Se-Derivatization of Functional RNAs for Structure Study

描述(申请人提供)：这项研究的目标是选择性地用硒原子取代核酸中的氧原子，特别是功能RNA，用于使用多波长反常色散(MAD)位相技术确定X射线晶体结构。类似于蛋氨酸残基中硫的硒同象取代蛋白质的相变，硒也可以用来替代核苷酸氧，因为在元素周期表中，硒(原子半径1.2埃)、硫(1.1埃)和氧(0.68埃)属于同一家族。由于核苷酸氧原子周围的空间通常可以容纳更大的原子，如硒，因此硒取代氧原子的空间问题应该不是问题。因此，可以假设，通过选择性地取代氧，可以化学地、稳定地将硒结合到核酸中，并且这种选择性取代在结构和化学上与其含氧化合物同构。与传统的溴衍生化仅限于5位嘧啶不同，硒可以选择性地结合到所有核苷酸构建块(核糖和脱氧核糖系列中的A、C、G、U和T)中，在不同的位置，如2‘-，3’-，5‘-核糖氧，磷酸非桥氧，和碱基氧。在替换方面有更大的选择可以避免对功能RNA的结构和功能扰动。由相应的含硒核苷衍生物在不同的位置设计和合成硒-亚磷酰胺和三磷酸构筑块。硒-亚磷酰胺和硒-三磷酸盐将用于含硒功能核糖核酸的化学和酶促衍生化。这种取代硒的策略通过标记核酸而不是蛋白质，在RNA-蛋白质和DNA-蛋白质复合体的衍生化方面也有很大的潜力。这种硒策略将极大地促进核酸及其与药物和蛋白质的络合物的三维晶体结构的确定。

DESCRIPTION (provided by applicant): The goal of this research is to selectively replace oxygen atoms of nucleic acids, especially functional RNAs, with selenium atoms for X-ray crystal structure determination using Multiwavelengh Anomalous Dispersion (MAD) phasing technique. Analogous to the selenium isomorphous substitution of the sulfur in the methonine residue for protein MAD phasing, selenium may also be used to substitute nucleotide oxygen, as selenium (atomic radii, 1.2 Angstroms), sulfur (1.1 Angstroms), and oxygen (0.68 Angstroms) are in the same Family VIA in the Periodic Table. Since the space surrounding nucleotide oxygen atoms is usually available to accommodate a larger atom, such as selenium, the steric issue of selenium replacing oxygen atom should not be a problem. Therefore, it is hypothesized that selenium can be chemically and stably incorporated into nucleic acid by selectively substituting oxygen, and such selective substitution is structurally and chemically isomorphous to its oxygen-containing counterpart. Unlike the conventional derivatization with bromine, which is limited to the 5-positions of the pyrimidines, selenium can be selectively incorporated into all nucleotide building blocks (A, C, G, U, and T, in both ribose and deoxyribose series) at various positions, such as, 2'-, 3'-, 5'-ribose oxygen, phosphate non-bridging oxygen, and nucleobase oxygen. A greater choice in the substitution allows avoidance of structural and functional perturbation on functional RNAs. The Se-phosphoramidite and triphosphate building blocks will be designed and synthesized from the corresponding nucleoside derivatives containing selenium at various positions. The Se-phosphoramidites and Se-triphosphates will be applied in chemical and enzymatic derivatization of functional RNAs with selenium. This selenium-substitution strategy also has great potential in derivatization of RNA-protein and DNA-protein complexes by labeling nucleic acids instead of the protein counterparts. This selenium strategy will greatly facilitate 3-D crystal structure determination of nucleic acids and their complexes with drugs and proteins.