Submolecular-scale Hairpin DNA Folding Dynamics Studied by High-Speed AFM with Optical Tweezers

Optical tweezers have contributed to elucidate the folding mechanisms associated with biomolecules. By combining single-molecule or super-resolution techniques, imaging can also be performed while measuring or inducing force coupling with biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this problem, here, we developed a technique that combines optical tweezers with high-speed atomic force microscopy (AFM). To solve the problem of incompatible instrumental configurations, we developed a unique optical tweezers measurement system that is specialized for high-speed AFM. Upon applying an external force to a synthesized DNA secondary structure, we successfully visualized the dissociation of the duplex structure. Furthermore, we succeeded in observing spontaneous reannealing of the duplex structure upon releasing the force, which demonstrates that the folding reaction can be reversibly controlled. We also reveal that along with duplex unfolding, a metastable secondary structure is generated and its topology changes transiently over time. The results indicate that this technique provides structural insights that cannot be obtained by conventional fluorescence techniques.