THE 2,6-DIAMINOPURINE RIBOSIDE-CENTER-DOT-5-METHYLISOCYTIDINE WOBBLE BASE-PAIR - AN ISOENERGETIC SUBSTITUTION FOR THE STUDY OF G-CENTER-DOT-U PAIRS IN RNA

Phylogenetically invariant G.U wobble pairs are present in a wide variety of RNA's. As a means to study the contribution of individual chemical groups within a G.U pair, we have synthesized and thermodynamically characterized oligoribonucleotides containing the unnatural nucleosides 2,6-diaminopurine riboside (DAP) and 5-methylisocytidine ((Me)iC). The DAP.(Me)iC pair at the end of an RNA duplex is as stable as a G.U pair, consistent with formation of a wobble base pair with two hydrogen bonds. DAP.(Me)iC is a valuable substitution for the study of G.U wobble pairs because it is conformationally similar to the G.U pair, but has a different array of functional groups in the major and minor grooves of the duplex and a reversed hydrogen bonding polarity between the bases. We also report the stability of several other terminal pairs proposed to be in a wobble configuration including inosine.U (I.U), A.(Me)iC, DAP.C, A.C, G.5-methyl-U, 2'-deoxyguanosine.U, and 2'-deoxy-7-deazaguanosine.U. These pairs present a diversity of functional group substitutions in the context of a wobble conformation. Comparison of wobble pairs with and without the N2 exocyclic amine, i.e., G.U vs I.U, DAP.(Me)iC vs A.(Me)iC, and DAP.C vs A.C, demonstrates that the amine does not contribute to base pairing stability when the pair is located at the terminal position of the RNA duplex. However, at a position internal to the duplex, the exocyclic amine does improve helix stability. An internal I.U pair is less stable (approximate to 1 kcal.mol(-1)) than an internal G.U pair, and substantially less stable (approximate to 2 kcal.mol(-1)) than an internal A-U pair. These data provide quantitation for the reduced duplex stability observed upon conversion of A-U to I.U pairs by double-stranded RNA adenosine deaminase (dsRAD). This collection of wobble pairs will help identify the contribution made by individual functional groups in RNA/protein interactions and in the tertiary folding of RNA.