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Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase

RNAs not only offer valuable information regarding our bodies but also regulate cellular functions, allowing for their specific manipulations to be extensively explored for many different biological and clinical applications. In particular, rather than temporary hybridization, permanent labeling is...

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Detalles Bibliográficos
Autores principales: Jo, Hyesung, Beon, Jiyun, Oh, Seung Soo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9605241/
https://www.ncbi.nlm.nih.gov/pubmed/36295109
http://dx.doi.org/10.3390/life12101674
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author Jo, Hyesung
Beon, Jiyun
Oh, Seung Soo
author_facet Jo, Hyesung
Beon, Jiyun
Oh, Seung Soo
author_sort Jo, Hyesung
collection PubMed
description RNAs not only offer valuable information regarding our bodies but also regulate cellular functions, allowing for their specific manipulations to be extensively explored for many different biological and clinical applications. In particular, rather than temporary hybridization, permanent labeling is often required to introduce functional tags to target RNAs; however, direct RNA labeling has been revealed to be challenging, as native RNAs possess unmodifiable chemical moieties or indefinable dummy sequences at the ends of their strands. In this work, we demonstrate the combinatorial use of RNA-compatible restriction endonucleases (REs) and RNA-extending polymerases for sequence-specific RNA cleavage and subsequent RNA functionalization. Upon the introduction of complementary DNAs to target RNAs, Type II REs, such as AvrII and AvaII, could precisely cut the recognition site in the RNA-DNA heteroduplexes with exceptionally high efficiency. Subsequently, the 3′ ends of the cleaved RNAs were selectively and effectively modified when Therminator DNA polymerase template-dependently extended the RNA primers with a variety of modified nucleotides. Based on this two-step RNA labeling, only the target RNA could be chemically labeled with the desired moieties, such as bioconjugation tags or fluorophores, even in a mixture of various RNAs, demonstrating the potential for efficient and direct RNA modifications.
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spelling pubmed-96052412022-10-27 Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase Jo, Hyesung Beon, Jiyun Oh, Seung Soo Life (Basel) Article RNAs not only offer valuable information regarding our bodies but also regulate cellular functions, allowing for their specific manipulations to be extensively explored for many different biological and clinical applications. In particular, rather than temporary hybridization, permanent labeling is often required to introduce functional tags to target RNAs; however, direct RNA labeling has been revealed to be challenging, as native RNAs possess unmodifiable chemical moieties or indefinable dummy sequences at the ends of their strands. In this work, we demonstrate the combinatorial use of RNA-compatible restriction endonucleases (REs) and RNA-extending polymerases for sequence-specific RNA cleavage and subsequent RNA functionalization. Upon the introduction of complementary DNAs to target RNAs, Type II REs, such as AvrII and AvaII, could precisely cut the recognition site in the RNA-DNA heteroduplexes with exceptionally high efficiency. Subsequently, the 3′ ends of the cleaved RNAs were selectively and effectively modified when Therminator DNA polymerase template-dependently extended the RNA primers with a variety of modified nucleotides. Based on this two-step RNA labeling, only the target RNA could be chemically labeled with the desired moieties, such as bioconjugation tags or fluorophores, even in a mixture of various RNAs, demonstrating the potential for efficient and direct RNA modifications. MDPI 2022-10-21 /pmc/articles/PMC9605241/ /pubmed/36295109 http://dx.doi.org/10.3390/life12101674 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jo, Hyesung
Beon, Jiyun
Oh, Seung Soo
Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase
title Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase
title_full Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase
title_fullStr Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase
title_full_unstemmed Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase
title_short Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase
title_sort selective rna labeling by rna-compatible type ii restriction endonuclease and rna-extending dna polymerase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9605241/
https://www.ncbi.nlm.nih.gov/pubmed/36295109
http://dx.doi.org/10.3390/life12101674
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