Probing the conformational dynamics of the frameshift-stimulatory pseudoknot of SARS-CoV-2 at the single-molecule level.

The virus causing the COVID-19 pandemic, SARS-CoV-2, uses a process called -1 programmed ribosomal frameshifting (-1 PRF) to express proteins essential for viral replication. -1 PRF involves a change in the way the viral genome is read that is triggered by a specific structure in the viral genome: a pseudoknot. Changing the level of -1 PRF in viruses can suppress infectivity, suggesting that the SARS-CoV-2 pseudoknot may be a good target for developing new drugs against the coronavirus. It is therefore essential to understand what features of the pseudoknot are important for its ability to stimulate -1 PRF. Work on other viruses has shown that specific interactions within pseudoknots play a key role, as do the dynamics of the structure under tension during -1 PRF. I will build on my previous published work on -1 PRF in SARS-CoV-2 showing that it can be inhibited by small-molecule drugs, aiming to study systematically the mechanisms by which the pseudoknot stimulates -1 PRF. Making mutations that knock out specific interactions within the pseudoknot, one, by one, I will measure both the functional effects these mutations have on -1 PRF, using enzymatic assays, and also the changes they cause in the structural dynamics of the pseudoknot under tension, using high-precision optical tweezers to apply force to single molecules. These studies will allow me to relate specific changes in the structure and dynamics of the RNA to their functional outcomes, and thereby map out the features of the pseudoknot that are essential to -1 PRF. I will also repeat these measurements in the presence of small molecules that inhibit -1 PRF, to deduce the modes of action by which different inhibitors act to change the -1 PRF level and determine which are most effective functionally. This work will provide crucial insights that can be used to target RNA pseudoknots for developing drugs against COVID-19 and possibly other viral diseases involving -1 PRF.