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

Abstract

The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed K d values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 μM, respectively, and similar K d values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.

Capture‐SELEX for a short aptamer for label‐free detection of salicylic acid

“…Upon target binding, a fraction of the ThT is displaced and decreased fluorescence is observed. Thioflavin T has been used in many non‐G‐quadruplex aptamers [32,33] . We mixed 1 μM SA1 aptamer and 2 μM ThT and then gradually titrated SA.…”

“…Thioflavin T has been used in many non-G-quadruplex aptamers. [32,33] We mixed 1 μM SA1 aptamer and 2 μM ThT and then gradually titrated SA. A gradual drop in the ThT fluorescence at 490 nm was observed, and eventually more than 50% fluorescence drop was achieved (Figure 2d, black trace).…”

Cross‐Binding of Adenosine by Aptamers Selected Using Theophylline

“…In another study, we found that two of adenosine binding aptamers can also bind theophylline and caffeine. [15] Given the physiological relationship between theophylline, caffeine, and adenosine in the regulation of sleep and their binding to the same protein receptors, our discovery may imply that nucleic acid versions of receptors can also bind to both molecules. While it is unknown that this point whether primitive organisms might have used RNA to regulate their biological processes such as sleep, nucleic acids certainly have the chemical capacity to perform this task.…”

“…We then tried a label-free fluorescent assay using thioflavin T (ThT) (Figure 3A). [15,[30][31] Using 100 nM Theo2201 aptamer and 2 μM ThT dye, we monitored the fluorescence of ThT at 490 nm. As shown in Figure 3B, a drop in the ThT fluorescence was observed upon titration of 100 μM theophylline.…”

“…We recently reported that the classic DNA aptamer for adenosine can also bind to theophylline and caffeine. [15] In the aptamer field, [9,11,[16][17] the theophylline RNA aptamer reported by Jenison et al in 1994 is probably the most mentioned example to articulate the high selectivity of aptamers, since its binding to caffeine was 10,000-fold lower even though these two molecules only differ by a single methyl group. [18][19] We recently reported a DNA aptamer for theophylline that showed even higher selectivity over caffeine.…”