Abstract
Two-dimensional proton NMR studies were undertaken on the d(C-G-A-G-A-A-T-T-C-C-C-G) duplex (designated A.C 12-mer) where the A at the mismatch site is flanked by G residues and the d(C-G-C-G-A-A-T-T-C-A-C-G) duplex (designated C.A 12-mer) where the A at the mismatch site is flanked by C residues in an attempt to elucidate the role of flanking base pairs on the structure of the A.C mismatch. The exchangeable and nonexchangeable proton spectra of these two dodecanucleotides have been completely characterized by two-dimensional nuclear Overhauser enhancement (NOE) experiments in H2O and D2O solution at acidic pH. The NOE distance connectivities demonstrate that both A and C at the mismatch site are stacked into a right-handed helix between flanking G.C base pairs and exhibit anti-glycosidic torsion angles. The proton chemical shifts and NOE patterns are consistent with Wobble A.C pairing for the A.C 12-mer and C.A 12-mer duplexes in solution and demonstrate that the A.C mismatches introduce local conformational perturbations that do not extend to the central AATT segment. We detect that amino protons of adenosine (approximately 9.2 ppm) but not of cytidine at the A.C mismatch site in both duplexes on lowering the pH below 6.
Highlights
Two-dimensional proton NMR studies were under- in Refs. 8 and 9) to elucidate the pairing at themismatch site taken on the d(C-G-&G-A-A-T-T-C-c-C-G) duplex and theresulting conformational perturbations
T-T-C-4-C-G)duplexwhere mismatches lower the helix to coil transition midpoint with the A at the mismatch site is flanked by C residues in an attemptto elucidate the roleof flanking base pairs the effect more pronounced for A.C compared to GST mismatches [6, 10,11,12]
The earlier proton NMR study on the C- A 12-mer was undertaken at pH 7.0 in H 2 0solution and at pH 7.2 in D 2 0 solution with the spectra characterized by somewhat broadened resonances [13]
Summary
Oligonucleotide Synthesis and Purificatwn-Protected nucleotides and protected 2-cyanoethylphosphoramiditesalong with DNA synthesis reagents and solvents were purchased from Beckman Instruments. Sequence synthesis tion of higher resolution techniques such as solution NMR was carried out on a 10-pmol scale on a Beckman System 1 Plus Two-dimensional magnitude correlated (COSY) and phase-sensitive nuclear Overhauser enhancement (NOESY) data sets were recorded on the A.C 12-mer and C. The magnitude COSY spectra were accumulated for 256 tl increments, and the data sets were apodized with an unshifted sine bell function zeroed to the 600th point in the tz dimension and to the 256th point in the tl dimension prior to Fourier transformation. The phase-sensitive NOESY spectra were accumulated for 256 complext, increments, and different window functions were used to process the data prior to Fourier transformation. The chemical shifts are referenced relative to the phosphorus resonance of external trimethyl phosphate
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