Cross-Binding of Adenosine by Aptamers Selected Using Theophylline

We recently reported that some adenosine binding aptamers can also bind caffeine and theophylline with around 20-fold lower affinities. This discovery led to the current work to examine the cross-binding of adenosine to theophylline aptamers. For the DNA aptamer for theophylline, cross-binding to adenosine was observed, and the affinity was 18 to 38-fold lower for adenosine based on assays using isothermal titration calorimetry and ThT fluorescence spectroscopy. The binding complexes were characterized using NMR spectroscopy, and both adenosine and theophylline showed an overall similar binding structure to the DNA theophylline aptamer, although small differences were also observed. In contrast, the RNA aptamer did not show binding to adenosine, although both aptamers have very similar relative selectivity for various methylxanthines including caffeine. After a negative selection, a few new aptamers with completely different primary sequences for theophylline were obtained and they did not show binding to adenosine. Thus, there are many ways for aptamers to bind theophylline and some can have cross-binding to adenosine. In biology, theophylline, caffeine, and adenosine can bind to the same protein receptors to regulate sleep, and their binding to the same DNA motifs may suggest an early role of nucleic acids in similar regulatory functions. The newly selected DNA aptamer for theophylline can also bind adenosine, whereas the classical RNA aptamer cannot. Theophylline and adenosine are related molecules in regulating sleep, suggesting that nucleic acids also have the chemical basis for a similar function.+image

Automated summary

This study reveals that some adenosine binding DNA aptamers can also bind caffeine and theophylline, albeit with lower affinities. The affinity of adenosine to theophylline DNA aptamer was found to be significantly lower compared to theophylline. NMR spectroscopy analysis showed similar binding structures for both adenosine and theophylline to the DNA theophylline aptamer, suggesting possible cross-binding. However, the RNA aptamer did not exhibit binding to adenosine. These findings highlight the diverse binding mechanisms of aptamers to theophylline and the potential role of nucleic acids in regulatory functions.