolayers for diverse applications. Investigation on the stability of thiol-gold interactions has thus become a hot topic. Here we use at. force microscopy to quantify the stability of individual thiol-gold contacts formed both by isolated single thiols and in self-assembled monolayers on gold surface. Our results show that the oxidized gold surface can enhance greatly the stability of gold-thiol contacts. In addn., the shift of binding modes from a coordinate bond to a covalent bond with the change in environmental pH and interaction time has been obsd. exptl. Furthermore, isolated thiol-gold contact is found to be more stable than that in self-assembled monolayers. Our findings revealed mechanisms to control the strength of thiol-gold contacts and will help guide the design of thiol-gold contacts for a variety of practical applications.
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    Generation of Biostable L-aptamers against Achiral Targets by Chiral Inversion of Existing D-aptamers
    Chen, Huapei; Xie, Sitao; Liang, Hao; Wu, Cuichen; Cui, Liang; Huan, Shuang-Yan; Zhang, Xiaobing
    Talanta (2017), 164 (), 662-667CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    Based on reciprocal chiral substrate specificity, taking achiral mols., ethanolamine (EA) and malachite green (MG) as two model targets, biostable L-DNA aptamers and L-RNA aptamers were generated resp. by chiral inversion of existing D-aptamers. In the detection of EA with L-DNA aptamer-based sensors, the feasibility of the authors' strategy was confirmed, while in the detection of MG with L-RNA aptamers, linear calibration curves were obtained at 0.1-5 μm with the detection limit of 0.065 μm under optimized exptl. conditions. The mirror-image L-aptamers have identical recognition capability as D-aptamers. Meanwhile, L-aptamers have superior biostability to resist nuclease digestion, protein binding interference and off-target effects, enabling their applications in complex practical samples, such as lake water and fish tissue extns. The authors' work provides a simple, yet universal and efficient way to develop biostable aptamers.
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    White, R. J.; Phares, N.; Lubin, A. A.; Xiao, Y.; Plaxco, K. W. Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry. Langmuir 2008, 24, 10513– 10518,  DOI: 10.1021/la800801v
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    Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry
    White, Ryan J.; Phares, Noelle; Lubin, Arica A.; Xiao, Yi; Plaxco, Kevin W.
    Langmuir (2008), 24 (18), 10513-10518CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Electrochem., aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. To further improve this technol. the authors have systematically studied the effects of probe (aptamer) packing d., the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small mol. cocaine and the protein thrombin. The authors find that, by controlling the concn. of aptamer employed during sensor fabrication, the authors can control the d. of probe DNA mols. on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with max. gain obsd. near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is obsd. at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, the authors find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher abs. sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the obsd. signal gain of the authors' cocaine sensor 10-fold. The authors demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.
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    Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Self-Assembled Monolayers That Resist the Adsorption of Proteins and the Adhesion of Bacterial and Mammalian Cells. Langmuir 2001, 17, 6336– 6343,  DOI: 10.1021/la010552a
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    Self-assembled monolayers that resist the adsorption of proteins and the adhesion of bacterial and mammalian cells
    Ostuni, Emanuele; Chapman, Robert G.; Liang, Michael N.; Meluleni, Gloria; Pier, Gerald; Ingber, Donald E.; Whitesides, George M.
    Langmuir (2001), 17 (20), 6336-6343CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    This paper examines the hypothesis that surfaces resistant to protein adsorption should also be resistant to the adhesion of bacteria (Staphylococcus aureus, Staphylococcus epidermidis) and the attachment and spreading of mammalian cells (bovine capillary endothelial (BCE) cells). The surfaces tested were those of self-assembled monolayers (SAMs) terminated with derivs. of tri(sarcosine) (Sarc), N-acetylpiperazine, permethylated sorbitol, hexamethylphosphoramide, phosphoryl choline, and an intramol. zwitterion (-CH2N+(CH3)2CH2CH2CH2SO3-) (ZW); all are known to resist the adsorption of proteins. There seems to be little or no correlation between the adsorption of protein (fibrinogen and lysozyme) and the adhesion of cells. Surfaces terminated with derivs. of Sarc and N-acetylpiperazine resisted the adhesion of S. aureus and S. epidermidis as well as did surfaces terminated with tri(ethylene glycol). A surface that presented Sarc groups was the only one that resisted the adhesion of BCE cells as well as did a surface terminated with tri(ethylene glycol). The attachment of BCE cells to surfaces could be patterned using SAMs terminated with derivs. of Sarc, N-acetylpiperazine, phosphoramide, and the ZW as the attachment-resistant component and methyl-terminated SAMs as the adhesive component.
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    Adsorption of proteins to hydrophobic sites on mixed self-assembled monolayers
    Ostuni, Emanuele; Grzybowski, Bartosz A.; Mrksich, Milan; Roberts, Carmichael S.; Whitesides, George M.
    Langmuir (2003), 19 (5), 1861-1872CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    This paper describes a technique that uses mixed self-assembled monolayers of two alkanethiolates (-S(CH2)11(OCH2CH2)6OR, R = a hydrophobic group, and -S(CH2)11(OCH2CH2)nOH, n = 3, 6, EGnOH), in combination with surface plasmon resonance spectroscopy, to study the influence of the size and shape of R, and its d. at the surface, on the hydrophobic adsorption of proteins at solid-liq. interfaces. Detailed results were obtained for β-galactosidase, carbonic anhydrase, lysozyme, and RNase A using R = C(C6H5)3, CH(C6H5)2, and CH2(C6H5). A hard-sphere model is used to rationalize the adsorption; this model, although very approx., helps to interpret qual. trends in the data. Using this model, the extent to which adsorbed proteins undergo conformational rearrangements appears to depend on the d. of the hydrophobic groups at the surface and on the concn. of protein in soln. This paper describes the first step toward the development of a system that will allow the study of hydrophobic interactions of proteins with surfaces presenting org. groups of well-defined shape.
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    Curtis, S. D.; Ploense, K. L.; Kurnik, M.; Ortega, G.; Parolo, C.; Kippin, T. E.; Plaxco, K. W.; Arroyo-Currás, N. Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data. Anal. Chem. 2019, 91, 12321– 12328,  DOI: 10.1021/acs.analchem.9b02553
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    Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data
    Curtis, Samuel D.; Ploense, Kyle L.; Kurnik, Martin; Ortega, Gabriel; Parolo, Claudio; Kippin, Tod E.; Plaxco, Kevin W.; Arroyo-Curras, Netzahualcoyotl
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12321-12328CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Electrochem. sensors are major players in the race for improved mol. diagnostics due to their convenience, temporal resoln., manufg. scalability, and their ability to support real-time measurements. This is evident in the ever-increasing no. of health-related electrochem. sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data vols. the authors describe here an open-source, easily customizable, multi-platform compatible program for the real-time control, processing and visualization of electrochem. data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which the authors have called Software for the Anal. and Continuous Monitoring of Electrochem. Systems (SACMES), also includes a graphical interface allowing the user to easily change anal. parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES the authors use it here to analyze the real-time data output by: (1) the electrochem., aptamer-based measurement of a specific small-mol. target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the detn. of the folding thermodn. of an electrode-attached, redox-reporter-modified protein.
