Probing the Response of Double-Stranded RNA to Force and Torque at the Single-Molecule Level

Double-stranded RNA (dsRNA) plays a number of roles in biological processes in which it often encounters mechanical strain; examples include the packaging of dsRNA viral genomes, deformations of the ribosome during translation, and conformational changes of RNAs while folding or due to interactions with proteins. While the response of dsDNA to applied forces and torques has been measured with exquisite precision, much less is known about dsRNA.We have developed a labeling method that allows us to generate fully double-stranded RNA constructs carrying multiple biotin and digoxigenin labels at opposite ends. Using the functionalized dsRNA constructs in a range of a complementary magnetic tweezers assays [1,2,3], we have probed the elastic properties of dsRNA and determined force and torque induced structural transitions that go beyond linear response behavior.From the force-extension response, we have determined the bending persistence length and the stretch (or Young's) modulus of dsRNA and find values overall similar to dsDNA. Employing our novel magnetic torque tweezers assays [1], we have probed the torsional response of dsRNA and again find a behavior that is generally similar to dsDNA. Surprisingly, measurements of the twist-stretch coupling reveal a striking difference between dsRNA and dsDNA. While DNA lengthens when overwound, RNA shortens. In addition, we have studied the dynamics of the buckling transition and discovered that the characteristic time scale of the transition is about two orders of magnitude slower for RNA than for DNA.We expect that these measurements of the fundamental properties of dsRNA can help refine our models for twist-storing polymers and inform quantitative models of RNA function in vivo.[1] Lipfert, et al. Nature Methods (2010)[2] Lipfert, Wiggin, et al., Nature Communications (2011)[3] Janssen, Lipfert, et al., Nano Lett. (2012)