A two unit antisense RNA cassette test system for silencing of target genes - PubMed (original) (raw)

A two unit antisense RNA cassette test system for silencing of target genes

H M Engdahl et al. Nucleic Acids Res. 1997.

Abstract

This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.

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References

    1. J Mol Biol. 1989 Dec 5;210(3):561-72 - PubMed
    1. Nucleic Acids Res. 1989 Jun 26;17(12):4441-54 - PubMed
    1. Biochemistry. 1993 Feb 2;32(4):1088-95 - PubMed
    1. Nucleic Acids Res. 1993 Mar 25;21(6):1381-7 - PubMed
    1. EMBO J. 1993 Jun;12(6):2567-73 - PubMed

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