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Nucleic Acids Research, 1994, Vol. 22, No. 19 3958-3965
© 1994

RNA

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design

Martin Tabler*,, Matthias Homann1, Sergia Tzortzakaki and Georg Sczakiel1

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas PO Box 1527, GR-71110 Heraklion, Crete, Greece 1Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany

*To whom correspondence should be addressed

Received June 16, 1994. Revised August 18, 1994. Accepted August 18, 1994.

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed ‘catalytic antisense RNAs’. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed.


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