Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 ± 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 ± 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.