This RO3 application is in response to a Program Announcement entitled Innovative Drug Discovery Research in AIDS Opportunistic Infections. The ultimate goal of this project is to discover principles that allow targeting of RNA with small oligonucleotide based compounds that inhibit function. Establishment of such principles could have wide ranging applications, including design of compounds to treat HIV and opportunistic infections. New paradigms to be explored are (1) binding enhancement by tertiary interactions (BETI), (2) suicide inhibition, and (3) oligonucleotide-catalyzed hydrolysis of splice sites. The group I self-splicing intron from the ribosomal RNA precursor in Pneumocystis carinii will serve as the model system for these studies. Correct splicing of this precursor is required for formation of active ribosomes. Preliminary work has shown that a nuclease stable, N3'-P5' phosphoramidate hexanucleotide exhibits binding enhanced by tertiary interactions in binding tightly to the sequence that normally aligns the 5' splice site. This hexanucleotide inhibits self-splicing via a suicide inhibition mechanism.
Specific aims for the proposed work are: (1) Determine if oligonucleotides with uncharged methylphosphonate linkages exhibit BETI and inhibition of self-splicing. (2) Determine if BETI is available at other sites on the group I intron. (3) Determine if in vitro oligonucleotide inhibitors are taken up by P. carinii cells. The results are expected to provide fundamental principles for designing inhibitors of many RNA functions. Because maturation of rRNA precursor is essential for proliferation of P. carinii, and no group I self-splicing introns are known in human genes, the results could also provide potential therapeutics for P. carinii pneumonia.
