Imino proton exchange in DNA catalyzed by ammonia and trimethylamine:: Evidence for a secondary long-lived open state of the base pair

The base-pair opening kinetics of the self-complementary oligomer d(CGCGAATTCGCG)(2) has been derived from NMR measurements of the imino proton exchange. In general, it has previously been found that imino proton exchange in duplex DNA is limited by the proton-transfer step from the open state and that the dependence of the exchange times on the inverse concentration of an added exchange catalyst is linear. In the present study, a curvature is observed for, in particular, the innermost AT base pair,with both ammonia and trimethylamine (TMA) as exchange catalysts. The two catalysts act on the same open states, but the accessibility of TMA is reduced by a factor of 2-3 compared to ammonia. Assuming that ammonia accesses the imino proton equally in the open state of the base pair and in the mononucleoside, the curvature is consistent with 7-9% of the openings ending in open states with lifetimes of-about 1 mu s while the bulk of open-state lifetimes fall in the nanosecond range. A curvature is also found for the exchange times of the imino protons in the A-tract sequence CGCA(8)CGC/GCGT(8)GCG. This curvature becomes increasingly pronounced from the 5'-end toward the center of the tract and hereby seems to be correlated with the contraction of the minor groove. Thus, while the base-pair lifetimes deduced from the present study are in accordance with previous measurements, a substantial fraction of the open states formed by the central AT-base pairs in the two oligomers,exhibits microsecond lifetimes in contrast to previous estimates in the nanosecond range. These findings:may be of relevance for the way sequence specific recognition is accomplished by proteins and ligands.