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 tweezer 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°/(°C·kbp), compared to ΔTwDNA = -11.0 ± 1.2°/(°C·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 the 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 parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure.

Department of Informatics and Chemistry University of Chemistry and Technology Prague Technická 5 166 28 Praha 6 Czech Republic

Department of Physics and Astronomy and LaserLaB Amsterdam Vrije Universiteit Amsterdam De Boelelaan 1081 Amsterdam 1081 HV The Netherlands

Junior Research Group 2 Interdisciplinary Center for Clinical Research Friedrich Alexander University Erlangen Nürnberg Cauerstr 3 Erlangen 91058 Germany

Soft Condensed Matter and Biophysics Department of Physics and Debye Institute Utrecht University Utrecht 3584 CC The Netherlands

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Comprehensive Assessment of Force-Field Performance in Molecular Dynamics Simulations of DNA/RNA Hybrid Duplexes