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Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing r...

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Autores principales: Hagelskamp, Felix, Borland, Kayla, Ramos, Jillian, Hendrick, Alan G, Fu, Dragony, Kellner, Stefanie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144906/
https://www.ncbi.nlm.nih.gov/pubmed/32083657
http://dx.doi.org/10.1093/nar/gkaa091
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author Hagelskamp, Felix
Borland, Kayla
Ramos, Jillian
Hendrick, Alan G
Fu, Dragony
Kellner, Stefanie
author_facet Hagelskamp, Felix
Borland, Kayla
Ramos, Jillian
Hendrick, Alan G
Fu, Dragony
Kellner, Stefanie
author_sort Hagelskamp, Felix
collection PubMed
description RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNA(Val)(AAC) is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNA(Val)(IAC)in vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.
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spelling pubmed-71449062020-04-13 Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro Hagelskamp, Felix Borland, Kayla Ramos, Jillian Hendrick, Alan G Fu, Dragony Kellner, Stefanie Nucleic Acids Res Methods Online RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNA(Val)(AAC) is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNA(Val)(IAC)in vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA. Oxford University Press 2020-04-17 2020-02-21 /pmc/articles/PMC7144906/ /pubmed/32083657 http://dx.doi.org/10.1093/nar/gkaa091 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Online
Hagelskamp, Felix
Borland, Kayla
Ramos, Jillian
Hendrick, Alan G
Fu, Dragony
Kellner, Stefanie
Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro
title Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro
title_full Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro
title_fullStr Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro
title_full_unstemmed Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro
title_short Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro
title_sort broadly applicable oligonucleotide mass spectrometry for the analysis of rna writers and erasers in vitro
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144906/
https://www.ncbi.nlm.nih.gov/pubmed/32083657
http://dx.doi.org/10.1093/nar/gkaa091
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