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In vitro adaptation of a ligase ribozyme for activity under a low‐pH condition
Author(s) -
Miyamoto Yoshitaka,
Teramoto Naozumi,
Imanishi Yukio,
Ito Yoshihiro
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10033
Subject(s) - ribozyme , vs ribozyme , ligase ribozyme , mammalian cpeb3 ribozyme , oligonucleotide , hairpin ribozyme , dna ligase , biology , rna ligase , rna , biochemistry , microbiology and biotechnology , chemistry , genetics , enzyme , dna , gene
A ligase ribozyme accelerating a ligation reaction with oligonucleotide under a low‐pH condition was selected by in vitro adaptation. A ribozyme active at pH 7 was randomly mutated, and the resultant RNA library was subjected to in vitro adaptation under a low‐pH reaction condition. At pH 4, the adapted RNAs reacted with the oligonucleotide substrates about 200 times faster than the original ribozyme. When the ribozyme was cloned and sequenced, 10 of the 30 clones sequenced had identical sequences. The differences in sequence from the original ribozyme were found at four positions in the middle region and at the 3′ end. A few sequential differences dominated the activity of the ribozyme under the extreme condition. The adapted ribozyme had one repeating sequence that was critical for the activity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 590–596, 2001.