Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level

Abstract

Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force–extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.

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

“…S2B) and an overstretching transition for torsionally unconstrained molecules (SI Appendix, Fig. S2C), in agreement with previous single-molecule studies (16,17). From fits of the extensible WLC model, we found S RNA = 350 ± 100 pN, about threefold lower than S DNA (SI Appendix, Fig.…”

“…S1G and Table S1). Our value for the S RNA is in reasonable agreement with, although slightly lower than, the value of S RNA ∼500 pN determined in single-molecule optical tweezers measurements (25), possibly due to subtle differences between magnetic and optical tweezers experiments. For torsionally unconstrained molecules, the overstretching transition is marked by a rapid increase in extension to 1.8 ± 0.1 times the crystallographic length over a narrow force range at F = 54 ± 5 pN (SI Appendix, Fig.…”

“…From fits of the WLC model, we determined the contour length L C = 1.15 ± 0.02 μm and the bending persistence length A RNA = 57 ± 2 nm in the presence of 100 mM monovalent salt (SI Appendix, Fig. S2A), in good agreement with the expected length (1.16 μm, assuming 0.28 nm per bp) (22,23) and previous single-molecule stretching experiments (15,16). A RNA decreases with increasing ionic strength (16) (SI Appendix, Fig.…”

“…1A). Although recent single-molecule stretching experiments using torsionally unconstrained dsRNA have revealed its bending persistence length (15,16), stretch modulus (16), and an overstretching transition (16,17), its response to torsional strains and structural transitions under forces and torques is unknown. This dearth of information on dsRNA is partially due to the relative difficulty, compared with dsDNA, of assembling RNA constructs suitable for single-molecule force and torque measurements.…”

Mechanical unfolding of long human telomeric RNA (TERRA)

“…2b). Single molecules were stretched with OT to forces exceeding the overstretching transition of the hybrid handles, 25,26 following the scheme of Fig. 2a.…”