Allosteric Regulation of Aptamer Affinity through Mechano-Chemical Coupling.

Abstract

The capacity to precisely modulate aptamer affinity is important for a wide variety of applications. However, most such engineering strategies entail laborious trial-and-error testing or require prior knowledge of an aptamer's structure and ligand-binding domain. We describe here a simple and generalizable strategy for allosteric modulation of aptamer affinity by employing a double-stranded molecular clamp that destabilizes aptamer secondary structure through mechanical tension. We demonstrate the effectiveness of the approach with a thrombin-binding aptamer and show that we can alter its affinity by as much as 65-fold. We also show that this modulation can be rendered reversible by introducing a restriction enzyme cleavage site into the molecular clamp domain and describe a design strategy for achieving even more finely-tuned affinity modulation. This strategy requires no prior knowledge of the aptamer's structure and binding mechanism and should thus be generalizable across aptamers.