Templating effect in DNA proximity ligation enables use of non-bioorthogonal chemistry in biological fluids

Spiropulos, Nicholas G.; Heemstra, Jennifer M.

doi:10.4161/adna.23842

Cited by 10 publications

(9 citation statements)

References 31 publications

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“…The addition of ligand reduced the k ex of imino protons close to the binding site, but it has little effect on resonances distant from the binding site. For example, G29 is located at the binding site of the aptamer, and its k ex value reduces from (50 5 6) s À1 in the free state of MN4 at 10 C to 15 When comparing quinine-and cocaine-bound MN4, we and others have shown that quinine binds $50-fold tighter than cocaine (15,23,25,27,(47)(48)(49). When looking at the k ex values of quinine-and cocaine-bound MN4, we generally observe little difference between these two bound aptamers at low temperatures.…”

Section: Discussionmentioning

confidence: 95%

Reduction in Dynamics of Base pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer

Churcher

Garaev

Hunter

et al. 2020

Biophysical Journal

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We have used magnetization transfer NMR experiments to measure the exchange rate constant (k ex ) of the imino protons in the unbound, cocaine-bound, and quinine-bound forms of the cocaine-binding DNA aptamer. Both long-stem 1 (MN4) and short-stem 1 (MN19) variants were analyzed, corresponding to structures with a prefolded secondary structure and ligandinduced-folding versions of this aptamer, respectively. The k ex values were measured as a function of temperature from 5 to 45 C to determine the thermodynamics of the base pair opening for MN4. We find that the base pairs close to the ligand-binding site become stronger upon ligand binding, whereas those located away from the binding site do not strengthen. With the buffer conditions used in this study, we observe imino 1 H signals in MN19 not previously seen, which leads us to conclude that in the free form, both stem 2 and parts of stem 3 are formed and that the base pairs in stem 1 become structured or more rigid upon binding. This is consistent with the k ex values for MN19 decreasing in both stem 1 and at the ligand-binding site. Based on the temperature dependence of the k ex values, we find that MN19 is more dynamic than MN4 in the free and both ligand-bound forms. For MN4, ligand-binding results in the reduction of dynamics that are localized to the binding site. These results demonstrate that an aptamer in which the base pairs are preformed also experiences a reduction in dynamics with ligand binding.

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Section: Discussionmentioning

confidence: 95%

Reduction in Dynamics of Base pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer

Churcher

Garaev

Hunter

et al. 2020

Biophysical Journal

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“…One feature of the cocaine-binding aptamer that sets it apart from other aptamers is its binding promiscuity. Though selected for cocaine binding, this aptamer also binds other alkaloids and even binds quinine with higher affinity than it binds cocaine. − Such versatility in ligand binding is uncommon among aptamers and is not a desirable characteristic for a biosensor, but it may be a useful property for the biotechnology applications of the cocaine-binding aptamer. The binding versatility of this aptamer leads to questions such as “How does the same sequence bind to different ligands?” and “Does the aptamer use the same binding site and binding mechanism for all ligands?” In this study, we investigate the interaction of the cocaine-binding aptamer with quinine.…”

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confidence: 99%

Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand

et al. 2013

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The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We 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 negative 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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“…Most aptasensors are designed to take advantage of the binding mechanism of the aptameric probe to generate a signal from target binding. − The majority of cocaine aptasensors reported in the literature rely on one of the structure-switching aptamer constructs for this reason. ,,− Our goal in the design of EMPAS-based aptasensors has always been to try and provide more universality in a sensing platform that would function with not only structure-switching aptamers but also rigid secondary structure variants. The main difference between our two EMPAS-based aptasensors, M6CA and M4CA, is the nature of the immobilized aptameric probe (Figure A).…”

Section: Discussionmentioning

confidence: 99%

“…Another structure-switching mechanism consists in splitting the aptamer into two or more strands that will come together in the presence of cocaine, inducing secondary structure formation and tertiary folding in the process. This mechanism is referred to as “split aptamer ligation” and is the foundation upon which most cocaine aptasensors are based. ,,− As evident with the cocaine aptamer example, there is a need for a more universal transduction technique that will function with different aptamer binding mechanisms.…”

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confidence: 99%

Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine

Neves

Blaszykowski²,

Thompson

2016

Anal. Chem.

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Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 μM and a 0.3 μM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the possibility to distinguish between the binding mechanisms of structure-switching (MN6) vs rigid (MN4) aptameric probes, an ability that could allow the EMPAS to provide a more universal aptasensing platform than what is ordinarily observed in the literature.

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Templating effect in DNA proximity ligation enables use of non-bioorthogonal chemistry in biological fluids

Cited by 10 publications

References 31 publications

Reduction in Dynamics of Base pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer

Reduction in Dynamics of Base pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer

Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand

Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine

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