Extended Data Fig. 5: SMFS of the C22G mutant.

a, Roughly 15% of unfolding FECs (black) show low forces consistent with secondary structure only, in state I₁′, which is akin to I₁ (Supplementary Fig. 2b) but with shorter P2 and longer P3. Dashed lines show WLC fits to each state, inset shows structure before unfolding. b, Roughly 17% of unfolding FECs (black) show a moderately high-force state I^m1 consistent with partial formation of 5′-TC and the non-native pseudoknot PK′ but without any 5′-end threading. Refolding FECs (red) show a pathway similar to I₁ (Supplementary Fig. 3a), although with shorter P2 and longer P3, indicating I^m1 derives from I₁′. c, Roughly 15% of unfolding FECs (black) show the same extreme stability as for the wild-type ring-knot, indicating this mutant forms a ring-knot analogous to the wild-type knot, with full 5′-TC and extreme mechanical resistance. d, Roughly 53% of unfolding (black) and refolding FECs (red) show low forces consistent with state I₁″ (inset), akin to I₁, containing secondary structure only but with lengthened P2. e, Proposed structures of the states identified in (a-d). Base-pairing of G22 with C44 prevents native 5′-TC but allows I₁′–I₄′ and I^m1 (with shorter P2 and longer P3) to form. Alternatively, base-pairing of G22 with C9 lengthens P2 but leaves C44 accessible for native 5′-TC formation, allowing formation of mutant states N^m1 and I_r^m1 containing a mechanically resistant ring-knot, in addition to intermediates I₁″, I₂″, and I₄″. Contour length changes expected from unfolding each structure are listed in Supplementary Table 4.