In vitro evolution and characterization of a ligase ribozyme adapted to acidic conditions: effect of further rounds of evolution.
A ligase ribozyme that accelerates the ligation reaction with an oligonucleotide under low pH conditions was identified by in vitro adaptation in a previous study. We examined the effects of further rounds of evolution to isolate a more active ribozyme. The ribozyme, which was obtained after four rounds of evolution, was randomly mutated, and the resultant RNA library was subjected to in vitro selection at low pH. One ribozyme isolated from the pool was found to react 8,000 times faster than the original b1 ribozyme at pH 4. The reaction rate of the isolated ribozyme was enhanced at various pH values, and its pH dependence was less than that of the original ribozyme or the ribozyme selected with four rounds of evolution. The reaction rate of the isolated ribozyme was reduced in the presence of 3' primer, the sequence of which is complementary to the 3' primer-binding site of the ligase ribozyme. This inhibition induced by the primer oligonucleotide binding to the ribozyme 3' region implies that the 3' region plays a role in the ligation reaction of the ribozyme.
Miyamoto
Yoshitaka
Y
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Teramoto
Naozumi
N
Imanishi
Yukio
Y
Ito
Yoshihiro
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011118:Polynucleotide Ligases; D015202:Protein Engineering / Q000379:methods*; D016337:RNA, Catalytic / Q000032:analysis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D016337:RNA, Catalytic; D011088:DNA Ligases; D011118:Polynucleotide Ligases