Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity. This approach is expensive due to the cost of chemical synthesis of RNAs. We report a technically simple, quick and cost-effective method for the production of siRNAs that makes use of in vitro transcription and deoxyribozyme digestion of the transcripts to produce the desired sequence and length. The method allows for several siRNAs to be produced in parallel at much reduced costs. The siRNAs produced with this method were tested in MDA-MB-231 human breast cancer cells for efficacy against the type 1 insulin-like growth factor receptor (IGF1R) mRNA and they caused dose-dependent inhibition of IGF1R expression comparable to that induced by chemically synthesised siRNAs of the same sequence. This method is also useful for producing long RNA fragments of defined length and sequence that may be difficult to synthesise chemically, and also for producing large quantities of RNAs for applications including structural studies and the study of interactions between RNA and other molecules, such as proteins, other nucleic acids and drugs.

The cytochrome P450 enzyme aromatase (P450arom) is an important target in breast cancer treatment. We have designed a 20-base pyrimidine oligodeoxynucleotide (ODN) which forms a sequence-specific triple helix (triplex) with a purine-rich tract in the P450arom coding sequence. The psoralen-linked ODN (Pso20T) formed photo-induced cross-linked products with target double-stranded DNA. Cross-linked adducts formed in vitro between ODNs and P450arom expression constructs were used to transfect COS and human MCF-7 breast cancer cells. Levels of aromatase transcripts and enzyme activity were significantly lower in cultures transfected with Pso20T-treated cDNA relative to controls. Pso20T had a lesser inhibitory effect on aromatase expression from a mutant P450arom construct, consistent with predicted effects of the mutations on triplex formation. These results are compatible with triplex-mediated interruption of transcription within intact cells.

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