Deoxyribozymes with 2′-5′ RNA ligase activity

Amber Flynn-Charlebois, Yangming Wang, Tracey K. Prior, Imran Rashid, Kelly A. Hoadley, Rebecca L. Coppins, Amanda C. Wolf, Scott K Silverman

Research output: Contribution to journalArticle

Abstract

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2′-5′ RNA phosphodiester linkage is created from a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. The new Mg2+-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 °C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 °C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA↓GGAA or UAUN↓GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2′-5′ linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg2+-dependent folding (ΔΔG°′ < 0.2 kcal/mol). This demonstrates that a 2′-5′ linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.

Original languageEnglish (US)
Pages (from-to)2444-2454
Number of pages11
JournalJournal of the American Chemical Society
Volume125
Issue number9
DOIs
StatePublished - Mar 5 2003

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Catalytic DNA
Ligases
RNA
Nucleotides
substrate
Substrates
folding
catalysis
Electrophoresis
Hydroxyl Radical
Introns

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Flynn-Charlebois, A., Wang, Y., Prior, T. K., Rashid, I., Hoadley, K. A., Coppins, R. L., ... Silverman, S. K. (2003). Deoxyribozymes with 2′-5′ RNA ligase activity. Journal of the American Chemical Society, 125(9), 2444-2454. https://doi.org/10.1021/ja028774y

Deoxyribozymes with 2′-5′ RNA ligase activity. / Flynn-Charlebois, Amber; Wang, Yangming; Prior, Tracey K.; Rashid, Imran; Hoadley, Kelly A.; Coppins, Rebecca L.; Wolf, Amanda C.; Silverman, Scott K.

In: Journal of the American Chemical Society, Vol. 125, No. 9, 05.03.2003, p. 2444-2454.

Research output: Contribution to journalArticle

Flynn-Charlebois, A, Wang, Y, Prior, TK, Rashid, I, Hoadley, KA, Coppins, RL, Wolf, AC & Silverman, SK 2003, 'Deoxyribozymes with 2′-5′ RNA ligase activity', Journal of the American Chemical Society, vol. 125, no. 9, pp. 2444-2454. https://doi.org/10.1021/ja028774y
Flynn-Charlebois A, Wang Y, Prior TK, Rashid I, Hoadley KA, Coppins RL et al. Deoxyribozymes with 2′-5′ RNA ligase activity. Journal of the American Chemical Society. 2003 Mar 5;125(9):2444-2454. https://doi.org/10.1021/ja028774y
Flynn-Charlebois, Amber ; Wang, Yangming ; Prior, Tracey K. ; Rashid, Imran ; Hoadley, Kelly A. ; Coppins, Rebecca L. ; Wolf, Amanda C. ; Silverman, Scott K. / Deoxyribozymes with 2′-5′ RNA ligase activity. In: Journal of the American Chemical Society. 2003 ; Vol. 125, No. 9. pp. 2444-2454.
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