Thermodynamics of RNA-RNA duplexes with 2-or 4-thiouridines: Implications for antisense design and targeting a group I intron

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick bas e pairing between the antisense compound and target provides the key recogn ition element. Uridine (U), however, not only stably base pairs with adenos ine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stabi lity of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interacti ons, binding to a group I intron ribozyme from mouse-derived Pneumocystis c arinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGAC s(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUCACU) is invol ved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of transsplicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, mod ified bases can enhance the specificity of base pairing while retaining oth er activities and, thus, increase the specificity of antisense compounds ta rgeting cellular RNA.