New Obligate Folding Intermediate of an RNA Pseudoknot Observed Using Atomic Force Microscopy Based Force Spectroscopy with 10 μs Resolution

Abstract

Programmed À1 frameshifting (À1 PRF) is an important biological process for the modification of gene expression enabled by the presence of RNA secondary and tertiary structures. However, the mechanism for this process is still under active study. Here, we investigate the role of mechanical force in À1 PRF by characterizing mechanically induced folding and unfolding of RNA pseudoknots using an enhanced atomic force microscopy (AFM) based singlemolecule force spectroscopy (SMFS) assay featuring $10 ms resolution. The pioneering SMFS study of RNA psuedoknots associated with À1 PRF used a custom-built optical trap. Unexpectedly, this study indicated PRF efficiency was correlated with the presence of alternative folding pathways (rather than with average unfolding force), indicating a complex role of RNA psuedoknots in À1 PRF involving folding dynamics. Here, we found a new folding intermediate in an RNA pseudoknot associated with the sugarcane yellow leaf virus (ScYLV) that was not observed in the original optical-trapping based assay. We speculate that the shorter linkers and stiffer force probe in our AFM assay (relative to an optical trap) are the primary reasons for this enhanced state resolution. Our initial measurements of contour length and folding kinetics indicate this folding intermediate is an RNA hairpin that is part of the overall pseudoknot structure. We observed this intermediate every time the pseudoknot folds, indicating that this intermediate is obligate for folding. Overall, our results indicate that the folding dynamics of RNA pseudoknots are significantly more complex than previously observed. Because of the role of the folding dynamics of RNA pseudoknots in À1 PRF, we expect these new insights into RNA pseudoknot folding dynamics will provide a deeper understanding into the mechanisms of À1 PRF.