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

Abstract

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.

Single-molecule junction spontaneously restored by DNA zipper

“…These junctions break at the interface between the constituent molecule and the electrode, because the bonding between the functional group of the molecule and the electrode, e.g., Au–S bond, is weakest in the junction 46 . It has been reported that the force required to break the Au–S or Au–Au bond is 1–2 nN 47 , while the DNA unzipping takes place under the force of 10–50 pN 22 – 24 . Thus, in the present junctions, the breakdown is attributed to be via DNA unzipping.…”

“…Sequencing analysis of DNA at the single-base resolution can be performed with nanopore devices during the constrained passage 18 – 21 . Also, precise control of DNA structures by atomic force microscopy (AFM) has enabled accurate mapping of DNA unzipping dynamics and a free-energy landscape 22 – 24 . This combination of electrical measurements and structural modulation of DNA could lead to the realization of the sophisticated functionality of electronic devices based on a single DNA molecule.…”

Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

“…Further theoretical treatments of this problem have been developed to reconstruct the entire one dimensional free-energy landscape from SMFS data (Rhee and Pande, 2005;Woodside and Block, 2014). By deconvoluting instrument effects (Walder et al, 2018b), such reconstruction approaches have been validated on DNA hairpins (Gupta et al, 2011) and proteins (Yu et al, 2012) and found agreement between various single-molecule manipulation techniques (Woodside and Block, 2014;Manuel et al, 2015). A full coverage of theoretical work covering this problem is, however, beyond the scope of this work.…”

Unveiling the druggable RNA targets and small molecule therapeutics

“…translation patterns, localization and stability. [166][167][168] A desirable feature of such compounds is that they interact with RNA not through intercalation, sequence, or electrostatic complementarity, but by means of specific molecular recognition events unique to the particular RNA target. The use of small molecules as RNA-specific drug-like compounds has increased due to their frequently desirable physicochemical characteristics, including their good hydrophobicity and selectivity, as well as cell and tissue permeability.…”