TETRANUCLEOTIDE GGGA MOTIF IN PRIMARY RNA TRANSCRIPTS - NOVEL TARGET SITE FOR ANTISENSE DESIGN

Selecting effective antisense target sites on a given mRNA molecule co nstitutes a major problem in antisense therapeutics. By trial-and-erro r, only 1 in 18 (6%) of antisense oligonucleotides designed to target the primary RNA transcript of tumor necrosis factor-alpha (TNF-alpha) strongly inhibited TNF-alpha synthesis. Subsequent studies showed that the area in RNA targeted by antisense oligonucleotides could be moved effectively 10-15 bases in either direction from the original area. W e observed that only molecules that incorporated a tetranucleotide mot if TCCC (complementary to GGGA on RNA) yielded potent antisense oligon ucleotides against TNF-alpha. A comprehensive literature survey showed that this motif is unwittingly present in 48% of the most potent anti sense oligonucleotides reported in the literature. This finding was pr ospectively used to predict the sequences of additional antisense olig onucleotides for the rat TNF-alpha primary RNA transcript. Over 50% of antisense constructs (13 of 22) containing the TCCC motif were found to effectively inhibit TNF-alpha synthesis. Marked reductions in mRNA were also observed. This motif was found to be most effective when tar geting introns in the primary RNA transcript, suggesting a nuclear loc alization for the antisense action. Predicting target sites based on t he presence of this motif in primary RNA transcripts should be of valu e in the development on new antisense pharmacotherapy.