RNA Folding and Unfolding Under Force: Single-Molecule Experiments and Their Analysis

We have previously described (Geffroy et al. Methods Mol Biol 1665:25-40, 2018) how to unfold (or fold) a single RNA molecule under force using a dual-beam optical trap setup. In this chapter, we complementarily describe how to analyze the corresponding data and how to interpret it in terms of RNA three-dimensional structure. As with all single-molecule methods, single RNA molecule force data often exhibit several discrete states where state-to-state transitions are blurred in a noisy signal. In order to cope with this limitation, we have implemented a novel strategy to analyze the data, which uses a hidden Markov modeling procedure. A representative example of such an analysis is presented.