A DNA Scaffold Approach Facilitates 5′ Labeling of the SARS‐CoV‐2 RNA Pseudoknot for Single‐Molecule Förster Resonance Energy Transfer Investigation

Single-molecule Förster resonance energy transfer (smFRET) studies of highly structured RNA molecules are often frustrated by issues with efficient dye conjugation. Here, a DNA scaffold-based labeling strategy is developed, and applied to the frameshift-stimulating RNA pseudoknot from the SARS-CoV-2 genome. FRET-active reporters were prepared containing both Cy3 (donor) and Cy5 (acceptor) molecules and measurements conducted on freely-diffusing single molecules, enabling the evaluation of conformational heterogeneity via smFRET population distributions. Freely diffusing pseudoknots, modified at the base of stem 1, display a broad range of NaCl-dependent FRET states in solution, consistent with conformational freedom that extends beyond the static X-ray and cryo-EM structures. This work is a proof-of-principle demonstration of the feasibility of our DNA scaffold approach in enabling smFRET studies on this important class of biomolecule. Together, this work outlines new biochemical and biophysical approaches toward the study of RNA conformational dynamics in pseudoknots, riboswitches, and other structured RNA elements.