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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-10-01
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| Series: | Life |
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| Online Access: | https://www.mdpi.com/2075-1729/12/10/1674 |
| _version_ | 1797471960016355328 |
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| author | Hyesung Jo Jiyun Beon Seung Soo Oh |
| author_facet | Hyesung Jo Jiyun Beon Seung Soo Oh |
| author_sort | Hyesung Jo |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T19:55:29Z |
| format | Article |
| id | doaj.art-d53ee40b9b8f4705936b72d52f45a9c6 |
| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-09T19:55:29Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Life |
| spelling | doaj.art-d53ee40b9b8f4705936b72d52f45a9c62023-11-24T00:58:16ZengMDPI AGLife2075-17292022-10-011210167410.3390/life12101674Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA PolymeraseHyesung Jo0Jiyun Beon1Seung Soo Oh2Department of Materials Science and Engineering, Pohang University of Science Technology (POSTECH), Pohang 37673, KoreaDepartment of Materials Science and Engineering, Pohang University of Science Technology (POSTECH), Pohang 37673, KoreaDepartment of Materials Science and Engineering, Pohang University of Science Technology (POSTECH), Pohang 37673, KoreaRNAs 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.https://www.mdpi.com/2075-1729/12/10/1674nucleic acidsRNA cleavageRNA modificationmodified nucleotidesmRNA detection |
| spellingShingle | Hyesung Jo Jiyun Beon Seung Soo Oh Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase Life nucleic acids RNA cleavage RNA modification modified nucleotides mRNA detection |
| 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 | nucleic acids RNA cleavage RNA modification modified nucleotides mRNA detection |
| url | https://www.mdpi.com/2075-1729/12/10/1674 |
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