Real-Time Sub-Molecular Visualization of DNA Folding Using a Hybrid High-Speed AFM and Optical Tweezers System

Optical tweezers have contributed to elucidating the folding mechanisms associated with biomolecules. By combining them with fluorescence imaging techniques, imaging can also be performed while measuring or applying forces that couple to biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this limitation, here, we developed a hybrid high-speed atomic force microscopy (HS-AFM) and optical tweezers system. To resolve the challenge of incompatible instrumental configurations, we designed a customized optical tweezers system optimized for HS-AFM. Using this platform, we applied external forces to synthetic DNA secondary structures and directly visualized duplex dissociation and spontaneous reannealing upon force release, demonstrating reversible control of folding. We also captured reversible DNA overstretching and transient secondary structure formation in ssDNA. These findings were further analyzed using molecular dynamics simulations and viscoelastic modeling. This integrated approach provides a powerful platform for investigating folding dynamics and force-coupled mechanisms in biomolecules.