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  2. Zach Watkins, Aleksandar Karajic, Thomas Young, Ryan White, Jason Heikenfeld. Week-Long Operation of Electrochemical Aptamer Sensors: New Insights into Self-Assembled Monolayer Degradation Mechanisms and Solutions for Stability in Serum at Body Temperature. ACS Sensors 2023, 8 (3) , 1119-1131. https://doi.org/10.1021/acssensors.2c02403
  3. Vincent Clark, Miguel Aller Pellitero, Netzahualcóyotl Arroyo-Currás. Explaining the Decay of Nucleic Acid-Based Sensors under Continuous Voltammetric Interrogation. Analytical Chemistry 2023, 95 (11) , 4974-4983. https://doi.org/10.1021/acs.analchem.2c05158
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  • Abstract

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    Figure 1

    Figure 1. Anatomy of E-AB sensors. (A) We fabricated sensors using aptamers of either naturally occurring d-DNA or the nuclease-resistant enantiomer l-DNA. These aptamers are modified on the 5′ end with an alkanethiol linker for self-assembly onto a gold electrode surface and on the 3′ end with a redox reporter for electrochemical interrogation. Both d- and l-DNA-functionalized sensors maintain similar signaling when interrogated by cyclic voltammetry (CV) (B) and square wave voltammetry (C). CV was measured in S1 buffer (5 mM HEPES, 50 mM NaCl, and 100 μΜ ZnCl2, pH ∼ 5.4) at 0.1 V/s. Square wave voltammetry was recorded in S1 buffer with a frequency of 80 Hz and an amplitude of 25 mV. Shaded areas represent the standard deviation calculated from four independent electrodes.

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    Figure 2

    Figure 2. Sensors employing d- or l-coc aptamers respond equivalently to achiral target additions. (A) We demonstrate this equivalence by, first, highlighting the fact that signal ON and OFF frequencies remain the same between sensor types. (B) By challenging both d-coc and l-coc-functionalized sensors with increasing concentrations of the achiral target procaine, we determine that the enantiomeric inversion of aptamers does not alter either signal gain (magnitude of current change at saturation, GainD-coc = 910% vs GainL-coc = 890%) or apparent target affinity (KD.d-DNA = 10.6 ± 0.6 mM vs KD.l-DNA = 10.3 ± 0.6 mM). These consistencies remain when sensors are challenged with targets other than procaine. Specifically, both d-coc and l-coc sensors respond similarly to the achiral target 6-hydroxyquinoline (C, Gaind-coc = 260% vs Gainl-coc = 300%; KD.d-DNA = 2.37 ± 0.10 mM vs KD.l-DNA = 2.29 ± 0.10 mM) and the chiral target quinine (D, Gaind-coc = 390% vs Gainl-coc = 360%; KD.d-DNA = 523 ± 40 μM vs KD.l-DNA = 686 ± 32 μM). The latter still binds to l-coc sensors because quinine contains an achiral quinoline backbone moiety. All SWV experiments were recorded from 0 to −0.5 V versus Ag/AgCl with an amplitude of 25 mV. Shaded areas in all panels represent the standard deviation calculated from eight independent sensors.

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    Figure 3

    Figure 3. l-DNA-functionalized sensors resist nuclease-driven hydrolysis. To illustrate this effect, we serially measured cyclic voltammograms on electrodes functionalized with d-DNA and l-DNA oligonucleotides, 18 nt long, every 25 s for 4 h. By monitoring the peak current from the oxidation of DNA-attached leucomethylene blue reporters, (A) we observed changes in the d-DNA electrode signal driven by both progressive monolayer desorption (constant, initial negative drift in all panels) and nuclease-driven hydrolysis across the range of physiological concentrations. The solid lines indicate the time at which we injected S1 nuclease into the electrochemical cell. (B) l-DNA-functionalized electrodes, in contrast, presented only slight signal loss upon exposure to added S1 nuclease. (C) This remains true even with a 50× excess of S1 nuclease relative to physiological levels of nucleases in human circulation. (D) If we first inactivate the nuclease by heating to 70 °C in the presence of ethylenediaminetetraacetic acid (EDTA), no additional degradation is seen on either form of DNA. Peak currents were extracted from cyclic voltammograms measured at scanning rates of 0.1 V s–1. Shaded areas for all traces represent the standard deviation calculated from at least four electrodes. 0 h in all graphs represents the time immediately after sensor fabrication, when electrodes are first placed into the cell without any prior electrochemical pre-treatment.

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    Figure 4

    Figure 4. E-AB signal decay in biological fluids. By interrogating d-coc and l-coc sensors with either MCH or HxSH monolayers in (A) unfiltered human serum, (B) saliva, (C) urine, (D) plasma, (E) CSF, or (F) whole blood, we demonstrate that signal decay is not driven by nuclease hydrolysis. While some difference in signal loss can be seen for sensors with HxSH monolayers before injection of excess nuclease (left of vertical lines), no significant difference is seen between d-coc and l-coc sensors with MCH monolayers in any of the biological fluids, either before or after injection of excess nuclease. This lack of nuclease-dependent signal decay likely indicates that the operational life of E-AB sensors is not limited by nuclease action but rather by monolayer desorption. Peak currents were extracted from cyclic voltammograms measured at scanning rates of 0.1 V s–1. Shaded areas for all traces represent the standard deviation calculated from at least four electrodes.

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  • References

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    This article references 42 other publications.

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      Electrochem. aptamer-based (E-AB) sensors achieve highly precise measurements of specific mol. targets in untreated biol. fluids. This unique ability, together with their measurement frequency of seconds or faster, has enabled the real-time monitoring of drug pharmacokinetics in live animals with unprecedented temporal resoln. However, one important weakness of E-AB sensors is that their bioelectronic interface degrades upon continuous electrochem. interrogation-a process typically seen as a drop in faradaic and an increase in charging currents over time. This progressive degrdn. limits their in vivo operational life to 12 h at best, a period that is much shorter than the elimination half-life of the vast majority of drugs in humans. Thus, there is a crit. need to develop novel E-AB interfaces that resist continuous electrochem. interrogation in biol. fluids for prolonged periods. In response, our group is pursuing the development of better packed, more stable self-assembled monolayers (SAMs) to improve the signaling and extend the operational life of in vivo E-AB sensors from hours to days. By invoking hydrophobicity arguments, we have created SAMs that do not desorb from the electrode surface in aq. physiol. solns. and biol. fluids. These SAMs, formed from 1-hexanethiol solns., decrease the voltammetric charging currents of E-AB sensors by 3-fold relative to std. monolayers of 6-mercapto-1-hexanol, increase the total faradaic current, and alter the electron transfer kinetics of the platform. Moreover, the stability of our new SAMs enables uninterrupted, continuous E-AB interrogation for several days in biol. fluids, like undiluted serum, at a physiol. temp. of 37°C.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFKqsro%253D&md5=5f10c2efa8960f892202a6184bf9d63a
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      Karlsen, Kasper K.; Wengel, Jesper
      Nucleic Acid Therapeutics (2012), 22 (6), 366-370CODEN: NATUCU; ISSN:2159-3337. (Mary Ann Liebert, Inc.)
