The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.
Conclusion: Acute knockdown of desmin in isolated adult rat ventricular myocytes reveals previously unidentified structural and functional roles of this IF. Further study must be done to identify how desmin upregulation affects disease and other roles desmin may play in mechanotransduction.
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.
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