BINDING OF GUANOSINE AND 3' SPLICE SITE ANALOGS TO A GROUP-I RIBOZYME- INTERACTIONS WITH FUNCTIONAL-GROUPS OF GUANOSINE AND WITH ADDITIONAL NUCLEOTIDES

Dissociation constants, K(d), were measured by equilibrium dialysis at 5-degrees-C for a series of substrates binding to the L-21 ScaI riboz yme derived from the Tetrahymena thermophila self-splicing large subun it (LSU) ribosomal RNA intron. These substrates are analogues for the 3' exon splice site, the cyclization site, and the exogenous G that in itiates group I splicing. UCG has a K(d) of 17 muM. Lengthening the su bstrate to GUCG and GGUCG enhances binding but by less than expected f rom potential base pairing. Functional groups on the 3'-terminal G of GUCG were replaced with H to test their effect on binding. GUC(2'dG) b inds slightly tighter than the all-ribose molecule but shows no reacti vity as a substrate. GUC(3'dG) binds weaker than GUCG. Inosine and 2-a minopurine ribonucleoside at the 3' position weaken binding by 16- and 26-fold, respectively, but both tetramers are reactive. Thus hydrogen bonds to Watson-Crick pairing positions of the 3'G of GUCG contribute 1-2 kcal/mol to the free energy change for binding. Similar results a re found in comparisons of UCG with UC(2'dG), UC(3'dG), and UCI. The n onreactive substrate GUCdGA includes a phosphodiester bond 3' to the g uanosine that is the site of chemistry for the all-ribose substrate GU CGA; GUCdGA binds 50 times more weakly than GUCdG. A similar result is obtained for GUCdGU. Competition experiments show that guanosine and guanosine 5'-monophosphate bind with dissociation constants of about 0 .9 mM. The monomers 2'dG and 3'dG have K(d)'s of 0.5 and greater-than- or-equal-to 3 mM, respectively. This suggests that sugar pucker and/or interactions with hydroxyl groups affect binding. Implications for ri bozyme catalysis, splicing, cyclization, and design of antisense oligo mers are discussed.