NMR investigation of DNA conformational changes on base protonation: use of copper(2+) and pyrazole as probes

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Abstract
In order to evaluate models for the acid denaturation of DNA and to assess the potential importance of protonated bases in mutations and gene expression, an NMR investigation of DNA and nucleotides in the pH range 7-2 has been conducted. The changes in the imino proton spectral region are readily observed and quite dramatic on lowering pH. At pH 7.0, calf thymus DNA has imino proton signals for AT (13.6 ppm, 56% area) and GC (12.6 ppm, 44% area) base pairs but no peaks in the 10-12 ppm region. At pH 5 a broad peak(s) between 10 and 11 ppm was (were) observed, and it narrowed and shifted to 10.9 ppm at pH 3.2. The original GC area was lost by pH 3.2 while the AT area was reduced by 50%. Below pH 3 the remainder of the AT signal was lost, and the area of the 10.9 ppm peak increased. Over this pH range the aromatic proton signals of DNA sharpened, and the cytosine amino protein signals in DNA narrowed and shifted downfield. Addition of pyrazole in the pH 4-6 range caused broadening of the new resonanance but had very little effect on the original signals. Addition of Cu2+ in the pH 4-6 range resulted in a large loss in area of GC and the new upfield peak(s). However, at lower pH, the upfield peak was not totally broadened by Cu2+. At pH below 7, the broad 31P signal of calf thymus DNA shifted slightly downfield and sharpened. This last result is inconsistent with a previously proposed model in which the deoxyguanosine adopts a syn conformatoin. The new results on DNA are explained by an alternate model in which initial protonation of C in the pH 5-7 range leads to the formation of non-base-repair CH+ and G. For such a disrupted base pair, the G imino protein resonates at approximately 10.6 ppm, and the G is probably stacked in the double helix but with enhanced breathing motions. The pKa of C in the DNA is 3.7. At pH below 5, there is continued protonation of C, initiation of protonation of A (pKa = 3.2), and shifting of the new peak from 10.6 to 10.9 ppm, indicating more severe disruption of the duplex.