DNAmoreDB - A Database of Deoxyribozymes

Published on 2003 in J. Am. Chem. Soc. volume 125 issue 9.

PubMed ID: 12603132

DOI:10.1021/ja028774y

Abstract:

n 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.



DNAzymes linked to this article:

Name Isolated sequence Length Reaction
9A5 AAGGGGAGGGCCGATTGGCATTATCGGCGTCTTAGCTCTA      40 RNA ligation
9A6 TAACCGTGGTTACCGTAAGCGCGGGGCTTATAGGGGATT      39 RNA ligation
9A12 CTTTACGGTAGGGTGCCTGTGATAATGATCAGGGGACGGC      40 RNA ligation
9A2 TGCGTTCTGATGAATTGCACATAGCTTATAGTTCCTTGCT      40 RNA ligation
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