DNA and RNA Dynamics: Probing Conformational Shifts with Single-Molecule FRET

Nucleic acids, which contain the essential code for life, are not static structures and a full understanding of their thermodynamic and kinetic properties is vital to understanding their biological functions. Ensemble measurements can give some insight into the impact of environmental variation on these systems but to extract precise kinetic and thermodynamic properties a single molecule approach is required. Single molecule Förster Resonance Energy Transfer (smFRET) was therefore employed to quantify the structure of two key nucleic acid systems. First the opening and closing dynamics of DNA hairpins were studied. These hairpins, formed of a doubled stranded stem and a single stranded loop appear in a range of biological processes and are used in bionanotechnological applications. pH, ionic strength, molecular crowding and temperature were all seen to be regulators of hairpin conformation. The impact of varying the loop composition and length was also investigated, showing changes to dynamics. The hairpin’s temperature dependence was exploited to extract entropic and enthalpic contributions to closing and entropic factors were seen to play a role in the loop length variation observed. Similar approaches were then applied to the SARS-CoV-2 RNA frameshifting element. A key feature of SARS-CoV-2 and many other viruses is -1 programmed ribosomal frameshifting. Essential for replication, it relies on the presence of a slippery sequence and stimulatory RNA secondary structure. This work demonstrates multiple strategies to doubly-label the SARS-Cov-2 RNA frameshifting element with a FRET pair, and evaluates the molecule’s conformation in solution, to reveal that the molecule exists in several environment-sensitive conformations, suggesting flexibility beyond the predicted structures. Overall, this work provides insights into the environmental sensitivity of nucleic acid structures, with direct implications for both fundamental biophysics and bionanotechnological applications. Therefore, contributing to the design of efficient synthetic nucleic acid systems and enhancing understanding of a potential anti-viral target.