RNA Translocation through Protein Nanopores: Unfolding of Secondary and Tertiary Structures

The translocation of RNA is greatly influenced by secondary and tertiary structures, making it challenging to track the structural changes during translation. We open up a new simulation framework that can capture the properties of the multi-level structures and the dynamical translocation of RNA. Our method is based on a combination of oxRNA model for RNA and Poisson–Nernst–Plank calculation for the electric field in protein nanopores. Through systemtic studies of the translocation behavior in three protein nanopores: α-hemolysin, CsgG, and MspA, three featured stages (pseudoknot, melting, and molten globule) have been explicitly identified based on the contact map and current traces. Further classification of the two translocation modes (fast and slow modes) based on the translocation time leads to the discovery of the following new physical phenomena: (1) In the fast mode, the average translocation speed is found to be independent of the nanopore geometry. Instead, it is primarily controlled by the electrical potential difference between the trans and cis sides of the nanopore. (2) In the slow mode, the molten globule stage is the key factor in slowing down the translocation of the hairpin RNA instead of the melting of the base pairs. Finally, we find that the electric field distribution is mainly responsible for the molten globule and not the geometry of the nanopore. These results provide a fundamental understanding of role of the secondary and tertiary structures on the translocation of RNA in direct RNA sequencing platform based on single-molecule electrophoresis. The framework for RNA translocation is promising for the exploration of advanced translocation problems and the design of the new protein nanopore.