In vitro adaptation of a ligase ribozyme for activity under a low-pH condition

A ligase ribozyme accelerating a ligation reaction with oligonucleotide und er a low-pH condition was selected by in vitro adaptation. A ribozyme activ e 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 ada pted RNAs reacted with the oligonucleotide substrates about 200 times faste r than the original ribozyme. When the ribozyme was cloned and sequenced, 1 0 of the 30 clones sequenced had identical sequences. The differences in se quence from the original ribozyme were found at four positions in the middl e region and at the 3' end. A few sequential differences dominated the acti vity of the ribozyme under the extreme condition. The adapted ribozyme had one repeating sequence that was critical for the activity. (C) 2001 John Wi ley & Sons, Inc.