      A review on locked nucleic acid (LNA) and aptamers in drug design including the topics LNA-contg. aptamers by post-SELEX modification, and LNA-contg. aptamers by de novo evolution.
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    12. 12
      Schmidt, K. S.; Borkowski, S.; Kurreck, J.; Stephens, A. W.; Bald, R.; Hecht, M.; Friebe, M.; Dinkelberg, L.; Erdmann, V. A. Application of Locked Nucleic Acids to Improve Aptamer in Vivo Stability and Targeting Function. Nucleic Acids Res. 2004, 32, 5757– 5765,  DOI: 10.1093/nar/gkh862
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      12
      Application of locked nucleic acids to improve aptamer in vivo stability and targeting function
      Schmidt, Kathrin S.; Borkowski, Sandra; Kurreck, Jens; Stephens, Andrew W.; Bald, Rolf; Hecht, Maren; Friebe, Matthias; Dinkelborg, Ludger; Erdmann, Volker A.
      Nucleic Acids Research (2004), 32 (19), 5757-5765CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Aptamers are powerful candidates for mol. imaging applications due to a no. of attractive features, including rapid blood clearance and tumor penetration. We carried out structure-activity relationship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding. We investigated the effect of thermal stabilization of the presumed non-binding double-stranded stem region on binding affinity and resistance against nucleolytic degrdn. To achieve maximal thermal stem stabilization melting expts. with model hexanucleotide duplexes consisting of unmodified RNA, 2'-O-Me RNA (2'-OMe), 2'-Fluoro RNA (2'-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temps. have been found for an LNA/LNA duplex. TTA1 derivs. with LNA and 2'-OMe modifications within the non-binding stem have subsequently been synthesized. Esp., the LNA-modified TTA1 deriv. exhibited significant stem stabilization and markedly improved plasma stability while maintaining its binding affinity to the target. In addn., higher tumor uptake and longer blood retention was found in tumor-bearing nude mice. Thus, our strategy to introduce LNA modifications after the selection procedure is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding properties.
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    13. 13
      Seth, P. P.; Jazayeri, A.; Yu, J.; Allerson, C. R.; Bhat, B.; Swayze, E. E. Structure Activity Relationships of α-l-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals. Mol. Ther.--Nucleic Acids 2012, 1, e47  DOI: 10.1038/mtna.2012.34
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      13
      Structure activity relationships of α-L-LNA modified phosphorothioate gapmer antisense oligonucleotides in animals
      Seth, Punit P.; Jazayeri, Ali; Yu, Jeff; Allerson, Charles R.; Bhat, Balkrishen; Swayze, Eric E.
      Molecular Therapy--Nucleic Acids (2012), 1 (Oct.), E47/1-E47/8CODEN: MTAOC5; ISSN:2162-2531. (Nature Publishing Group)
      We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifiations targeting phosphatase and tensin homolog (PTEN) mRNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN mRNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen wts. suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ wts. Examn. of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clin. setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.
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    14. 14
      Swayze, E. E.; Siwkowski, A. M.; Wancewicz, E. V.; Migawa, M. T.; Wyrzykiewicz, T. K.; Hung, G.; Monia, B. P.; Bennett, C. F. Antisense Oligonucleotides Containing Locked Nucleic Acid Improve Potency but Cause Significant Hepatotoxicity in Animals. Nucleic Acids Res. 2007, 35, 687– 700,  DOI: 10.1093/nar/gkl1071
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      14
      Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals
      Swayze, Eric E.; Siwkowski, Andrew M.; Wancewicz, Edward V.; Migawa, Michael T.; Wyrzykiewicz, Tadeusz K.; Hung, Gene; Monia, Brett P.; Bennett, C. Frank
      Nucleic Acids Research (2007), 35 (2), 687-700CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A series of antisense oligonucleotides (ASOs) contg. either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA contg. oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE contg. ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ wts. and body wts. This toxicity was evident for multiple sequences targeting three different biol. targets, as well as in mismatch control sequences having no known mRNA targets. Histopathol. evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was obsd. as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.
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    15. 15
      Shen, W.; De Hoyos, C. L.; Sun, H.; Vickers, T. A.; Liang, X.-h.; Crooke, S. T. Acute Hepatotoxicity of 2′ Fluoro-Modified 5–10–5 Gapmer Phosphorothioate Oligonucleotides in Mice Correlates with Intracellular Protein Binding and the Loss of DBHS Proteins. Nucleic Acids Res. 2018, 46, 2204– 2217,  DOI: 10.1093/nar/gky060
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      15
      Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins
      Shen, Wen; De Hoyos, Cheryl L.; Sun, Hong; Vickers, Timothy A.; Liang, Xue-Hai; Crooke, Stanley T.
      Nucleic Acids Research (2018), 46 (5), 2204-2217CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      We reported previously that a 2' fluoro-modified (2' F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5-10-5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2'-O-(2-methoxyethyl) (2' MOE) or 2',4'-constrained 2'-O-Et (cEt) did. Rapid degrdn. of these proteins and cytotoxicity were obsd. in cells treated with 2' F PS-ASO. Here, we report that 2' F gapmer PS-ASOs of different sequences caused redn. in levels of DBHS proteins and hepatotoxicity in mice. 2'F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were obsd. as early as 24 to 48 h after a single administration of the 2' F PS-ASO. The obsd. effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2' F nucleoside metabolites. Instead, we found that 2' F PS-ASO assocd. with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates pos. with the 2' F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2' F PS-ASO in vivo.
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    16. 16
      Lee, E. J.; Lim, H. K.; Cho, Y. S.; Hah, S. S. Peptide Nucleic Acids Are an Additional Class of Aptamers. RSC Adv. 2013, 3, 5828– 5831,  DOI: 10.1039/c3ra40553b
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      16
      Peptide nucleic acids are an additional class of aptamers
      Lee, Eun Jeong; Lim, Hyun Kyung; Cho, Yea Seul; Hah, Sang Soo
      RSC Advances (2013), 3 (17), 5828-5831CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      Peptide nucleic acids (PNAs) have been known for their similar or superior properties to DNA or RNA since 1991, but not regarded as a sep. class of aptamers. We report here an interesting addnl. property of PNAs revealed by graphene oxide (GO)-based assays, suggesting that nucleobases in certain aptamers play more important roles in strong and selective binding to the target mols.
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    17. 17
      Young, B. E.; Kundu, N.; Sczepanski, J. T. Mirror-Image Oligonucleotides: History and Emerging Applications. Chem.─Eur. J. 2019, 25, 7981– 7990,  DOI: 10.1002/chem.201900149
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      17
      Mirror-Image Oligonucleotides: History and Emerging Applications
      Young, Brian E.; Kundu, Nandini; Sczepanski, Jonathan T.
