Direct Observation of Activated Kinetics and Downhill Dynamics in DNA Dehybridization

We have studied two model DNA oligonucleotide sequences that display contrasting degrees of heterogeneous melting using an optical temperature jump to trigger dehybridization and a nonlinear infrared (IR) spectroscopy probe to track the response of the helix ensemble. This approach offers base-sensitive structural insight through the unique vibrational fingerprint characteristic of each nucleobase as well as time resolution capable of following unfolding across nanoseconds to milliseconds. We observe predissociation unzipping of the helical termini, loss of final dimer contacts, and rehybridization of the dissociated strands all in a single measurement. Complete dissociation of the dimer is found to be well described by Arrhenius kinetics for both sequences, with dissociation barriers in the range of 160-190 kJ/mol. A sequence with terminal adenine-thymine (AT) base pairs and a guanine-cytosine core returns a large-amplitude fast response ranging from 70 to 170 ns, originating only from the AT base pairs. Variable temperature jump (T-jump) experiments in which the final temperature (T-f) is fixed and the initial temperature (T-i) is varied such that different starting ensembles all evolve on the same final free-energy surface were employed to explore the features of the underlying potential that dictates hybridization. These experiments reveal that the unzipping of the AT termini is an essentially barrierless process and that both activated and downhill events are necessary to describe the dehybridization mechanism. Although our results are largely consistent with the classic nucleation-zipper picture, new insights regarding the nature of base pair zippering refine the mechanistic details of the fastest DNA hybridization dynamics.