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Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing
dc.contributor.author | Athapattu, Uditha S. | |
dc.contributor.author | Amarasekara, Charuni A. | |
dc.contributor.author | Immel, Jacob R. | |
dc.contributor.author | Bloom, Steven | |
dc.contributor.author | Barany, Francis | |
dc.contributor.author | Nagel, Aaron C. | |
dc.contributor.author | Soper, Steven A. | |
dc.date.accessioned | 2022-01-04T20:37:03Z | |
dc.date.available | 2022-01-04T20:37:03Z | |
dc.date.issued | 2021-01-28 | |
dc.identifier.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 | en_US |
dc.identifier.uri | http://hdl.handle.net/1808/32327 | |
dc.description.abstract | 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. | en_US |
dc.publisher | Oxford University Press | en_US |
dc.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. | en_US |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | en_US |
dc.subject | Nucleic acid modification | en_US |
dc.subject | RNA characterisation and manipulation | en_US |
dc.title | Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing | en_US |
dc.type | Article | en_US |
kusw.kuauthor | Athapattu, Uditha S. | |
kusw.kuauthor | Amarasekara, Charuni A. | |
kusw.kuauthor | Immel, Jacob R. | |
kusw.kuauthor | Bloom, Steven | |
kusw.kuauthor | Soper, Steven A. | |
kusw.kudepartment | Chemistry | en_US |
kusw.kudepartment | Medicinal Chemistry | en_US |
kusw.kudepartment | Mechanical Engineering and Bioengineering | en_US |
dc.identifier.doi | 10.1093/nar/gkab001 | en_US |
kusw.oaversion | Scholarly/refereed, publisher version | en_US |
kusw.oapolicy | This item meets KU Open Access policy criteria. | en_US |
dc.identifier.pmid | PMC8053086 | en_US |
dc.rights.accessrights | openAccess | en_US |