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Determination of RNA Orientation during Translocation through a Biological Nanopore

Tom Z. Butler ^*, Jens H. Gundlach ^* and Mark A. Troll  

^* Departments of Physics, Electrical Engineering, and Microbiology, University of Washington, Seattle, Washington

Correspondence: Address reprint requests to Tom Z. Butler, E-mail: twb2{at}u.washington.edu.

We investigate single-molecule electrophoretic translocation of A₅₀, C₅₀, A₂₅C₅₀, and C₅₀A₂₅ RNA molecules through the -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.

This article has been cited by other articles:

				T. Z. Butler, J. H. Gundlach, and M. Troll Ionic Current Blockades from DNA and RNA Molecules in the {alpha}-Hemolysin Nanopore Biophys. J., November 1, 2007; 93(9): 3229 - 3240. [Abstract] [Full Text] [PDF]