Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins.

Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively.

Probing microscopic conformational dynamics in folding reactions by measuring transition paths.

Transition paths comprise those parts of a folding trajectory where the molecule passes through the high-energy transition states separating folded and unfolded conformations. The transition states determine the folding kinetics and mechanism but are difficult to observe because of their brief duration. Single-molecule experiments have in recent years begun to characterize transition paths in folding reactions, allowing the microscopic conformational dynamics that occur as a molecule traverses the energy barriers to be probed directly.

Measuring the average shape of transition paths during the folding of a single biological molecule.

Transition paths represent the parts of a reaction where the energy barrier separating products and reactants is crossed. They are essential to understanding reaction mechanisms, yet many of their properties remain unstudied. Here, we report measurements of the average shape of transition paths, studying the folding of DNA hairpins as a model system for folding reactions.

Transition-path properties for folding reactions in the limit of small barriers.

Transition paths are of great interest because they encapsulate information about the mechanisms of barrier-crossing reactions. Analysis of experiments measuring biomolecular folding reactions has relied on expressions for properties of transition paths such as transition-path times and velocities that hold in the limit of large harmonic barriers, but real molecules often have relatively small barriers. Recent theoretical work presented more general expressions for transition-path properties.

Measuring the Local Velocity along Transition Paths during the Folding of Single Biological Molecules.

Transition paths are the most interesting part of folding reactions but remain little studied. We measured the local velocity along transition paths in DNA hairpin folding using optical tweezers. The velocity distribution agreed well with diffusive theories, yielding the diffusion coefficient.

Testing Kinetic Identities Involving Transition-Path Properties Using Single-Molecule Folding Trajectories.

Recent advances in single-molecule assays have allowed individual transition paths during the folding of single molecules to be observed directly. We used the transition paths of DNA hairpins having different sequences, measured with high-resolution optical tweezers, to test theoretical relations between the properties of the transition paths and the folding kinetics. We showed that folding and unfolding rates were related to the average transition-path times, as expected from theory, for all hairpins studied.