A PNEUMOCYSTIS-CARINII GROUP-I INTRON RIBOZYME THAT DOES NOT REQUIRE 2'-OH GROUPS ON ITS 5'-EXON MIMIC FOR BINDING TO THE CATALYTIC CORE

The recent increase in the population of immunocompromised patients ha s led to an insurgence of opportunistic human fungal infections. The l ack of effective treatments against some of these pathogens makes it i mportant to develop new therapeutic strategies. One such strategy is t o target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting o f group I self-splicing introns in fungal pathogens. The group I intro n from the large ribosomal subunit RNA of mouse-derived Pneumocystis c arinii has been isolated and characterized. This intron self-splices i n vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for p otential antisense agents. At 37 degrees C, in 50 mM Hepes (25 mM Na+) , 15 mM MgCl2, and 135 mM KCl at pH 7.5, the exogenous 5' exon mimic r (AUGACU) binds about 60 000 times more tightly to this ribozyme than t o r(GGUCAU), a mimic of its complementary binding site on the ribozyme . This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2' OH groups of the 5' exon mimic do not f orm stabilizing tertiary interactions with the P-8/4x ribozyme, in con trast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37 degree s C, the exogenous 5' exon mimic d(ATGACT) binds nearly 32 000 times m ore tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligo nucleotides without 2' OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possibl e from base pairing. These results suggest a new paradigm for antisens e targeting: targeting the tertiary interactions of structural RNAs wi th short antisense oligonucleotides.