Mechanical Stability of Single DNA Molecules

AbstractUsing a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In λ-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (≤10mM NaCl), while high ionic strength buffers (1M NaCl) stabilize the double-helical conformation. The mechanical energy that can be deposited in the DNA double helix before force induced melting occurs was found to decrease with increasing temperature. This energy correlates with the base-pairing free enthalpy ΔGbp(T) of DNA. Experiments with pure poly(dG-dC) and poly(dA-dT) DNA sequences again revealed a close correlation between the mechanical energies at which these sequences melt with base pairing free enthalpies ΔGbp(sequence): while the melting transition occurs between 65 and 200 pN in λ-phage DNA, depending on the loading rate, the melting transition is shifted to ∼300pN for poly(dG-dC) DNA, whereas poly(dA-dT) DNA melts at a force of 35pN