RNA Translocation through\nProtein Nanopores: Interlude\nof the Molten RNA Globule

Direct translocation of RNA with secondary structures\nusing single-molecule\nelectrophoresis through protein nanopores shows significant fluctuations\nin the measured ionic current, in contrast to unstructured single-stranded\nRNA or DNA. We developed a multiscale model combining the oxRNA model\nfor RNA with the 3-dimensional Poisson–Nernst–Planck\nformalism for electric fields within protein pores, aiming to map\nRNA conformations to ionic currents as RNA translocates through three\nprotein nanopores: α-hemolysin, CsgG, and MspA. Our findings\nreveal three distinct stages of translocation (pseudoknot, melting,\nand molten globule) based on contact maps and current values. Two\ntranslocation modes emerge: fast and slow. In the fast mode, the speed\nis determined by the electric field, independent of pore geometry.\nIn the slow mode, the molten globule stage is the rate-determining\nfactor in slowing the translocation, instead of the previous paradigm\nof melting of the base pairs. Using these insights, we propose a neural\nnetwork framework to identify and reconstruct RNA secondary structures\nfrom ionic current windows. We find that the electric field distribution,\nnot the nanopore geometry, drives the molten globule stage. Our results\nexplain the large current fluctuations. These results provide a fundamental\nunderstanding of the role of secondary and tertiary structures in\nthe translocation of RNA in direct RNA translocation platforms based\non single-molecule electrophoresis. This work offers design rules\nfor new protein pores and real-time imaging of the secondary structures\nof RNA.