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A procedure for rapidly generating a library of antisense-accessible sites on native mRNAs (mRNA antisense-accessible sites library [MASL]) is described that involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific polymerase chain reaction (PCR). Antisense phosphorothioate oligodeoxynucleotides (ODNs), peptide nucleic acids (PNAs), and small interfering RNAs (siRNAs) can then be identified by screening against the antisense-accessible sites. The utility of this methodology is demonstrated for the identification of more effective inhibitors of inducible nitric oxide synthase (iNOS) induction than have previously been reported. This method may also be useful for constraining folding calculations of native mRNAs and for designing mRNA imaging probes.

Native mRNA antisense-accessible sites library for the selection of antisense oligonucleotides, PNAs, and siRNAs