Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity

Abstract

The classical DNA aptamer for adenosine and ATP has been the most used small molecule binding aptamer for biosensing, imaging, and DNA nanotechnology. This sequence has recurred multiple times in previous aptamer selections, and all previous selections used a high concentration of ATP as the target. Herein, two separate selections were performed using adenosine and ATP as targets. By pushing the target concentrations down to the low micromolar range, two new aptamers with K d as low as 230 nM were obtained, showing around 30-fold higher affinity compared to the classical aptamer. The classical aptamer sequence still dominated the library in the early rounds of the selections, but it was suppressed in the later rounds. The new aptamers bind to one target molecule instead of two. Mutation studies confirmed their secondary structures and specific binding. Using the deep sequencing data from the selections, long-standing questions such as the existence of one-site aptamers and mutation distribution in the classical aptamer were addressed. Comparisons were made with previously reported DNA aptamers for ATP. Finally, a strand-displacement biosensor was tested showing selectivity for adenosine and its nucleotides.

Capture-SELEX for Chloramphenicol Binding Aptamers for Labeled and Label-Free Fluorescence Sensing

“…A K d of 9.8 μM was quite reasonable for a molecule like CAP containing a single aromatic ring and a few hydrogen bond donors/acceptors. For comparison, the classical adenosine aptamer has a K d of around 6 μM. , We reason that reports of K d values down to 0.03 μM CAP might not be from aptamer binding to CAP but from some other events. − …”

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines

“…The buffer contained 50 mM NaCl and 4 mM MgCl 2 . Panel (A) adapted from ref with permission. Copyright 2023 American Chemical Society.…”

“…We recently selected two new DNA aptamers, both with around 200 nM affinity for adenosine . An interesting question is whether they can bind to methylxanthines.…”

“…The structure of the Ade1304 aptamer is shown in Figure B . We then tested Ade1304 using the ThT method, and adenosine caused a 90% fluorescence drop with a K d of 0.99 μM.…”

“…By immobilizing a DNA library that can fold to a hairpin structure, − we recently used adenosine and ATP as targets and also observed this classical aptamer especially in the early rounds of the selections when the target concentrations were high . In the late rounds, when the target concentrations were dropped to as low as 1 μM, this sequence was suppressed and two higher affinity aptamers ( K d ∼ 200 nM adenosine) dominated.…”