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. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy–landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding.

生物分子折叠涉及在天然构象的无数可能性中进行搜索，但从未直接检测到从理论中预期的这种搜索的基本步骤。我们使用光学镊子探测了高分辨率的折叠动力学，测量了核酸发夹通过支配动力学和定义折叠机制的高能过渡态时的单个轨迹。我们观察到过渡态的短暂但无处不在的停顿，其停留时间分布与定量折叠的微观理论相匹配。序列依赖性表明，在每个核苷酸残基搜索天然碱基配对构象期间，停顿主要由来自非天然构象的微屏障控制。此外，停顿取决于位置，揭示能量景观粗糙度的细微局部变化，并允许在不重建能量景观形状的情况下找到描述屏障内微观动力学的扩散系数。这些结果表明高分辨率测量如何阐明折叠过程中的关键微观事件，以测试折叠的基本理论。