Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstret ched, and the mechanical stability of the DNA double helix was investigated . In lambda-phage DNA the previously reported B-S transition at 65 piconewt ons (pN) is followed by a second conformational transition, during which th e DNA double helix melts into two single strands. Unlike the B-S transition , the melting transition exhibits a pronounced force-loading-rate dependenc e and a marked hysteresis, characteristic of a nonequilibrium conformationa l transition. The kinetics of force-induced melting of the double helix, it s reannealing kinetics, as well as the influence of ionic strength, tempera ture, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabiliz ed under low salt buffer conditions (less than or equal to 10 mM NaCl), whi le high ionic strength buffers(1 M NaCl) stabilize the double-helical confo rmation. The mechanical energy that can be deposited in the DNA double heli x before force induced melting occurs was found to decrease with increasing temperature. This energy correlates with the base-pairing free enthalpy De lta G(bp)(T) of DNA. Experiments with pure poly(dG-dC) and poly(dA-dT) DNA sequences again revealed a close correlation between the mechanical energie s at which these sequences melt with base pairing free enthalpies Delta G(b p)(sequence): while the melting transition occurs between 65 and 200 pN in lambda-phage DNA, depending on the loading rate, the melting transition is shifted to similar to 300 pN for poly(dG-dC) DNA, whereas poly(dA-dT) DNA m elts at a force of 35 pN.