RRM
2013 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Nanopores are under investigation for its key role in designing rapid, low cost nucleic acid sequencing platforms. They have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological nanopores are extremely reproducible with uniform channel size. The bacterial virus phi29 DNA-packaging motor is a naturally occurring biological channel for the transportation of dsDNA, and has the largest diameter among other well-studied biological channels, thus offering more space for further channel modifications and conjugation. Interestingly, this large channel, which translocates dsDNA, cannot detect single stranded nucleic acids, ssDNA or ssRNA. Here, we reengineered this motor channel to detect the real time translocation of ssDNA or RNA. One internal loop segment of the channel was removed, which results in two classes of channels, one with the same size as wild-type channel and other with cross-sectional area of 60% of the wild-type. The loop-deleted connector was able to translocate ssRNA with equal competencies and the translocation was observed to be bi-directional. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA-packaging motor in sensing of RNA structure and folding. We demonstrate the robust characteristics of single pore electrophysiological assays. The conductance of each class of pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by RNA are consistent with the dimensions of the channel and nucleic acids. The connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for RNA quantification and characterization. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of nucleic acid oligomers passing through the channel. The fingerprint of RNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of RNA.
References:
Wendell, D.; Jing, P.; Geng, J.; Subramaniam, V.; Lee, T. J.; Montemagno, C.; Guo, P. Nat. Nanotechnol. 2009, 4, 765
Geng J, Wang S, Fang H, Guo P. ACS Nano. 2013, 7, 3315
Keywords: Nanopore, Nanomotor
