Inosine — Adenosine base pairing in d-GGTACIAGTACC: Duplex and hairpin with a two-base loop

An oligonucleotide dodecamer d-GGTACIAGTACC containing two inosine-adenosine mismatched base-pairs has been studied by1H and31P NMR spectroscopy. Unique assignments of1H and31P spins have been achieved by using a recently proposed two-dimensionalheteronuclearlong rangecorrelation (2D-HELCO) technique. The 2D nuclear Overhauser enhancement spectroscopy (NOESY) spectrum recorded in a mixed solvent of 90% H2O+10%2H2O has been used to assign all the exchangeable imino and amino proton resonances. NMR results indicate that at concentrations above 5 mM, the molecule adopts a duplex structure where the inosine imino protons are hydrogen bonded. Cross-strand NOEs between the imino proton of I6 and H2 proton of A7 and that between H2 of I6 and H2 of A7 provide evidence for inosine-adenosine base-pairing, with both bases I6 and A7 inanti conformation. The observed NOE connectivitiesd1,d2 andd3 from almost the middle of the molecule to the two ends are consistent with a right handed B-DNA conformation. However, the molecule adopts a more extended conformation in the mismatch region to accomodate the bulky purine-purine (IA) base pairs. NMR experiments at lower concentrations indicate the coexistence of a monomeric hairpin with a two base loop, consisting of I6 and A7 units, in equilibrium with the duplex. 2D-ROESY experiment at such a concentration shows that the conformation of the stem of the hairpin does not deviate much from the corresponding region of the duplex and four nucleotides C5, I6, A7 and G8 undergo significant structural changes during the formation of hairpin loop.