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Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil. [ABSTRACT FROM AUTHOR]

Electrochemical aptamer‐based (E‐AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode‐bound, redox‐reporter‐modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding‐induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance‐dependent change in electron transfer rates in E‐AB sensors. In response, we investigate here the signal transduction of the well‐studied cocaine‐binding aptamer with different analytical methods and found that this sensor relies on a redox‐reporter ‐ ligand competition mechanism rather than a ligand‐induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine‐binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine‐binding aptamer, given the prevalence of methylene blue in E‐AB sensors, a similar competition‐based may occur in other systems. [ABSTRACT FROM AUTHOR]

Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART. [ABSTRACT FROM AUTHOR]

Intrinsic fluorescence analysis is a sensitive technique to gain insight about aptamer-small molecule interactions. Employing fluorescence properties of the ligand, the binding affinities of the aptamer-ligand complex can be quantified in the nM to mM range with great accuracy and precision. Here, we present a detection method for aptamer-ligand binding analysis that is based on the inherent fluorescence of the ligand. Further, we discuss how to optimize and resolve some common experimental challenges. [ABSTRACT FROM AUTHOR]

Aptamers were first described in 1990 and since then many aptamers have been reported in the literature for numerous applications in both diagnostics and therapeutics. However, as with most fields, missing or unclear information presented in the publication makes it difficult to replicate some of the work described in the literature. To increase the reproducibility of the data and facilitate academic laboratories and industrial companies to develop reliable aptamer work, essential guidelines should be proposed and followed in any aptamer publication, especially in those that highlight de novo aptamer sequences. Here, we provide suggestions for authors, reviewers, and editors to follow when performing and reporting their aptamer work to ensure that we meet the minimum standards for publication of future aptamer sequences. [ABSTRACT FROM AUTHOR]

A bifunctional cocaine and deoxycholic acid-binding aptamer was constructed from the individual ligand binding aptamers and the binding affinity and thermodynamics were measured using isothermal titration calorimetry. We show that the bifunctional aptamer binds its ligands with positive cooperativity, having a Hill coefficient of 1.2 -1.5, whether the ligands are added individually or as an equimolar mixture. When the helix between the binding sites is decreased from eight to six base pairs the Hill coefficient increased indicating a greater degree of cooperativity. Reducing the distance between the binding sites to four base pairs resulted in the bifunctional aptamer displaying negative cooperativity as the two sites are now too close to function efficiently. A mechanism where molecular motion, or dynamics, at one ligand-binding site are affected by the presence of the ligand at the other is proposed to account for the observed cooperative binding. [ABSTRACT FROM AUTHOR]

Nuclear Magnetic Resonance (NMR) Spectroscopy is a powerful technique for studying aptamer structure and function. Important information about ligand binding and how binding affects aptamer structure can be obtained relatively quickly using ¹H NMR spectra focused on the imino resonances. Here we summarise some practical information for preparing aptamer samples for NMR analysis, designing experiments, and interpreting results, with an emphasis on aptamer-small molecule interactions. [ABSTRACT FROM AUTHOR]

Isothermal titration calorimetry (ITC) is a powerful technique for studying binding interactions. From a single ITC experiment it is possible to quantify the affinity and thermodynamics of a binding event. Here, we outline an experimental approach for performing ITC experiments with a focus on aptamer-small molecule interactions. We also discuss some common problems that can be encountered and how to resolve these issues. [ABSTRACT FROM AUTHOR]

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The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods. This aptamer follows a structure-switching binding mechanism and is unusual in that it binds two copies of its ligand. We have used isothermal titration calorimetry methods to study the binding of ATP, ADP, AMP and adenosine to the ATP-binding aptamer. Using both individual and global fitting methods, we show that this aptamer follows a positive cooperative binding mechanism. We have determined the binding affinity and thermodynamics for both ligand-binding sites. By separating the ligand-binding sites by an additional four base pairs, we engineered a variant of this aptamer that binds two adenosine ligands in an independent manner. Together with NMR and thermal stability experiments, these data indicate that the ATP-binding DNA aptamer follows a population-shift binding mechanism that is the source of the positive binding cooperativity by the aptamer. [ABSTRACT FROM AUTHOR]

Aptamers are short oligonucleotides capable of binding specifically to various targets (i.e. , small molecules, proteins, and whole cells) which have been introduced in biosensors such as in the electrochemical aptamer-based (E-AB) sensing platform. E-AB sensors are comprised of a redox-reporter-modified aptamer attached to an electrode that undergoes, upon target addition, a binding-induced change in electron transfer rates. To date, E-AB sensors have faced a limitation in the translatability of aptamers into the sensing platform presumably because sequences obtained from Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are typically long (>80 nucleotides) and that obtaining structural information remains time and resource consuming. In response, we explore the utility of aptamer base truncations and in silico docking to improve their translatability into E-AB sensors. Here, we first apply this to the glucose aptamer, which we characterize in solution using NMR methods to guide design and translate truncated variants in E-AB biosensors. We further investigated the applicability of the truncation and computational approaches to four other aptamer systems (vancomycin, cocaine, methotrexate and theophylline) from which we derived functional E-AB sensors. We foresee that our strategy will increase the success rate of translating aptamers into sensing platforms to afford low-cost measurements of molecules directly in undiluted complex matrices. [Display omitted] [ABSTRACT FROM AUTHOR]

