Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG.cntdot.dA mismatch site

Abstract

NMR was used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (12-mer GA) that contains a dG-dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (12-mer) containing standard Watson-Crick base pairs at position 3. The dG-dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. Base-pair formation between anti orientations of the guanosine and adenosine rings is demonstrated on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti).cntdot.dA(anti) pairing should result in H-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG.cntdot.dA pair remains intact at low temperature, but these dG.cntdot.dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. The H exchange kinetics at positions 4-6 were estimated from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5-40.degree. C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of .apprx. 2 compared to the corresponding values in the 12-mer duplex. This implies that H exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G.cntdot.T wobble pair at position 3. The nonexchangeable base protons were assigned by intra and inter base pair NOE experiments and these assigned markers were monitored through the 12-mer GA duplex to strand transition. Replacing 2 dG.cntdot.dC by 2 dG.cntdot.dA base pairs reduces the melting temperature of the dodecanucleotide by .apprx. 17.degree. C. The phosphorus spectrum of the 12-mer GA differs significantly from that of the 12 mer, indicative of changes in the phosphodiester backbone in order to accommodate the dG (anti).cntdot.dA(anti) base-pair formation in the interior of a DNA duplex.