Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.

Under a tension of ~65 pN, double-stranded (ds) DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10-25°C). The shortening transient following a 20-35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses due to reannealing of melted segments. The lengthening transients following a 2-35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is 1/3 of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the inter-strand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition.