Temperature-dependent twist of double-stranded RNA probed by magnetic tweezers experiments and molecular dynamics simulations

Abstract

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions and the structure of double-stranded RNA (dsRNA) has been revealed at high-resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezers measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = −14.4 +/- 0.7 deg/(degC.kbp), compared to ΔTwDNA = −11.0 +/- 1.2 deg/(degC.kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature, but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA-RNA hybrid duplexes, reveal a linear correlation between twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested, or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parameterizations for RNA simulations. They may also inspire physical models of temperature-dependent dsRNA structure.

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step

“…Applying a low number of positive (negative) rotations overwinds (underwinds) and twists the DNA. Beyond a buckling point( 34 ), twist is converted into writhe which absorbs the applied turns into plectonemic supercoils, resulting in the shortening of the DNA end-to-end extension ( 29, 30, 32, 35 ). Monitoring the DNA end-to-end extension versus positive and negative rotations thus generates a rotation curve which is symmetric around zero rotations (Δ L k = 0) at low forces (≤ 0.6 pN, i.e., where DNA remains in its B-form ( 29, 30, 32, 35 )) (Fig.…”

“…With the objective to probe the degree of DNA twist induced by SMCs during single DNA loop extrusion steps, we turned to magnetic tweezers as this single-molecule technique has been proven to be able to resolve both single loop-extrusion steps of yeast condensin and human cohesion (27,28), as well as minute changes in DNA twist (29)(30)(31)(32). Torsionally constrained 3.6 kbp-long dsDNA molecules were tethered between a superparamagnetic 1 µm bead and an antidigoxygenin-functionalized glass surface via handles on the end of DNA molecules containing either multiple biotin or digoxygenin labels, respectively (Methods).…”