Minimal sequence requirements for ribozyme activity.

The hammerhead ribozyme, as engineered by J. Haseloff and W. L. Gerlach [(1988) Nature (London) 334, 585-591], is an RNA molecule containing two regions of conserved nucleotides, a double helix, called helix II, which connects the two conserved regions, and flanking arms of variable sequence, which hybridize the ribozyme to its specific target. Here we show that this ribozyme may be reduced in size and still retain cleavage activity by replacing helix II with just a few nucleotides that cannot form Watson-Crick base pairs between themselves. Furthermore, the nucleotides replacing helix II and the nucleotides in the flanking arms may be substituted with DNA, and this small, DNA-containing ribozyme is fully as active as the original, full-size ribozyme. Cleavage activity of the minimized ribozyme depends on the number and sequence of the few nucleotides that replace helix II; optimal activity, thus far, is achieved by four or five deoxyribopyrimidines. The minimized ribozyme, or "minizyme," is active as a monomer, as shown by its nearly constant activity over a concentration range varying 25,000-fold, by the mobility of the minizyme-substrate complex in nondenaturing polyacrylamide gels as compared with other nucleic acid molecules of known size, and by other observations. These minizymes provide an excellent model system for studying the structure and mechanism of catalytic RNA; they might also be useful in a variety of biological applications.