ANTISENSE BINDING ENHANCED BY TERTIARY INTERACTIONS - BINDING OF PHOSPHOROTHIOATE AND N3'-]P5' PHOSPHORAMIDATE HEXANUCLEOTIDES TO THE CATALYTIC CORE OF A GROUP-I RIBOZYME FROM THE MAMMALIAN PATHOGEN PNEUMOCYSTIS-CARINII

Pneumocystis carinii is the most common lethal opportunistic pathogen infecting Acquired Immune Deficiency Syndrome (AIDS) patients, and mor e effective therapeutics for it are needed. P. carinii, but not humans , contain RNA self-splicing group I introns, so these functionally imp ortant RNAs are potential anti-fungal targets. In vitro, d(ATGACT), wh ich mimics the 3' end of the 5' exon of a conserved ribosomal RNA grou p I intron from mouse-derived Pneumocystis carinii binds to a ribozyme that is a truncated form of this intron. The binding is about 30,000 times tighter than expected for simple base-pairing because binding is enhanced by tertiary interactions. Here we report the effects of modi fying the phosphodiester backbone of d(ATGACT) with phosphorothioate a nd of d(ATGAC)rU with N3'-->P5' phosphoramidate linkages. The enhancem ent of binding by tertiary interactions is not substantially decreased , and in some cases is increased when single R-p and S-p phosphorothio ate substitutions are made, although overall binding is weaker by up t o 6-fold. A mixture of 5' exon mimic isomers that each contain five ph osphorothioate linkages binds to the ribozyme at least 14-fold less ti ghtly than the corresponding phosphodiester mimic. In contrast, the 5' exon mimic with five internal N3'-->P5' phosphoramidate linkages bind s 4-fold more tightly than d(ATGAC)rU. This increased binding is large ly due to more favorable base-pairing, but tertiary interactions still enhance binding by more than 2,000-fold. These results indicate that chemically modified, nuclease stable 5' exon mimics can act as antisen se agents with binding enhanced by tertiary interactions (BETI). This strategy permits design of short antisense agents with high specificit y.