Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Abstract

RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a forcetorque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed "P-RNA," and a highly underwound, left-handed state denoted "L-RNA." Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA-protein interactions at the single-molecule level.RNA | nucleic acids | magnetic tweezers | force | torque

Understanding the mechanical response of double-stranded DNA and RNA under constant stretching forces using all-atom molecular dynamics

“…Direct evidence of the interplay between mechanical stress and biological activity has been provided via single-molecule experiments (13,14). Indeed, techniques such as optical and magnetic tweezers (1,13,14) have emerged as essential tools in the characterization of the mechanical properties of dsDNA (1) and, most recently, dsRNA (5,6,15).…”

“…The analysis of single-molecule force extension data with a continuum mechanics approach, the so-called elastic rod model (17,18), reveals two particularly striking results: the threefold softer stretching response of dsRNA compared with dsDNA (6) and the opposite sign of the twist-stretch coupling (2,3,5,19). Ad hoc model extensions, such as the addition of an outer helical wire (2), and several models at the base-pair level (5,20) have been proposed to rationalize some of these results.…”

A Guide to Magnetic Tweezers and Their Applications

“…The slope of the hat curves, about 40 nm per turn, can significantly increase at low stretching force [80,81]. The critical level of DNA supercoiling required to extrude the plectoneme increases with increasing tension.…”

“…Production of labeled RNA molecules has been achieved with the help of in vitro transcription [52,81]. To this end, dsDNA templates were constructed that give rise to complementary RNA strands with unique leader sequences to facilitate further cloning.…”

“…The DNA was then cut elsewhere and ligated to digoxigenin-and biotin-labeled DNA handles to produce a molecule with a bubble [101]. (C) Production of dsRNA [81]. End labeled ssRNA substrates were prepared using in vitro transcription.…”