Step size of the first unfolding transition. (a–c) When KC30 RNA was unfolded at constant forces, the first transitions were unkiss alone, double transition, or triple transition. (b) At ≈16 pN, after the double transition, extension of the molecule hopped frequently, indicating the reversible unfolding of hairpin 2. (c) Similarly, hairpin 1 hopped at 17.7 pN once the triple transition occurred. (d) ΔX of the first unfolding transition as a function of force. Each value represents at least 100 observations. ▴, ●, and ■ represent unkiss alone, double transition, or triple transition, respectively. Dashed curves are ΔX calculated by using the worm-like-chain interpolation formula (20). In these calculations, the persistence and contour length of a single strand are 1 nm and 0.59 nm per nucleotide, respectively; and the contour length of each base pair in the hairpin was assumed to be 0.3 nm.