Synthetic RNA and DNA duplexes. Premelting, melting and postmelting transitions of alternating inosine-cytosine polynucleotides in solution.

Abstract

The duplex-to-strand transition of poly(I-C) and poly(dI-dC) has been monitored at the nonexchangeable base and sugar protons and the Watson-Crick imino and amino protons by 360-MHz nuclear magnetic resonance (NMR) spectrocopy in 0.1 M phosphate solution. The four base and two sugar H-1′ protons of the inosine-cytosine polynucleotides shift upfield on duplex formation with the magnitude of these melting transition shifts reflecting differences in the base pair overlap geometries for the synthetic DNA and the synthetic RNA duplexes in solution. The poly(I-C) nonexchangeable protons shift as average peaks during the melting transition and the protons distributed at the base and the sugar H-1′ positions can be plotted on a common melting curve representing the fraction of duplex as a function of temperature. The nonexchangeable protons which undergo large shifts during the melting transition exhibit uncertainty broadening contributions in the fast exchange region. The magnitude of the duplex dissociation rates (103–104 s−1) are consistent with the folding of the alternating purine-pyrimidine ribopolynucleotides into smaller duplexes which melt independently of each other. The exchange between duplex and strand states for poly(dI-dC) is slow on the NMR time scale with dissociation rates ≦0.1 × 103 s−1. We have outlined methods to correlate individual resonances in the duplex and strand states by extending the studies to high salt solution and comparing the chemical shift parameters for poly(dI-dC) with those previously reported for poly(dA-dT). The magnitude and direction of the temperature-dependent chemical shift changes at the base and sugar H-1′ resonances of poly(I-C) and poly(dI-dC) in the duplex state demonstrate a common structural change during the premelting conformational transition for the synthetic RNA and DNA duplexes. The synthetic DNA duplexes exhibit a predominant C-3′exo sugar pucker geometry in the strand state which appears to be maintained on duplex formation. By contrast, the synthetic RNA duplexes exhibit an equilibrium between C-3′exo and C-3′endo pucker geometries in the strand state, with the equilibrium shifting predominantly towards the latter on duplex formation. The Watson-Crick imino inosine H-1 proton, characteristic of the duplex state, is observed at 15.1 ppm in the spectra of poly(I-C) and poly(dI-dC) in H2O solution at 25 °C. These inosine H-1 resonances are about 2ppm downfield relative to the guanine H-1 resonances in alternating (dG-dC) oligonucleotide duplexes, indicative of the shielding effect of the purine 2-amino group on the purine 1-imino proton in a base pair. The cytosine H-4 amino proton in a Watson-Crick hydrogen bond resonates downfield from the cytosine H-4 amino proton exposed to solvent for poly(I-C) and poly(dI-dC) in the duplex state. We observe chemical shift changes for the poly(dI-dC) resonances in the duplex state on addition of 4M NaCl indicative of a conformational transition in high salt solution.