Se-Derivatization of Functional RNAs for Structure Study

描述（由申请人提供）：本研究的目的是利用多波长异常色散（MAD）相位技术，选择性地用硒原子取代核酸，特别是功能性rna的氧原子，用于x射线晶体结构的测定。类似于硒同形取代蛋氨酸残基中的硫以取代蛋白质的MAD相，硒也可以用来取代核苷酸氧，因为硒（原子半径为1.2埃），硫（1.1埃）和氧（0.68埃）在元素周期表中属于同一个家族VIA。由于核苷酸氧原子周围的空间通常可以容纳更大的原子，例如硒，因此硒取代氧原子的空间问题应该不是问题。因此，我们假设硒可以通过选择性取代氧在化学上稳定地掺入核酸中，并且这种选择性取代在结构和化学上与含氧对应物是同构的。与传统的溴衍生化反应不同，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）的不同位置，如2'-,3'-,5'-核糖氧，磷酸盐非桥接氧和核碱基氧。更大的替代选择允许避免对功能性rna的结构和功能扰动。将设计和合成含有不同位置硒的相应核苷衍生物的硒磷酰胺和三磷酸基。硒-磷酰胺和硒-三磷酸将应用于含硒的功能性rna的化学和酶衍生化。这种硒替代策略也有很大的潜力，在rna -蛋白质和dna -蛋白质复合物的衍生化标记核酸而不是相应的蛋白质。这种硒策略将极大地促进核酸及其与药物和蛋白质配合物的三维晶体结构测定。

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.