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Translocating kilobase RNA through the Staphylococcal ?-hemolysin nanopore.

ABSTRACT: The electrophoretic translocation of polynucleotides through nanopores may permit direct single-molecule nucleic acid sequencing. Here we describe the translocation of ssRNA heteropolymers (91-6083 bases) through the ?-hemolysin nanopore. Translocation of these long ssRNAs is characterized by surprisingly long, almost complete ionic current blockades with durations averaging milliseconds per base (at +180 mV). The event durations decrease exponentially with increased transmembrane potential but are largely unaffected by the presence of urea. When the ssRNA is coupled at the 3' end to streptavidin, which cannot translocate through the pore, permanent blockades are observed, supporting our conclusion that the transient blockades arise from ssRNA translocation.

SUBMITTER: Cracknell JA

PROVIDER: S-EPMC3712197 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Translocating kilobase RNA through the Staphylococcal α-hemolysin nanopore.

Cracknell James A JA Japrung Deanpen D Bayley Hagan H

Nano letters 20130523 6

The electrophoretic translocation of polynucleotides through nanopores may permit direct single-molecule nucleic acid sequencing. Here we describe the translocation of ssRNA heteropolymers (91-6083 bases) through the α-hemolysin nanopore. Translocation of these long ssRNAs is characterized by surprisingly long, almost complete ionic current blockades with durations averaging milliseconds per base (at +180 mV). The event durations decrease exponentially with increased transmembrane potential but ...[more]

PMID: 23678965

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Urea facilitates the translocation of single-stranded DNA and RNA through the alpha-hemolysin nanopore.

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2015-04-01 | GSE60543 | GEO

Unzipping of A-Form DNA-RNA, A-Form DNA-PNA, and B-Form DNA-DNA in the ?-Hemolysin Nanopore.

Project description:Unzipping of double-stranded nucleic acids by an electric field applied across a wild-type ?-hemolysin (?HL) nanopore provides structural information about different duplex forms. In this work, comparative studies on A-form DNA-RNA duplexes and B-form DNA-DNA duplexes with a single-stranded tail identified significant differences in the blockage current and the unzipping duration between the two helical forms. We observed that the B-form duplex blocks the channel 1.9 ± 0.2 pA more and unzips ?15-fold more slowly than an A-form duplex at 120 mV. We developed a model to describe the dependence of duplex unzipping on structure. We demonstrate that the wider A-form duplex (d = 2.4 nm) is unable to enter the vestibule opening of ?HL on the cis side, leading to unzipping outside of the nanopore with higher residual current and faster unzipping times. In contrast, the smaller B-form duplexes (d = 2.0 nm) enter the vestibule of ?HL, resulting in decreased current blockages and slower unzipping. We investigated the effects of varying the length of the single-stranded overhang, and studied A-form DNA-PNA duplexes to provide additional support for the proposed model. This study identifies key differences between A- and B-form duplex unzipping that will be important in the design of future probe-based methods for detecting DNA or RNA.

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Unzipping kinetics of duplex DNA containing oxidized lesions in an ?-hemolysin nanopore.

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Characterization of Interstrand DNA-DNA Cross-Links Using the ?-Hemolysin Protein Nanopore.

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?-Hemolysin nanopore studies reveal strong interactions between biogenic polyamines and DNA hairpins.

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| S-EPMC4874251 | biostudies-literature

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