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Direct measurement of sequence-dependent transition path times and conformational diffusion in DNA duplex formation

Significance The transition path time in folding reactions—the time required to cross through the high-energy transition states—offers a powerful tool for studying folding mechanisms because it is uniquely sensitive to the microscopic motions along the transition paths that dominate the folding dynamics. We used transition path times to probe helix formation in nucleic acids and elucidate differences between the formation of canonical G:C and A:T base pairs. Stem-loop hairpins of the same size but increased G:C content had faster transition path times, revealing that the conformational diffusion coefficient determining the timescale for molecular dynamics was higher for formation of G:C base pairs than A:T base pairs. Such sequence dependence, not previously observed, reflects differences in the internal friction for reconfiguring the bases. The conformational diffusion coefficient, D, sets the timescale for microscopic structural changes during folding transitions in biomolecules like nucleic acids and proteins. D encodes significant information about the folding dynamics such as the roughness of the energy landscape governing the folding and the level of internal friction in the molecule, but it is challenging to measure. The most sensitive measure of D is the time required to cross the energy barrier that dominates folding kinetics, known as the transition path time. To investigate the sequence dependence of D in DNA duplex formation, we measured individual transition paths from equilibrium folding trajectories of single DNA hairpins held under tension in high-resolution optical tweezers. Studying hairpins with the same helix length but with G:C base-pair content varying from 0 to 100%, we determined both the average time to cross the transition paths, τtp, and the distribution of individual transit times, PTP(t). We then estimated D from both τtp and PTP(t) from theories assuming one-dimensional diffusive motion over a harmonic barrier. τtp decreased roughly linearly with the G:C content of the hairpin helix, being 50% longer for hairpins with only A:T base pairs than for those with only G:C base pairs. Conversely, D increased linearly with helix G:C content, roughly doubling as the G:C content increased from 0 to 100%. These results reveal that G:C base pairs form faster than A:T base pairs because of faster conformational diffusion, possibly reflecting lower torsional barriers, and demonstrate the power of transition path measurements for elucidating the microscopic determinants of folding.