DNA folding and melting observed in real time redefine the energy landscape.

We report real-time observations of the folding and melting of DNA by probing two active sites of a hairpin structure, the bases and the stem end, and using an ultrafast T-jump. Studies at different initial temperatures (before, during, and after melting) provide the time scale of water heating (<20 ps), single-strand destacking (700 ps to 2 ns), and hairpin destacking (microseconds and longer) in solutions of various ionic strengths and pH values. The behavior of transient changes gives direct evidence to the existence of intermediate collapsed structures, labile in destacking but compact in nature, and indicates that melting is not a two-state process. We propose a landscape that is defined by these nucleation structures and destacking for efficient folding and melting.