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Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device

Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and m...

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Autores principales: Smith, Andrew M., Abu-Shumays, Robin, Akeson, Mark, Bernick, David L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478443/
https://www.ncbi.nlm.nih.gov/pubmed/26157798
http://dx.doi.org/10.3389/fbioe.2015.00091
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author Smith, Andrew M.
Abu-Shumays, Robin
Akeson, Mark
Bernick, David L.
author_facet Smith, Andrew M.
Abu-Shumays, Robin
Akeson, Mark
Bernick, David L.
author_sort Smith, Andrew M.
collection PubMed
description Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNA(fMet) and tRNA(Lys), we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device.
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spelling pubmed-44784432015-07-08 Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device Smith, Andrew M. Abu-Shumays, Robin Akeson, Mark Bernick, David L. Front Bioeng Biotechnol Bioengineering and Biotechnology Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNA(fMet) and tRNA(Lys), we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device. Frontiers Media S.A. 2015-06-24 /pmc/articles/PMC4478443/ /pubmed/26157798 http://dx.doi.org/10.3389/fbioe.2015.00091 Text en Copyright © 2015 Smith, Abu-Shumays, Akeson and Bernick. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Smith, Andrew M.
Abu-Shumays, Robin
Akeson, Mark
Bernick, David L.
Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device
title Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device
title_full Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device
title_fullStr Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device
title_full_unstemmed Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device
title_short Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device
title_sort capture, unfolding, and detection of individual trna molecules using a nanopore device
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478443/
https://www.ncbi.nlm.nih.gov/pubmed/26157798
http://dx.doi.org/10.3389/fbioe.2015.00091
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