Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore

Abstract

Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozen-hydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system.