QUANTITATION OF HYDROGEN BONDS OF DNA AND RNA BY NMR

DNA和RNA发挥着许多功能作用：它们的各种结构调节基因调控、翻译、有丝分裂和染色体稳定性等过程。 理解核酸的结构和功能，以及故障，依赖于它们的局部和全局结构的清晰图像。 氢键（H-键）在核酸的结构和功能中具有核心的重要性。 具有讽刺意味的是，目前还没有一种方法可以直接和定量地测量DNA和RNA分子中氢键的基本方面。 今天，核酸分子的氢键信息最多是在使用其他约束解决结构之后从空间邻近性推断出来的。 由于与修饰碱基、蛋白质、药物、核酸和金属的相互作用是由不同角度和长度的特定氢键介导的，因此有必要获得氢键的定量参数，以便更清楚地了解DNA、RNA及其复合物的结构与功能之间的关系。 本文提出，可以通过以下呈现的核磁共振（NMR）实验以定量方式测量核酸中的H键长度和角度。 第一个实验测量在已知的H2O/D2 O混合物中平衡的核酸分子的亚氨基和氨基位点处的所谓分馏值（phi）。 比方说，在50%H2O和50%D2O中平衡的DNA分子的亚氨基位点的phi值是该位点上氘代态相对于质子化态的总体，并且将取决于亚氨基位点相对于溶剂的振动力常数。 在此提出，phi值将对核酸分子的氢键角敏感，可以通过NMR以准确和精确的方式测量，并且可以在phi值和核酸氢键几何形状之间找到经验关系。 这里提出的第二个实验将允许四极耦合常数（QCC）值的测量（氘代）亚氨基和氨基位点的DNA和RNA溶解在D2 O。 QCC对局域电对称性敏感。 相对于较长的H-键，短的强H-键在氨基或亚氨基位点产生更电对称的环境。 在此提出，在这些网站上的氘核的QCC值将反映在DNA和RNA的氢键距离，它们可以被准确和精确地测量NMR，并可以建立一个强大的经验QCC和核酸氢键长度之间的关系。 Phi和QCC值一起将提供有关DNA和RNA中氢键结构的宝贵信息，它们将首次提供直接和定量研究这些分子中氢键几何形状的机会。 该项目的一个具体目标是开发NMR实验，以最准确和精确的方式测量phi和QCC值。 长期目标是建立氢键几何形状和NMR观测值之间的关系。

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.