FIGURE 4. Unwinding during overstretching, and the global force–torque phase diagram of DNA.

a, Unwinding during overstretching. Constructs were pulled to forces (black trace) above 65 pN. Cumulative rotations (red) of a 400 nm rotor bead showed continuous constant-torque untwisting while tension was maintained at 85 pN (Supplementary Movie 4). Concurrent decrease in twist (red) and increase in extension (blue) reflect S-DNA formation. When the force was relaxed to 15 pN, the molecule rewound, returning to B-form twist and extension. b, The global force–torque phase diagram of DNA. A single molecule was unwound by pulling to successively higher forces (relaxing at 15 pN between each run) to measure the torque of the B–S transition (judged from _{B S}) as a function of force (red). The critical torque of the P–B transition was also measured as a function of force by relaxation of overwound tethers under varying tensions (green), and all critical torque data (orange: _crit,- = 9.6 pN nm) were found to agree with a simple model¹⁰ in which DNA can access five distinct structural states differing only in extension, twist, and free energy. scP, P-DNA that has been shortened by supercoiling^6,7.