      Chemistry - A European Journal (2019), 25 (34), 7981-7990CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. As chiral mols., naturally occurring D-oligonucleotides have enantiomers, L-DNA and L-RNA, which are comprised of L-(deoxy)ribose sugars. These mirror-image oligonucleotides have the same phys. and chem. properties as that of their native D-counterparts, yet are highly orthogonal to the stereospecific environment of biol. Consequently, L-oligonucleotides are resistant to nuclease degrdn. and many of the off-target interactions that plague traditional D-oligonucleotide-based technologies; thus making them ideal for biomedical applications. Despite a flurry of interest during the early 1990s, the inability of D- and L-oligonucleotides to form contiguous Watson-Crick base pairs with each other has ultimately led to the perception that L-oligonucleotides have only limited utility. Recently, however, scientists have begun to uncover novel strategies to harness the bio-orthogonality of L-oligonucleotides, while overcoming (and even exploiting) their inability to Watson-Crick base pair with the natural polymer. Herein, a brief history of L-oligonucleotide research is presented and emerging L-oligonucleotide-based technologies, as well as their applications in research and therapy, are presented.
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    18. 18
      Cui, L.; Peng, R.; Fu, T.; Zhang, X.; Wu, C.; Chen, H.; Liang, H.; Yang, C. J.; Tan, W. Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems. Anal. Chem. 2016, 88, 1850– 1855,  DOI: 10.1021/acs.analchem.5b04170
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      18
      Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems
      Cui, Liang; Peng, Ruizi; Fu, Ting; Zhang, Xiaobing; Wu, Cuichen; Chen, Huapei; Liang, Hao; Yang, Chaoyong James; Tan, Weihong
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (3), 1850-1855CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      DNAzymes, an important type of metal ion-dependent functional nucleic acid, are widely applied in bioanal. and biomedicine. However, the use of DNAzymes in practical applications has been impeded by the intrinsic drawbacks of natural nucleic acids, such as interferences from nuclease digestion and protein binding, as well as undesired intermol. interactions with other nucleic acids. On the basis of reciprocal chiral substrate specificity, the enantiomer of D-DNAzyme, L-DNAzyme, could initiate catalytic cleavage activity with the same achiral metal ion as a cofactor. Meanwhile, by using the advantage of nonbiol. L-DNAzyme, which is not subject to the interferences of biol. matrixes, as recognition units, a facile and stable L-DNAzyme sensor was proposed for sensing metal ions in complex biol. samples and live cells.
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    19. 19
      Ke, G.; Wang, C.; Ge, Y.; Zheng, N.; Zhu, Z.; Yang, C. J. L-DNA Molecular Beacon: A Safe, Stable, and Accurate Intracellular Nano-Thermometer for Temperature Sensing in Living Cells. J. Am. Chem. Soc. 2012, 134, 18908– 18911,  DOI: 10.1021/ja3082439
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      19
      L-DNA Molecular Beacon: A Safe, Stable, and Accurate Intracellular Nano-thermometer for Temperature Sensing in Living Cells
      Ke, Guoliang; Wang, Chunming; Ge, Yun; Zheng, Nanfeng; Zhu, Zhi; Yang, Chaoyong James
      Journal of the American Chemical Society (2012), 134 (46), 18908-18911CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Noninvasive and accurate measurement of intracellular temp. is of great significance in biol. and medicine. This paper describes a safe, stable, and accurate intracellular nano-thermometer based on an L-DNA mol. beacon (L-MB), a dual-labeled hairpin oligonucleotide built from the optical isomer of naturally occurring D-DNA. Relying on the temp.-responsive hairpin structure and the FRET signaling mechanism of MBs, the fluorescence of L-MBs is quenched below the melting temp. and enhanced with increasing temp. Because of the excellent reversibility and tunable response range, L-MBs are very suitable for temp. sensing. More importantly, the non-natural L-DNA backbone prevents the L-MBs from binding to cellular nucleic acids and proteins as well as from being digested by nucleases inside the cells, thus ensuring excellent stability and accuracy of the nano-thermometer in a complex cellular environment. The L-MB nano-thermometer was used for the photothermal study of Pd nanosheets in living cells, establishing the nano-thermometer as a useful tool for intracellular temp. measurement.
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    20. 20
      Kim, K.-R.; Lee, T.; Kim, B.-S.; Ahn, D.-R. Utilizing the Bioorthogonal Base-Pairing System of l-DNA to Design Ideal DNA Nanocarriers for Enhanced Delivery of Nucleic Acid Cargos. Chem. Sci. 2014, 5, 1533– 1537,  DOI: 10.1039/c3sc52601a
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      20
      Utilizing the bioorthogonal base-pairing system of l-DNA to design ideal DNA nanocarriers for enhanced delivery of nucleic acid cargos
      Kim, Kyoung-Ran; Lee, Taemin; Kim, Byeong-Su; Ahn, Dae-Ro
      Chemical Science (2014), 5 (4), 1533-1537CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      DNA nanoconstructs are a potential drug carrier with high biocompatibility. They are promising particularly when therapeutic nucleic acids are the cargos to be delivered, since both the carrier and the cargo are nucleic acids which can be designed, synthesized and assembled in a seamless feature without using post-synthetic conjugation chem. However, the unwanted base-paring events between the cargo and the carrier may potentially disturb the desired structure of the cargo-loaded carrier. To address this concern, we propose a DNA nanocarrier composed of l-DNA strands having a bioorthogonal base-paring system. The study presented here provides useful properties of l-DNA as a backbone for the DNA nanocarrier and demonstrates superiority of l-DNA over the natural d-DNA backbone in the delivery of an anti-proliferative aptamer as well as in the construction of the cargo-carrier assembly.
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    21. 21
      Phares, N.; White, R. J.; Plaxco, K. W. Improving the Stability and Sensing of Electrochemical Biosensors by Employing Trithiol-Anchoring Groups in a Six-Carbon Self-Assembled Monolayer. Anal. Chem. 2009, 81, 1095– 1100,  DOI: 10.1021/ac8021983
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      21
      Improving the Stability and Sensing of Electrochemical Biosensors by Employing Trithiol-Anchoring Groups in a Six-Carbon Self-Assembled Monolayer
      Phares, Noelle; White, Ryan J.; Plaxco, Kevin W.
      Analytical Chemistry (Washington, DC, United States) (2009), 81 (3), 1095-1100CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Alkane thiol self-assembled monolayers (SAMs) have seen widespread utility in the fabrication of electrochem. biosensors. Their utility, however, reflects a potentially significant compromise. While shorter SAMs support efficient electron transfer, they pack poorly and are thus relatively unstable. Longer SAMs are more stable but suffer from less efficient electron transfer, thus degrading sensor performance. Here the authors use the electrochem. DNA (E-DNA) sensor platform to compare the signaling and stability of biosensors fabricated using a short, six-carbon monothiol with those employing either of two com. available trihexylthiol anchors (a flexible Letsinger type and a rigid adamantane type). The authors find that all three anchors support efficient electron transfer and E-DNA signaling, with the gain, specificity, and selectivity of all three being effectively indistinguishable. The stabilities of the three anchors, however, vary significantly. Sensors anchored with the flexible trithiol exhibit enhanced stability, retaining 75% of their original signal and maintaining excellent signaling properties after 50 days storage in buffer. Likewise these sensors exhibit excellent temp. stability and robustness to electrochem. interrogation. The stability of sensors fabricated using the rigid trithiol anchor, by comparison, are similar to those of the monothiol, with both exhibiting significant (>60%) loss of signal upon wet storage or thermocycling. Employing a flexible trithiol anchor in the fabrication of SAM-based electrochem. biosensors may provide a means of improving sensor robustness without sacrificing electron transfer efficiency or otherwise impeding sensor performance.
