Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post synthetically modified oligonucleotides

We introduced 4-thio- (U-4S), 2-thio- (U-2S), 4-O methyluridine (U-4Me) 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, U-4S+2 Or U-4Me+2 at similar to 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a U-2S+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 U-4S+2 and U-2S+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.