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Nucleic Acids Research, Vol 24, Issue 22 4401-4406, Copyright © 1996 by Oxford University Press

ARTICLES

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

JB Thomson, ST Sigurdsson, A Zeuch and F Eckstein
Max-Planck-Institut fur Experimentelle Medizin, Gottingen, Germany.

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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