Internal motions of nucleic acid structures and the determination of base-pair lifetimes

Over the years, imino proton exchange measurements have provided a description of base-pair opening and of properties of internal motions of nucleic acids such as the individual opening of base pairs and the insensitivity of base-pair lifetimes to the nature and stability of neighboring pairs. A recent determination of base-pair lifetimes in d(CGCGATCGCG) conflicts with the original measurements and their interpretation. This question is analyzed in the present work. We emphasize the importance of high concentrations of exchange catalyst (eg 1 M NH3) for the accurate determination of base-pair lifetimes. These concentrations entail a high ionic strength, which can lead to aggregation, enhanced magnetic relaxation and underestimation of base-pair lifetimes if exchange is measured by its effect on the proton relaxation rate. Magnetization transfer which provides a more direct method for the measurement of proton exchange rates is therefore preferred. We show that the lack of measurements at high catalyst concentration is responsible for the discrepancy mentioned above. Measurements by the magnetization transfer method validate the original interpretation and inversion recovery experiments illustrate the effect of the ionic strength on the relaxation rate of the imino protons.