Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues.

The parasitic nature of the SARS-CoV-2 virus demands selective packaging of its RNA genome (gRNA) from the abundance of other nucleic acids present in infected cells. Despite increasing evidence that stem-loop 4 (SL4) of the gRNA 5' UTR is involved in the initiation of this process by binding the nucleocapsid (N) protein, little is known about its conformational dynamics. Here, we unravel the stability, dynamics and (un)folding pathways of SL4 using optical tweezers and a base analogue, tCO , that provides a local and subtle increase in base stacking without perturbing hydrogen bonding. We find that SL4 (un)folds mainly in a single step or through an intermediate, encompassing nucleotides from the central U bulge to the hairpin loop. Due to an upper-stem CU mismatch, SL4 is prone to misfold, the extent of which can be tuned by incorporating tCO at different positions. Our study contributes to a better understanding of SARS-CoV-2 packaging and the design of drugs targeting SL4. We also highlight the generalizability of using base analogues in optical tweezers experiments for probing intramolecular states and conformational transitions of various nucleic acids at the level of single molecules and with base-pair resolution.