Single-molecule insights into SAM-I riboswitch ligand binding using scaled magnetic force spectroscopy with comparison to conventional biophysical methods

Abstract

ABSTRACT Biophysical methods are widely used for drug discovery, with surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) being amongst the most used techniques. However, these types of bulk or ensemble methods do not provide single-molecule resolution or structural and mechanistic insights, so that researchers are forced to rely upon a battery of complementary techniques. Here, we used a scaled magnetic force spectroscopy (MFS) instrument system capable of analysing hundreds of single molecules in parallel and obtaining dynamic conformational insights. Taking the S-adenosylmethionine-I (SAM-I) riboswitch as a model system, we demonstrated that MFS can be used to characterize the RNA structure in conjunction with the published crystal structures. Next, we compared the performance of MFS to SPR and ITC for measuring binding affinities of two ligands with highly different affinities, SAM and SAH in both high and low magnesium concentrations. MFS gave similar binding affinities to SPR and ITC but was more closely aligned to ITC. We then demonstrated the power of MFS to study the molecular dynamics of SAM-I and the mechanisms of SAM and S-adenosylhomocysteine (SAH) binding. By subjecting the RNA to constant forces for extended periods, we were able to observe that the conformation dynamics of SAM-I are impacted differently by SAM and SAH binding, pointing to different stabilization mechanisms between the two ligands.