We introduced 4-thio- ((4S)U), 2-thio- ((2S)U), 4-O-methyluridine ((4Me)U) and cytidine substitutions for U+2, which is an important base for cleavage in a substrate RNA. Oligonucleotides containing 4-thio- and 4-O-methyluridine were prepared by a new convenient post-synthetic modification method using a 4-O-p-nitrophenyl-uridine derivative. A hairpin ribozyme cleaved the substrate RNA with either C+2, (4S)U+2 or (4Me)U+2 at ~ 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a (2S)U+2 was cleaved with the same activity as the natural substrate. These results suggest that the O4 of U+2 is involved in hydrogen bonding at loop A, but the O2 of U+2 is not recognized by the active residues. Circular dichroism data of the ribozymes containing (4S)U+2 and (2S)U+2, as well as the susceptibility of the thiocarbonyl group to hydrogen peroxide, suggest that a conformational change of U+2 occurs during the domain docking in the cleavage reaction. We propose here the conformational change of U+2 from the ground state to the active molecule during the reaction.
CITATION STYLE
Komatsu, Y., Kumagai, I., & Ohtsuka, E. (1999). Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides. Nucleic Acids Research, 27(22), 4314–4323. https://doi.org/10.1093/nar/27.22.4314
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