Label-Free, High-Time-Resolution, Single-Molecule Studies Of Riboswitch Folding

Riboswitches are genetic control elements that are located within the 5’ untranslated region of predominantly bacterial mRNAs and that undergo metabolite-dependent structural rearrangements that regulate mRNA transcription, splicing, translation, or stability in response to specific metabolites. A molecular-resolution understanding of the mechanisms through which riboswitches fold, specifically recognize and bind their target metabolites, and subsequently influence cellular processes is essential for development of next-generation antibiotics that target riboswitches and for engineering riboswitches for use in synthetic biology applications. Motivated by this, previous studies using single-molecule fluorescence and force spectroscopy methods have provided insights into the metabolite-responsive folding and function of several riboswitches. Despite the power of these methods, however, they suffer from intrinsic disadvantages (e.g., fluorophore labeling, invasive forces, etc.) that can corrupt and/or restrict the mechanistic information that they provide. Additionally, the resolution of these methods is generally limited to tens-to-hundreds of milliseconds, resulting in time-averaging of RNA dynamics that occur on faster timescales. To overcome these limitations, we have developed carbon nanotube-based, single-molecule field-effect transistors (smFETs) that enable label-free, high-time-resolution, single-molecule studies of RNA folding and dynamics. Using these novel smFET devices, we have investigated the dynamics of the pbuE adenine-responsive riboswitch at the single-molecule level and a time resolution of tens of microseconds. Our results show that the adenine-free riboswitch exists in a conformational equilibrium between at least four states, some of which exchange on timescales too fast to have been previously observed. Using a combination of adenine, adenine analogs, and riboswitch mutants, we have detected the formation of an initial, highly transient riboswitch-metabolite complex whose evolution to the final, stably bound complex we are currently investigating. Taken together with previous studies, our results are providing unprecedented insights into the structure, dynamics, and function of this paradigmatic metabolite-responsive riboswitch.