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    22. 22
      Ricci, F.; Zari, N.; Caprio, F.; Recine, S.; Amine, A.; Moscone, D.; Palleschi, G.; Plaxco, K. W. Surface Chemistry Effects on the Performance of an Electrochemical DNA Sensor. Bioelectrochemistry 2009, 76, 208– 213,  DOI: 10.1016/j.bioelechem.2009.03.007
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      22
      Surface chemistry effects on the performance of an electrochemical DNA sensor
      Ricci, Francesco; Zari, Nadia; Caprio, Felice; Recine, Simona; Amine, Aziz; Moscone, Danila; Palleschi, Giuseppe; Plaxco, Kevin W.
      Bioelectrochemistry (2009), 76 (1-2), 208-213CODEN: BIOEFK; ISSN:1567-5394. (Elsevier B.V.)
      E-DNA sensors are a reagentless, electrochem. oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. The authors have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. The authors find that, among the thiols tested, the pos. charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, resp., the neg. charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chem. can lead to significant improvements in the performance of this broad class of electrochem. DNA sensors.
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    23. 23
      Reinstein, O.; Yoo, M.; Han, C.; Palmo, T.; Beckham, S. A.; Wilce, M. C. J.; Johnson, P. E. Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an off-Target Ligand. Biochemistry 2013, 52, 8652– 8662,  DOI: 10.1021/bi4010039
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      23
      Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand
      Reinstein, Oren; Yoo, Mina; Han, Chris; Palmo, Tsering; Beckham, Simone A.; Wilce, Matthew C. J.; Johnson, Philip E.
      Biochemistry (2013), 52 (48), 8652-8662CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The cocaine-binding aptamer is unusual in that it tightly binds mols. other than the ligand it was selected for. Here, the authors study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titrn. calorimetry was used to quantify the quinine-binding affinity and thermodn. of a set of sequence variants of the cocaine-binding aptamer. The authors find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more neg. change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle x-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.
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    24. 24
      Slavkovic, S.; Altunisik, M.; Reinstein, O.; Johnson, P. E. Structure-Affinity Relationship of the Cocaine-Binding Aptamer with Quinine Derivatives. Bioorg. Med. Chem. 2015, 23, 2593– 2597,  DOI: 10.1016/j.bmc.2015.02.052
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      24
      Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives
      Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E.
      Bioorganic & Medicinal Chemistry (2015), 23 (10), 2593-2597CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      In addn. to binding its target mol., cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titrn. calorimetry-based binding expts. to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic arom. ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliph. portion of quinine, quinuclidine, does not show detectable binding. Compds. with three fused arom. rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alc. or an amino group all result in lower binding affinity. For all ligands that bind, assocn. is driven by a neg. enthalpy compensated by unfavorable binding entropy.
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    25. 25
      Dey, S.; Sczepanski, J. T. In Vitro Selection of L-DNA Aptamers That Bind a Structured d-RNA Molecule. Nucleic Acids Res. 2020, 48, 1669– 1680,  DOI: 10.1093/nar/gkz1236
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      25
      In vitro selection of L-DNA aptamers that bind a structured D-RNA molecule
      Dey, Sougata; Sczepanski, Jonathan T.
      Nucleic Acids Research (2020), 48 (4), 1669-1680CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      The development of structure-specific RNA binding reagents remains a central challenge in RNA biochem. and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve L-RNA aptamers that bind tightly to structured D-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of L-DNA, which has several practical advantages compared to L-RNA, remains unknown. Here, we report the discovery and characterization of the first L-DNA aptamers against a structured RNA mol., precursor microRNA-155, thereby establishing the capacity of DNA and RNA mols. of the opposite handedness to form tight and specific 'cross-chiral' interactions with each other. L-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of L-DNA to interact with native D-RNA via Watson- Crick base pairing. Furthermore, L-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of L-DNA aptamers are distinct from previously reported L-RNA aptamers against pre-miR-155, indicating that L-DNA and L-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that L-DNA may be pursued as an alternative to L-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.
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    26. 26
      Feng, X.-N.; Cui, Y.-X.; Zhang, J.; Tang, A.-N.; Mao, H.-B.; Kong, D.-M. Chiral Interaction Is a Decisive Factor to Replace d-DNA with l-DNA Aptamers. Anal. Chem. 2020, 92, 6470– 6477,  DOI: 10.1021/acs.analchem.9b05676
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      26
      Chiral Interaction Is a Decisive Factor To Replace D-DNA with L-DNA Aptamers
      Feng, Xue-Nan; Cui, Yun-Xi; Zhang, Jing; Tang, An-Na; Mao, Han-Bin; Kong, De-Ming
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (9), 6470-6477CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Nucleic acid aptamers have been widely used in various fields such as biosensing, DNA chip, and medical diagnosis. However, the high susceptibility of nucleic acids to ubiquitous nucleases reduces the biostability of aptamers and limits their applications in biol. contexts. Therefore, improving the biostability of aptamers becomes an urgent need. Herein, the authors present a simple strategy to resolve this problem by directly replacing the D-DNA-based aptamers with left-handed L-DNA. By testing several reported aptamers against resp. targets, the authors found that the proposed strategy stood up well for nonchiral small mol. targets (e.g., Hemin and cationic porphyrin) and chiral targets whose interactions with aptamers are chirality-independent (e.g., ATP). The authors also found that the L-DNA aptamers were indeed endowed with greatly improved biostability due to the extraordinary resistance of L-DNA to nuclease digestion. With respect to other small-mol. targets whose interactions with aptamers are chirality-dependent (e.g., kanamycin) and biomacromols. (e.g., tyrosine kinase-7), however, the proposed strategy was not entirely effective likely due to the participation of the DNA backbone chirality into the target recognition. In spite of this limitation, this strategy indeed paves an easy way to screen highly biostable aptamers important for the applications in many fields.
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    27. 27
      Lovrić, M.; Komorsky-Lovric, Š. Square-Wave Voltammetry of an Adsorbed Reactant. J. Electroanal. Chem. 1988, 248, 239– 253,  DOI: 10.1016/0022-0728(88)85089-7
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      Square-wave voltammetry of an adsorbed reactant
      Lovric, Milivoj; Komorsky-Lovric, Sebojka
      Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1988), 248 (2), 239-53CODEN: JEIEBC; ISSN:0022-0728.
