Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing
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Issue Date
2021-01-28Author
Athapattu, Uditha S.
Amarasekara, Charuni A.
Immel, Jacob R.
Bloom, Steven
Barany, Francis
Nagel, Aaron C.
Soper, Steven A.
Publisher
Oxford University Press
Type
Article
Article Version
Scholarly/refereed, publisher version
Rights
Copyright: The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s−1 and a processivity of >10.5 kb at 25°C.
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Citation
Uditha S Athapattu, Charuni A Amarasekara, Jacob R Immel, Steven Bloom, Francis Barany, Aaron C Nagel, Steven A Soper, Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing, Nucleic Acids Research, Volume 49, Issue 7, 19 April 2021, Page e41, https://doi.org/10.1093/nar/gkab001
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