PMID: 8948630Nov 15, 1996Paper

In vitro selection of hammerhead ribozymes containing a bulged nucleotide in stem II

Nucleic Acids Research
J B ThomsonF Eckstein

Abstract

Hammerhead ribozymes were transcribed from a dsDNA template containing four random nucleotides between stems II and III, which replace the naturally occurring GAA nucleotides. In vitro selection was used to select hammerhead ribozymes capable of in cis cleavage using denaturing polyacrylamide gels for the isolation of cleaving sequences. Self-cleaving ribozymes were cloned after the first and second rounds of selection, sequenced and characterised. Only sequences containing 5'-HGAA-3', where H is A, C or U, between stems II and III were active; G was clearly not tolerated at this position. Thus, only three sequences out of the starting pool of 256 (4(4)) were active. The Michaelis-Menten parameters were determined for the in trans cleaving versions of these ribozymes and indicate that selected ribozymes are less efficient than the native sequence. We propose that the selected ribozymes accommodate the extra nucleotide as a bulge in stem II.

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Citations

May 10, 2000·Chemistry & Biology·A BeaudryL Beigelman
Jul 17, 1998·Current Opinion in Chemical Biology·S VermaF Eckstein
Apr 16, 1998·Proceedings of the National Academy of Sciences of the United States of America·N K VaishF Eckstein
May 24, 2000·Proceedings of the National Academy of Sciences of the United States of America·J Tang, R R Breaker
Aug 20, 2011·Wiley Interdisciplinary Reviews. RNA·Katarzyna Matylla-KulinskaRenée Schroeder
Jun 28, 2014·Nucleosides, Nucleotides & Nucleic Acids·Mituhiro KuriyamaYoshiyuki Tanaka

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