PCR-Based Construction of Long Hammerhead Ribozymes
With the advent of antisense technology, there has been much interest in the use of long RNAs expressed in vivo to inhibit the expression of target genes. More recently, there have been numerous reports that the incorporation of either hairpin or hammerhead ribozyme motifs (catalytic antisense) into such RNAs increases their effectiveness (1, 2, 3). This section will describe a generally applicable, simple, PCR-based method to construct catalytic antisense RNA containing the hammerhead catalytic core that will cleave the target of interest at a GUC sequence. All discussion will focus on the hammerhead motif, although, in principle, the hairpin motif could be incorporated instead.
KeywordsMicrocentrifuge Tube Hammerhead Ribozyme Hairpin Motif Final Insert Incomplete Denaturation
- 1.De Young, M. B., Kincade-Denker, J., Boehm, C. A., Riek, R. P., Mamone, J. A., McSwiggen, J. A., and Graham, R. M. (1994) Functional characterization of ribozymes expressed using U1 and T7 vectors for the intracellular cleavage of ANF mRNA. Biochemistry 33, 12,127–12,138.PubMedCrossRefGoogle Scholar
- 2.Homann, M., Tabler, M., Tzortzakaki, S., and Sczakiel, G. (1994) Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in having cells. Nucleic Acids Res. 22, 3951–3957.PubMedCrossRefGoogle Scholar
- 15.Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Ehrlich, H. (1986) Specific enzymatic amplification of DNA in vitro the polymerase chain reaction. Cold Spring Harbor Sym. 51, 263–273.Google Scholar