Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins
Abstract Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5′-O-triphosphates bearing a reactive squaramate group attached to position...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Communications Chemistry |
| Online Access: | https://doi.org/10.1038/s42004-024-01399-6 |
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| author | Ivana Ivancová Tania Sánchez Quirante Marek Ondruš Radek Pohl Marta Vlková Eva Žilecká Evžen Bouřa Michal Hocek |
| author_facet | Ivana Ivancová Tania Sánchez Quirante Marek Ondruš Radek Pohl Marta Vlková Eva Žilecká Evžen Bouřa Michal Hocek |
| author_sort | Ivana Ivancová |
| collection | DOAJ |
| description | Abstract Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5′-O-triphosphates bearing a reactive squaramate group attached to position 5 of cytidine or position 7 of 7-deazaadenosine and used them as substrates for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using TGK polymerase was used for synthesis of squaramate-modified RNA probes which underwent covalent bioconjugations with amine-linked fluorophore and lysine-containing peptides and proteins including several viral RNA polymerases or HIV reverse transcriptase. Inhibition of RNA-depending RNA polymerases from Japanese Encephalitis virus was observed through formation of covalent cross-link which was partially identified by MS/MS analysis. Thus, the squaramate-linked NTP analogs are useful building blocks for the synthesis of reactive RNA probes for bioconjugations with primary amines and cross-linking with lysine residues. |
| format | Article |
| id | doaj-art-b26558414009433f96fa3d3739440194 |
| institution | Kabale University |
| issn | 2399-3669 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Chemistry |
| spelling | doaj-art-b26558414009433f96fa3d37394401942025-01-05T12:10:51ZengNature PortfolioCommunications Chemistry2399-36692025-01-018111010.1038/s42004-024-01399-6Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteinsIvana Ivancová0Tania Sánchez Quirante1Marek Ondruš2Radek Pohl3Marta Vlková4Eva Žilecká5Evžen Bouřa6Michal Hocek7Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000 Prague 6Abstract Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5′-O-triphosphates bearing a reactive squaramate group attached to position 5 of cytidine or position 7 of 7-deazaadenosine and used them as substrates for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using TGK polymerase was used for synthesis of squaramate-modified RNA probes which underwent covalent bioconjugations with amine-linked fluorophore and lysine-containing peptides and proteins including several viral RNA polymerases or HIV reverse transcriptase. Inhibition of RNA-depending RNA polymerases from Japanese Encephalitis virus was observed through formation of covalent cross-link which was partially identified by MS/MS analysis. Thus, the squaramate-linked NTP analogs are useful building blocks for the synthesis of reactive RNA probes for bioconjugations with primary amines and cross-linking with lysine residues.https://doi.org/10.1038/s42004-024-01399-6 |
| spellingShingle | Ivana Ivancová Tania Sánchez Quirante Marek Ondruš Radek Pohl Marta Vlková Eva Žilecká Evžen Bouřa Michal Hocek Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins Communications Chemistry |
| title | Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins |
| title_full | Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins |
| title_fullStr | Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins |
| title_full_unstemmed | Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins |
| title_short | Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins |
| title_sort | enzymatic synthesis of reactive rna probes containing squaramate linked cytidine or adenosine for bioconjugations and cross linking with lysine containing peptides and proteins |
| url | https://doi.org/10.1038/s42004-024-01399-6 |
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