Determination of RNA Orientation during Translocation through a Biological Nanopore

We investigate single-molecule electrophoretic translocation of A50, C50, A25C50, and C50A25 RNA molecules through the α-hemolysin transmembrane protein pore. We observe pronounced bilevel current blockages during translocation of A25C50 and C50A25 molecules. The two current levels observed during these bilevel blockages are very similar to the characteristic current levels observed during A50 and C50 translocation. From the temporal ordering of the two levels within the bilevel current blockages, we infer whether individual A25C50 and C50A25 molecules pass through the pore in a 3′ → 5′ or 5′ → 3′ orientation. Correlation between the level of current obstruction and the inferred A25C50 or C50A25 orientation indicates that 3′ → 5′ translocation of a poly C segment causes a significantly deeper current obstruction than 5′ → 3′ translocation. Our analysis also suggests that the 3′ ends of C50 and A25C50 RNA molecules are more likely to initiate translocation than the 5′ ends. Orientation dependent differences in a smaller current blockage that immediately precedes many translocation events suggest that this blockage also contains information about RNA orientation during translocation. These findings emphasize that the directionality of polynucleotide molecules is an important factor in translocation and demonstrate how structure within ionic current signals can give new insights into the translocation process.