Imino Proton Exchange and Base-Pair Kinetics in RNA Duplexes

Abstract

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton, we have measured the base-pair lifetimes and the dissociation constants of six RNA duplexes: [r(CGCGAUCGCG)]₂, [r(CGCGAAUUCGCG)]₂, [r(CCUUUCGAAAGG)]₂, [r(CGCACGUGCG)]₂, [r(GGU₈CC)·r(GGA₈CC)], and [poly(rA)·poly(rU)], and we compare them with those of their DNA homologues. As predicted by a two-state (closed/open) model of the pair, the imino proton exchange times decrease linearly vs. the inverse of catalyst concentration. As in DNA duplexes, base pairs open one at a time, and the kinetics is in most cases insensitive to the nature of the adjacent residues. The lifetime of the r(G·C) pairs, 40 to 50 ms, is longer than that of the equivalent in the corresponding oligodeoxynucleotides, and the dissociation constants, about 10^-7, are slightly smaller. The r(A·U) opening and closing rates are much larger than those of the d(A·T) pairs, but the stabilities are comparable.