In vitro and in vivo targeting of RNA in fungal pathogens with oligonucleotides and small-molecules

The increasing number of RNAs that are known to play critical roles in essential biochemical events makes RNA a potential target for drug discovery efforts. This work aims to develop a better understanding of how RNAs can be targeted specifically using oligonucleotides and small-molecules. For in vitro studies, two strategies were used to design oligonucleotides to target group I self-splicing introns from the human pathogens Pneumocystis carinii and Candida albicans. These strategies are B&barbelow;inding E&barbelow;nhancement by T&barbelow;ertiary I&barbelow;nteractions (BETI) and suicide inhibition. BETI allows for specific targeting of RNA by exploiting tertiary interactions used by the intron to recognize its splice site; oligonucleotides that use BETI are shown to bind more than a 100000-fold more tightly to a target site that accommodates tertiary interactions than one that only binds the oligonucleotide by base pairing. Suicide inhibition uses the catalytic potential of RNA to trick group I introns into reacting with a short oligonucleotide mimic of their substrate rather than their endogenous substrate. Several oligonucleotides are discussed that use these strategies to inhibit self-splicing. To allow for testing of these strategies in cell culture, uptake of oligonucleotides into C. albicans was studied. Results show that oligonucleotides are internalized with, in some cases, greater than 100-fold higher intracellular concentrations than extracellular concentrations. An oligonucleotide inhibitor of C. albicans growth was discovered that was designed to mimic an essential hairpin in the ribosome. Evidence is presented that the oligonucleotide affects both the structure and activity of the ribosome in vivo. For small-molecules, a library of acridine conjugates was screened for inhibition of group I intron self-splicing and several molecules were found that are potent and specific inhibitors of self-splicing. Another small-molecule, Hoechst 33258, was found to inhibit group I intron self-splicing both in vitro and in vivo. The structural ramifications of Hoechst 33258 binding to the intron were determined by chemical probing of the intron with Hoechst 33258. Chemical probing of the intron also indicated that the binding site for Hoechst 33258 is an important internal loop in the intron.