      Many electroanal. methods employ the adsorptive accumulation at the hanging Hg drop electrode combined with different stripping voltammetric techniques. One of the more sensitive techniques is square-wave voltammetry (SWV). The theory of SWV responses of adsorbed reactants is presented. The pronounced sensitivity of the SWV to quasi-reversible redox reactions from the adsorbed state is demonstrated. The relations between the properties of the SW response and the parameters of a charge transfer and of the excitation signal are discussed. The SW voltammograms of methylene blue are analyzed according to theor. predictions.
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    28. 28
      Komorsky-Lovrić, Š.; Lovrić, M. Measurements of Redox Kinetics of Adsorbed Azobenzene by “a Quasireversible Maximum” in Square-Wave Voltammetry. Electrochim. Acta 1995, 40, 1781– 1784,  DOI: 10.1016/0013-4686(95)00097-X
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      Measurements of redox kinetics of adsorbed azobenzene by "a quasireversible maximum" in square-wave voltammetry
      Komorsky-Lovric, Sebojka; Lovric, Milivoj
      Electrochimica Acta (1995), 40 (11), 1781-4CODEN: ELCAAV; ISSN:0013-4686. (Pergamon)
      The std. redox reaction rate consts. of azobenzene adsorbed to the mercury electrode are ks = 350 ± 25 s-1 in the acidic perchlorate medium (pH 2) and ks = 175 ± 20 s-1 in the acetate buffer pH 4.7. Under the influence of 0.2%, 0.5% and 1% acetonitrile in these electrolytes, the rate consts. are reduced to 265 ± 5 s-1, 160 ± 20 s-1 and 77 ± 10 s-1, at pH 2, and to 150 ± 5 s-1 and 56 ± 13 s-1 at pH 4.7.
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    29. 29
      Dauphin-Ducharme, P.; Plaxco, K. W. Maximizing the Signal Gain of Electrochemical-DNA Sensors. Anal. Chem. 2016, 88, 11654– 11662,  DOI: 10.1021/acs.analchem.6b03227
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      29
      Maximizing the Signal Gain of Electrochemical-DNA Sensors
      Dauphin-Ducharme, Philippe; Plaxco, Kevin W.
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (23), 11654-11662CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Electrochem. DNA (E-DNA) sensors have emerged as a promising class of biosensors capable of detecting a wide range of mol. analytes (nucleic acids, proteins, small mols., inorg. ions) without the need for exogenous reagents or wash steps. In these sensors a binding-induced conformational change in an electrode-bound "probe" (a target-binding nucleic acid or nucleic-acid-peptide chimera) alters the location of an attached redox reporter, leading to a change in electron transfer that is typically monitored using square-wave voltammetry. Because signaling in this class of sensors relies on binding-induced changes in electron transfer rate, the signal gain of such sensors (change in signal upon the addn. of satg. target) is dependent on the frequency of the square-wave potential pulse used to interrogate them, with the optimal square-wave frequency depending on the structure of the probe, the nature of the redox reporter, and other features of the sensor. Because it alters the driving force of the redox reaction and thus electron transfer kinetics, signal gain in this class of sensors is also strongly dependent on the amplitude of the square-wave potential pulse. Specifically, the authors show here that the simultaneous optimization of square-wave frequency and amplitude produces large (often more than two-fold) increases in the signal gain of a wide range of E-DNA-type sensors.
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    30. 30
      Ali, M. S.; Farah, M. A.; Al-Lohedan, H. A.; Al-Anazi, K. M. Comprehensive Exploration of the Anticancer Activities of Procaine and Its Binding with Calf Thymus DNA: A Multi Spectroscopic and Molecular Modelling Study. RSC Adv. 2018, 8, 9083– 9093,  DOI: 10.1039/c7ra13647a
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      Comprehensive exploration of the anticancer activities of procaine and its binding with calf thymus DNA: a multi spectroscopic and molecular modelling study
      Ali, Mohd. Sajid; Abul Farah, Mohammad; Al-Lohedan, Hamad A.; Al-Anazi, Khalid Mashay
      RSC Advances (2018), 8 (17), 9083-9093CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      Procaine is an anesthetic drug commonly administrated topically or i.v. for use in local anesthesia. Promisingly, some anticancer activities of procaine have also been reported. Therefore, the mechanism of interaction between anesthetic drug procaine with ct-DNA was detd. collectively by means of various spectroscopic and mol. docking methods. Minor groove 1 : 1 binding of procaine to the ct-DNA was evidenced from absorption spectroscopy, fluorescence quenching, DNA melting, competitive binding measurements with EB and DAPI dyes, viscosity and CD spectroscopy together with mol. docking simulations and DFT calcns. Mol. docking on five different B-DNA structures (taken from the Protein Data Bank) shows that procaine binds in the AT rich region of all five B-DNA structures. Thermodn. parameters, evaluated using van't Hoff's isotherm, shown that the interaction was feasible and the binding forces involved were hydrophobic as well as hydrogen bonding which were, further, confirmed by mol. docking. The frontier MOs (HOMO and LUMO) of procaine and DNA bases have been calcd. by DFT method and the chem. potential (μ), chem. hardness (η) and fraction no. of electrons (ΔN) from procaine to DNA bases were evaluated, which have shown that procaine acts as an electron donor to the DNA bases. Simultaneously, anticancer activities of procaine alone and in combination with doxorubicin were obsd. on the MCF-7 breast cancer cell line. The results showed that the combined treatment with both procaine and doxorubicin enhanced the cytotoxic and apoptotic inducing potential of doxorubicin.
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    31. 31
      Reddy, L. G.; Shankar, V. Immobilization of Single-Strand Specific Nuclease (S1 Nuclease) from Aspergillus Oryzae. Appl. Biochem. Biotechnol. 1987, 14, 231– 240,  DOI: 10.1007/BF02800310
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      Immobilization of single-strand specific nuclease (S1 nuclease) from Aspergillus oryzae
      Reddy, L. Gurucharan; Shankar, V.
      Applied Biochemistry and Biotechnology (1987), 14 (3), 231-40CODEN: ABIBDL; ISSN:0273-2289.
      S1 nuclease from A. oryzae (EC 3.1.30.1) was coupled to gelatin-alginate composite matrix by using the residual free aldehyde groups on the surface of glutaraldehyde-crosslinked matrix. The immobilized enzyme retained ∼10% activity of the sol. enzyme. When partially purified enzyme was bound to the matrix, the immobilized prepn. did not show any detectable enzyme activity. However, the activity could be restored when the coupling was carried out in the presence of a coprotein or substrate. The optimum pH of the immobilized S1 nuclease shifted to 3.8 from 4.3 for the sol. enzyme. Also, the optimum temp. increased to 65° after immobilization. Immobilized S1 nuclease showed increased pH and temp. stabilities. Immobilization brought about a 2-fold decrease in the Km.