We have developed a new cocaine-binding aptamer variant that has a significantly higher melt temperature when bound to a ligand than the currently used sequence. Retained in this new construct is the ligand-induced structure-switching binding mechanism that is important in biosensing applications of the cocaine-binding aptamer. Isothermal titration calorimetry methods show that the binding affinity of this new sequence is slightly tighter than the existing cocaine-binding aptamer. The improved thermal performance, a T m increase of 4 °C for the cocaine-bound aptamer and 9 °C for the quinine-bound aptamer, was achieved by optimizing the DNA sequence in stem 2 of the aptamer to have the highest stability based on the nearest neighbor thermodynamic parameters and confirmed by UV and fluorescence spectroscopy. The sequences in stem 1 and stem 3 were unchanged in order to retain the structure switching and ligand binding functions. The more favorable thermal stability characteristics of the OR3 aptamer should make it a useful construct for sensing applications employing the cocaine-binding aptamer system. [ABSTRACT FROM AUTHOR]

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method’s ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. [ABSTRACT FROM AUTHOR]

Fluorescent dyes that bind specifically to DNA aptamers with turn-on emission serve as powerful tools for biosensor development. DNA three-way junctions (3WJs) consisting of a Y-shaped hydrophobic branch point connecting three double-stranded stems serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. Herein, we highlight the tunability of phenolic hemicyanine dyes for host-guest fluorescent aptasensor applications using the cocaine-binding aptamer (MN4) as a representative 3WJ-aptamer substrate. The DNA aptamer MN4 exhibits ligand binding promiscuity and binds the alkaloid quinine with ∼26-fold greater affinity than its intended cocaine target. Combining the cationic N -methylbenzothiazolium (Btz) acceptor with a 2-fluoro-phenolic (FPhO) donor through a vinyl-linkage creates the FPhOBtz hemicyanine as a universal fluorescent turn-on probe for DNA 3WJs. The FPhOBtz dye binds MN4 with higher affinity than cocaine, but with weaker affinity than quinine. Consequently, FPhOBtz displacement from the 3WJ with turn-off fluorescence is effectively mediated by quinine, but not cocaine, resulting in poor detection of cocaine by the dye-displacement assay. Our current results demonstrate superior cocaine detection by replacing the planar cationic Btz group with the non-planar indolenium (Ind) acceptor to afford FPhOInd as the signaling fluorescent probe. This simple change in dye structure strongly diminishes MN4 binding affinity (19-fold) with cocaine now possessing stronger affinity for the 3WJ of MN4 compared to FPhOInd. Consequently, FPhOInd displacement from MN4 is effectively mediated by cocaine to provide a 13-fold improvement in cocaine detection in undiluted urine using the host-guest biosensing platform. [Display omitted] • Phenolic hemicyanine dyes serve as universal DNA three-way junction fluorescent light-up probes. • They can be used in host-guest biosening through target mediated dye displacement. • Phenolic hemicyanines are tunable through synthetic manipulation to alter their affinity for the DNA three-way junction. • Tunability affords a greater range of targets to be assayed using host-guest biosensing. [ABSTRACT FROM AUTHOR]

Uncoupling the protein-protein interaction between collapsin response mediator protein 2 (CRMP2) and N-type voltage-gated calcium channel (CaV2.2) with an allosteric CRMP2-derived peptide (CBD3) is antinociceptive in rodent models of inflammatory and neuropathic pain. We investigated the efficacy, duration of action, abuse potential, and neurobehavioral toxicity of an improved mutant CRMP2 peptide. A homopolyarginine (R9)-conjugated CBD3-A6K (R9-CBD3-A6K) peptide inhibited the CaV2.2-CRMP2 interaction in a concentration-dependent fashion and diminished surface expression of CaV2.2 and depolarization-evoked Ca influx in rat dorsal root ganglia neurons. In vitro studies demonstrated suppression of excitability of small-to-medium diameter dorsal root ganglion and inhibition of subtypes of voltage-gated Ca channels. Sprague-Dawley rats with tibial nerve injury had profound and long-lasting tactile allodynia and ongoing pain. Immediate administration of R9-CBD3-A6K produced enhanced dopamine release from the nucleus accumbens shell selectively in injured animals, consistent with relief of ongoing pain. R9-CBD3-A6K, when administered repeatedly into the central nervous system ventricles of naive rats, did not result in a positive conditioned place preference demonstrating a lack of abusive liability. Continuous subcutaneous infusion of R9-CBD3-A6K over a 24- to 72-hour period reversed tactile allodynia and ongoing pain, demonstrating a lack of tolerance over this time course. Importantly, continuous infusion of R9-CBD3-A6K did not affect motor activity, anxiety, depression, or memory and learning. Collectively, these results validate the potential therapeutic significance of targeting the CaV-CRMP2 axis for treatment of neuropathic pain. [ABSTRACT FROM AUTHOR]

In addition to binding its target molecule, 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 titration calorimetry-based binding experiments 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 aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic 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 alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy. [ABSTRACT FROM AUTHOR]