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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]