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Biophys. J. BioFAST: First Published October 7, 2005. doi:10.1529/biophysj.105.068957
© 2005 by the Biophysical Society.

A more recent version of this article appeared on January 1, 2006.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

Determination of RNA Orientation during Translocation through a Biological Nanopore

Tom Z Butler 1*, Jens H Gundlach 1 and Mark A Troll 1

1 University of Washington

* To whom correspondence should be addressed. E-mail: twb2{at}u.washington.edu.

Submitted on June 22, 2005
Revised on July 25, 2005
Accepted on 28 September 2005


  Abstract
We investigate single-molecule electrophoretic translocation of A50, C50, A25C50, and C50A25 RNA molecules through the {alpha}-hemolysin transmembrane protein pore. We observe pronounced bi-level current blockages during translocation of A25C50 and C50A25 molecules. The two current levels observed during these bi-level 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 bi-level 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 gives new insights into the translocation process.

Key Words: RNA, alpha hemolysin, bio-sensor, nanopore, single molecule, translocation




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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]


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Copyright © 2005 by the Biophysical Society.