Se-Derivatization of Functional RNAs for Structure Study

描述（由申请人提供）：本研究的目的是使用多波长异常色散（MAD）定相技术，用硒原子选择性地取代核酸（特别是功能性RNA）的氧原子，用于X射线晶体结构测定。 与用于蛋白质MAD定相的蛋氨酸残基中硫的硒同晶取代类似，硒也可用于取代核苷酸氧，因为硒（原子半径，1.2埃）、硫（1.1埃）和氧（0.68埃）在周期表中的相同族VIA中。 由于核苷酸氧原子周围的空间通常可用于容纳更大的原子，如硒，因此硒取代氧原子的空间问题应该不是问题。 因此，假设硒可以通过选择性取代氧而化学地和稳定地掺入核酸中，并且这种选择性取代在结构上和化学上与其含氧对应物同晶。 与传统的溴衍生化（仅限于嘧啶的5位）不同，硒可以选择性地掺入所有核苷酸结构单元（核糖和脱氧核糖系列中的A、C、G、U和T）的各个位置，例如，2 '-、3'-、5 '-核糖氧、磷酸非桥氧和核碱基氧。 取代中的更大选择允许避免对功能RNA的结构和功能扰动。 Se-亚磷酰胺和三磷酸盐结构单元将由相应的在不同位置含有硒的核苷衍生物设计和合成。 硒-亚磷酰胺和硒-三磷酸盐将被应用于功能RNA与硒的化学和酶促衍生化。 这种硒取代策略在通过标记核酸而不是蛋白质对应物来衍生RNA-蛋白质和DNA-蛋白质复合物方面也具有很大的潜力。 这种硒策略将极大地促进核酸及其与药物和蛋白质的复合物的3-D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.