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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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Unfoldase-mediated protein translocation through an α-hemolysin nanopore.

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| S-EPMC3772521 | biostudies-literature

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Redirecting Pore Assembly of Staphylococcal α-Hemolysin by Protein Engineering.

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Translatome and transcriptome profiling of SH-SY5Y cells treated with staphylococcal alpha-hemolysin oligomers

2015-04-01 | GSE60543 | GEO

Translatome and transcriptome profiling of SH-SY5Y cells treated with staphylococcal alpha-hemolysin oligomers

Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. This study uncovers the reshaping undergoing in the translational control system of the host in response to staphylococcal α-hemolysin oligomers (rAHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to staphylococcal α-hemolysin oligomers (rAHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with 3nM of extracted oligomers (rAHL) for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.

2015-04-01 | E-GEOD-60543 | biostudies-arrayexpress

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

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| S-EPMC4826734 | biostudies-literature

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

Project description:The α-hemolysin (α-HL) nanopore analyzes DNA as it is electrophoretically driven through the pore. The respective current vs. time (i-t) traces depends on the DNA sequence, its secondary structures, or on the physical conditions of the analysis. The current study describes analysis of a DNA hairpin with a 5'-extension with the α-HL nanopore in the presence of the polyamines spermine (Spm), spermidine (Spd), and putrescine (Put). These studies identified a new i-t trace characteristic of the DNA-polyamine complex. Voltage-dependent studies determined that the hairpin-Spm complex formed with excess Spm was not unzipped and translocated through the pore even when the voltage was increased to 180 mV. The DNA hairpin sample was titrated with Spm, Spd, or Put that showed a dose-dependent response in the characteristic event patterns for hairpins bound to Spm or Spd, but not for Put. Plots of the event types vs. count were used to calculate binding constants for the Spm or Spd hairpin interactions under these conditions. The titration also demonstrated that the event rate decreased ~10-fold when the Spm or Spd concentration was increased from 0 to 4 mM. These observations impose practical limitations on the ability to use Spm or Spd for DNA studies with the α-HL nanopore.

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Project description:The effect of acidic pH on the translocation of single-stranded DNA through the ?-hemolysin pore is investigated. Two significantly different types of events, i.e. deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, whereas the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to being used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing.

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