QUANTITATION OF HYDROGEN BONDS OF DNA AND RNA BY NMR

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DNA and RNA play many functional roles: their various structures modulate processes such as gene regulation, translation, mitosis, and chromosome stability. Understanding nucleic acid structure and function, and malfunction, relies on a clear picture of their local and global structures. It is universally accepted that the hydrogen bond (H-bond) is of central importance in nucleic acid structure and function. Ironically, there is at present no method that can directly and quantitatively measure essential aspects of the H-bond in DNA and RNA molecules. Today, H-bond information on nucleic acid molecules is, at best, inferred from spatial proximity after the structure has been solved using other constraints. As interactions with modified bases, proteins, drugs, nucleic acids, and metals are mediated by specific H-bonds of varying angles and lengths, it is necessary to obtain quantitative parameters on H-bonds for a clearer understanding of the relationship between the structure of DNA, RNA, their complexes, and function. It is proposed here that H-bond length and angle can be measured In nucleic acids In a quantitative manner by the nuclear magnetic resonance (NMR) experiments presented below. The first experiment measures so called fractionation values (phi) at imino and amino sites of a nucleic acid molecule equilibrated in a known mixture of H2O/D2O. The phi value of, say, an imino site of a DNA molecule equilibrated in 50 percent H2O, 50 percent D2O is the population of deuterated over protonated states at that site, and will depend on the vibrational force constant of the imino site relative to that of the solvent. It is proposed here that phi values will be sensitive to H-bond angles of nucleic acid molecules, can be measured in an accurate and precise manner by NMR, and that an empirical relationship can be found between phi values and nucleic acid H-bond geometry. The second experiment presented here will allow the measurement of quadrupole coupling constant (QCC) values at (deuterated) imino and amino sites of DNA and RNA dissolved in D2O. QCC is sensitive to the local electrical symmetry. A short strong H-bond produces a more electrically symmetric environment at the amino or imino site relative to a longer H-bond. It is proposed here that the QCC values of deuterons at these sites will reflect the H-bond distance in DNA and RNA, that they can be measured accurately and precisely by NMR, and that a robust empirical relationship between QCC and nucleic acid H-bond length can be established. Together, phi and QCC values will provide invaluable information on the structure of the H-bond in DNA and RNA, and they will provide the first opportunity to investigate H-bond geometries directly and quantitatively in these molecules. A specific aim of this project is to develop the NMR experiments which will be able to measure phi and QCC values in the most accurate and precise manner. The long term objective is to establish the relationships between H-bond geometries and the NMR observables.