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    32. 32
      Tamkovich, S. N.; Cherepanova, A. V.; Kolesnikova, E. V.; Rykova, E. Y.; Pyshnyi, D. V.; Vlassov, V. V.; Laktionov, P. P. Circulating DNA and DNase Activity in Human Blood. Ann. N.Y. Acad. Sci. 2006, 1075, 191– 196,  DOI: 10.1196/annals.1368.026
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      Circulating DNA and DNase activity in human blood
      Tamkovich, Svetlana N.; Cherepanova, Anna V.; Kolesnikova, Elena V.; Rykova, Elena Y.; Physhnyi, Dmitrii V.; Vlassov, Valentin V.; Laktionov, Pavel P.
      Annals of the New York Academy of Sciences (2006), 1075 (), 191-196CODEN: ANYAA9; ISSN:0077-8923. (Blackwell Publishing, Inc.)
      The concn. of circulating DNA (cirDNA) and DNase activity in blood plasma of healthy donors and patients with colon or stomach cancer were analyzed. The concn. of DNA was measured using Hoechst 33258 fluorescent assay after the isolation by the glass-milk protocol. A 1-kbp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers was used as a substrate for DNase. DNase activity was estd. from the data of immunochem. detection of the nonhydrolyzed amplicon. The av. concn. of cirDNA in the plasma of healthy donors was low (34±34 ng/mL), and was accompanied with high DNase activity (0.356±0.410 U/mL). The increased concns. of cirDNA in blood plasma of patients with colon and stomach cancer were accompanied by a decrease in DNase activity below the detection level of the assay. The data obtained demonstrate that low DNase activity in blood plasma of cancer patients can cause an increase in the concn. of cirDNA.
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    33. 33
      Ershova, E.; Sergeeva, V.; Klimenko, M.; Avetisova, K.; Klimenko, P.; Kostyuk, E.; Veiko, N.; Veiko, R.; Izevskaya, V.; Kutsev, S. Circulating Cell-Free DNA Concentration and DNase I Activity of Peripheral Blood Plasma Change in Case of Pregnancy with Intrauterine Growth Restriction Compared to Normal Pregnancy. Biomed. Rep. 2017, 7, 319– 324,  DOI: 10.3892/br.2017.968
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      Circulating cell-free DNA concentration and DNase I activity of peripheral blood plasma change in case of pregnancy with intrauterine growth restriction compared to normal pregnancy
      Ershova, Elizaveta; Sergeeva, Vasilina; Klimenko, Maria; Avetisova, Kristina; Klimenko, Peter; Kostyuk, Edmund; Veiko, Natalia; Veiko, Roman; Izevskaya, Vera; Kutsev, Sergey; Kostyuk, Svetlana
      Biomedical Reports (2017), 7 (4), 319-324CODEN: BREIB7; ISSN:2049-9442. (Spandidos Publications Ltd.)
      The level of apoptosis is increased during pregnancy. Dying cells emit DNA that remains in blood circulation and is known as cell free DNA (cfDNA). The concn. of cfDNA can reflect the level of cell death. The present article is the result of studying cfDNA concn. and DNase I activity in the blood plasma of 40 non pregnant women (control), 40 healthy pregnant women (over 37 wk) and 40 pregnant women with a diagnosis of intrauterine growth restriction (IUGR). In order to explain the obtained results, a program modeling the change of cfDNA concn. under the influence of different internal and external factors was written. It was reported that, despite the fact that the level of cell death is increased, cfDNA concn. in blood can be decreased due to activation of cfDNA elimination system. A significant increase of DNase I activity has been reported in cases of IUGR. Increase in DNase I activity over a certain threshold indicates presence of pathol. processes in the organism. CfDNA circulating in blood cannot be a reliable marker of increased cell death during pregnancy. Thus, assessment of the level of cell death during pregnancy should be done by simultaneous anal. of cfDNA level and DNase I activity.
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      Zhong, W.; Sczepanski, J. T. Direct Comparison of D-DNA and L-DNA Strand-Displacement Reactions in Living Mammalian Cells. ACS Synth. Biol. 2020, 10, 209– 212,  DOI: 10.1021/acssynbio.0c00527
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      Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. How Strong Is a Covalent Bond?. Science 1999, 283, 1727– 1730,  DOI: 10.1126/science.283.5408.1727
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      How strong is a covalent bond?
      Grandbois, Michel; Beyer, Martin; Rief, Matthias; Clausen-Schaumann, Hauke; Gaub, Hermann E.
      Science (Washington, D. C.) (1999), 283 (5408), 1727-1730CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The rupture force of single covalent bonds under an external load was measured with an at. force microscope (AFM). Single polysaccharide mols. were covalently anchored between a surface and an AFM tip and then stretched until they became detached. By using different surface chemistries for the attachment, it was found that the silicon-carbon bond ruptured at 2.0 ± 0.3 nanonewtons, whereas the sulfur-gold anchor ruptured at 1.4 ± 0.3 nanonewtons at force-loading rates of 10 nanonewtons per s. Bond rupture probability calcns. that were based on d. functional theory corroborate the measured values.
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      Xue, Y.; Li, X.; Li, H.; Zhang, W. Quantifying Thiol-Gold Interactions towards the Efficient Strength Control. Nat. Commun. 2014, 5, 4348,  DOI: 10.1038/ncomms5348
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      Quantifying thiol-gold interactions towards the efficient strength control
      Xue, Yurui; Li, Xun; Li, Hongbin; Zhang, Wenke
      Nature Communications (2014), 5 (), 4348CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      The strength of the thiol-gold interactions provides the basis to fabricate robust self-assembled monolayers for diverse applications. Investigation on the stability of thiol-gold interactions has thus become a hot topic. Here we use at. force microscopy to quantify the stability of individual thiol-gold contacts formed both by isolated single thiols and in self-assembled monolayers on gold surface. Our results show that the oxidized gold surface can enhance greatly the stability of gold-thiol contacts. In addn., the shift of binding modes from a coordinate bond to a covalent bond with the change in environmental pH and interaction time has been obsd. exptl. Furthermore, isolated thiol-gold contact is found to be more stable than that in self-assembled monolayers. Our findings revealed mechanisms to control the strength of thiol-gold contacts and will help guide the design of thiol-gold contacts for a variety of practical applications.
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      Chen, H.; Xie, S.; Liang, H.; Wu, C.; Cui, L.; Huan, S.-Y.; Zhang, X. Generation of Biostable L-Aptamers against Achiral Targets by Chiral Inversion of Existing D-Aptamers. Talanta 2017, 164, 662– 667,  DOI: 10.1016/j.talanta.2016.11.001
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      Generation of Biostable L-aptamers against Achiral Targets by Chiral Inversion of Existing D-aptamers
      Chen, Huapei; Xie, Sitao; Liang, Hao; Wu, Cuichen; Cui, Liang; Huan, Shuang-Yan; Zhang, Xiaobing
      Talanta (2017), 164 (), 662-667CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      Based on reciprocal chiral substrate specificity, taking achiral mols., ethanolamine (EA) and malachite green (MG) as two model targets, biostable L-DNA aptamers and L-RNA aptamers were generated resp. by chiral inversion of existing D-aptamers. In the detection of EA with L-DNA aptamer-based sensors, the feasibility of the authors' strategy was confirmed, while in the detection of MG with L-RNA aptamers, linear calibration curves were obtained at 0.1-5 μm with the detection limit of 0.065 μm under optimized exptl. conditions. The mirror-image L-aptamers have identical recognition capability as D-aptamers. Meanwhile, L-aptamers have superior biostability to resist nuclease digestion, protein binding interference and off-target effects, enabling their applications in complex practical samples, such as lake water and fish tissue extns. The authors' work provides a simple, yet universal and efficient way to develop biostable aptamers.
