High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy

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.