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- ((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 approximately 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.
Komatsu
Y
Y
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. komatsu@pharm.hokudai.ac.jp
Kumagai
I
I
Ohtsuka
E
E
D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D012313:RNA / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013891:Thiouridine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
C105273:2-thiouridine; D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D016337:RNA, Catalytic; D013891:Thiouridine; D012313:RNA; D014529:Uridine