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    38. 38
      White, R. J.; Phares, N.; Lubin, A. A.; Xiao, Y.; Plaxco, K. W. Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry. Langmuir 2008, 24, 10513– 10518,  DOI: 10.1021/la800801v
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      38
      Optimization of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing Density and Surface Chemistry
      White, Ryan J.; Phares, Noelle; Lubin, Arica A.; Xiao, Yi; Plaxco, Kevin W.
      Langmuir (2008), 24 (18), 10513-10518CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Electrochem., aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. To further improve this technol. the authors have systematically studied the effects of probe (aptamer) packing d., the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small mol. cocaine and the protein thrombin. The authors find that, by controlling the concn. of aptamer employed during sensor fabrication, the authors can control the d. of probe DNA mols. on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with max. gain obsd. near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is obsd. at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, the authors find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher abs. sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the obsd. signal gain of the authors' cocaine sensor 10-fold. The authors demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.
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    39. 39
      Arroyo-Currás, N.; Dauphin-Ducharme, P.; Scida, K.; Chávez, J. L. From the Beaker to the Body: Translational Challenges for Electrochemical, Aptamer-Based Sensors. Anal. Methods 2020, 12, 1288– 1310,  DOI: 10.1039/d0ay00026d
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      Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Self-Assembled Monolayers That Resist the Adsorption of Proteins and the Adhesion of Bacterial and Mammalian Cells. Langmuir 2001, 17, 6336– 6343,  DOI: 10.1021/la010552a
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      40
      Self-assembled monolayers that resist the adsorption of proteins and the adhesion of bacterial and mammalian cells
      Ostuni, Emanuele; Chapman, Robert G.; Liang, Michael N.; Meluleni, Gloria; Pier, Gerald; Ingber, Donald E.; Whitesides, George M.
      Langmuir (2001), 17 (20), 6336-6343CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      This paper examines the hypothesis that surfaces resistant to protein adsorption should also be resistant to the adhesion of bacteria (Staphylococcus aureus, Staphylococcus epidermidis) and the attachment and spreading of mammalian cells (bovine capillary endothelial (BCE) cells). The surfaces tested were those of self-assembled monolayers (SAMs) terminated with derivs. of tri(sarcosine) (Sarc), N-acetylpiperazine, permethylated sorbitol, hexamethylphosphoramide, phosphoryl choline, and an intramol. zwitterion (-CH2N+(CH3)2CH2CH2CH2SO3-) (ZW); all are known to resist the adsorption of proteins. There seems to be little or no correlation between the adsorption of protein (fibrinogen and lysozyme) and the adhesion of cells. Surfaces terminated with derivs. of Sarc and N-acetylpiperazine resisted the adhesion of S. aureus and S. epidermidis as well as did surfaces terminated with tri(ethylene glycol). A surface that presented Sarc groups was the only one that resisted the adhesion of BCE cells as well as did a surface terminated with tri(ethylene glycol). The attachment of BCE cells to surfaces could be patterned using SAMs terminated with derivs. of Sarc, N-acetylpiperazine, phosphoramide, and the ZW as the attachment-resistant component and methyl-terminated SAMs as the adhesive component.
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      Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Adsorption of Proteins to Hydrophobic Sites on Mixed Self-Assembled Monolayers. Langmuir 2003, 19, 1861– 1872,  DOI: 10.1021/la020649c
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      41
      Adsorption of proteins to hydrophobic sites on mixed self-assembled monolayers
      Ostuni, Emanuele; Grzybowski, Bartosz A.; Mrksich, Milan; Roberts, Carmichael S.; Whitesides, George M.
      Langmuir (2003), 19 (5), 1861-1872CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      This paper describes a technique that uses mixed self-assembled monolayers of two alkanethiolates (-S(CH2)11(OCH2CH2)6OR, R = a hydrophobic group, and -S(CH2)11(OCH2CH2)nOH, n = 3, 6, EGnOH), in combination with surface plasmon resonance spectroscopy, to study the influence of the size and shape of R, and its d. at the surface, on the hydrophobic adsorption of proteins at solid-liq. interfaces. Detailed results were obtained for β-galactosidase, carbonic anhydrase, lysozyme, and RNase A using R = C(C6H5)3, CH(C6H5)2, and CH2(C6H5). A hard-sphere model is used to rationalize the adsorption; this model, although very approx., helps to interpret qual. trends in the data. Using this model, the extent to which adsorbed proteins undergo conformational rearrangements appears to depend on the d. of the hydrophobic groups at the surface and on the concn. of protein in soln. This paper describes the first step toward the development of a system that will allow the study of hydrophobic interactions of proteins with surfaces presenting org. groups of well-defined shape.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjvFeksg%253D%253D&md5=314907dcf6f3eb27079d6ce25fa83d27
    42. 42
      Curtis, S. D.; Ploense, K. L.; Kurnik, M.; Ortega, G.; Parolo, C.; Kippin, T. E.; Plaxco, K. W.; Arroyo-Currás, N. Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data. Anal. Chem. 2019, 91, 12321– 12328,  DOI: 10.1021/acs.analchem.9b02553
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      42
      Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data
      Curtis, Samuel D.; Ploense, Kyle L.; Kurnik, Martin; Ortega, Gabriel; Parolo, Claudio; Kippin, Tod E.; Plaxco, Kevin W.; Arroyo-Curras, Netzahualcoyotl
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (19), 12321-12328CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Electrochem. sensors are major players in the race for improved mol. diagnostics due to their convenience, temporal resoln., manufg. scalability, and their ability to support real-time measurements. This is evident in the ever-increasing no. of health-related electrochem. sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data vols. the authors describe here an open-source, easily customizable, multi-platform compatible program for the real-time control, processing and visualization of electrochem. data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which the authors have called Software for the Anal. and Continuous Monitoring of Electrochem. Systems (SACMES), also includes a graphical interface allowing the user to easily change anal. parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES the authors use it here to analyze the real-time data output by: (1) the electrochem., aptamer-based measurement of a specific small-mol. target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the detn. of the folding thermodn. of an electrode-attached, redox-reporter-modified protein.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1KksL%252FI&md5=8a2fb45b4fd84748c8c38c8754c1db12

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.1c00166.

    • Aptamer secondary structure and target chemical structures, quasi-reversible maximum maps for hydroxyquinoline and quinine, deconvoluted mass spectrum of the synthesiszed l-cocaine aptamer, and deconvoluted mass spectrum of the synthesiszed d-cocaine aptamer (PDF)


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