Structural and biochemical characterization of DSL ribozyme.
We recently reported on the molecular design and synthesis of a new RNA ligase ribozyme (DSL), whose active site was selected from a sequence library consisting of 30 random nucleotides set on a defined 3D structure of a designed RNA scaffold. In this study, we report on the structural and biochemical analyses of DSL. Structural analysis indicates that the active site, which consists of the selected sequence, attaches to the folded scaffold as designed. To see whether DSL resembles known ribozymes, a biochemical assay was performed. Metal-dependent kinetic studies suggest that the ligase requires Mg2+ ions. The replacement of Mg2+ with Co(NH3)6(3+) prohibits the reaction, indicating that DSL requires innersphere coordination of Mg2+ for a ligation reaction. The results show that DSL has requirements similar to those of previously reported catalytic RNAs.
Horie
Souta
S
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Ikawa
Yoshiya
Y
Inoue
Tan
T
D001483:Base Sequence; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011130:RNA Ligase (ATP) / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*
D016337:RNA, Catalytic; D003035:Cobalt; D011130:RNA Ligase (ATP); D008274:Magnesium