Hydrogen Exchange Rates of the DNA Oligomer Duplex, d[CGCGCGCCATAAC]/d[GTTATGGCGCGCG] Determined by NMR Spectroscopy

Abstract

The measurement of hydrogen exchange rates has been an important tool for studying the macromolecular conformation or dynamics. The imino proton resonances of nucleic acids are good probes of the hydrogen exchange study for studying the dynamics of each base pair. The exchange rates of imino protons depend on not only the base pair stability but also solvent accessibility. Hydrogendeuterium (H/D) exchange method is widely used to measure the exchange rates of slowly exchanging imino protons, whereas the exchange rates of fast-exchanging imino protons can be determined by water magnetization transfer method of NMR. NMR hydrogen exchange experiment can provide the information on the thermodynamics and kinetics for basepair opening in the nucleic acids. Lots of results of these experiments determined in various DNAs, RNAs and DNA-protein complexes were reported. Analysis of the hydrogen exchange of imino protons generally was based in the two-state (open/closed) model for the base pair where hydrogen exchange only occurs from the open state. The opening/closing rate constants and/or equilibrium constant for base-pair opening can often be determined by measuring the exchange as a function of the concentration of external catalysts such as ammonia or TRIS. Left-handed Z-DNA is a higher-energy form of the double helix produced at an alternating CG sequence region and is stabilized by high salt concentration or negative super-coiling. Recently, X-ray crystal structure of the B-Z junction induced by the Z-DNA binding protein, where the alternating CGCGCG exhibited left-handed Z-DNA whereas a nonalternating AT-rich region maintained B-form helix, was reported. They also found the continuous stacking of bases between B-DNA and Z-DNA segments with the breaking of one base pair at the junction. This result indicates that the one base pair at the B-Z junction should be destabilized and then its bases might be extruded to allow the B-Z conformational change at the CGCGCG region. Here, we measured the imino proton exchange rates for a bzDNA-13 DNA duplex formed by an 15-nt bzA-15mer, d[GTCGCGCGCCATAAC] and a 13-nt bzB-13mer, d[GTTATGGCGCGCG] (Fig. 1A) by water magnetization transfer method to understand the dynamic character of base pairs. Experimental Section