Selenium-derivatized New Reagents for Nucleic Acid X-ray Crystallography

 描述(申请人提供)：为了了解疾病分子水平的机制和发现创新的药物，对核酸及其蛋白质复合体的三维结构测定的需求越来越大。X射线结晶学是确定这些大分子及其络合物结构的最直接、最有力的工具之一。然而，结晶和物相确定(两个长期存在的瓶颈问题)在很大程度上减缓了新结构和褶皱的结构确定。因此，开发新的技术来促进结晶和物相确定具有巨大的价值。最近，Pi的小组开创并成功地展示了一种新的衍生化策略，即用硒(Se)取代核酸的氧。他们的研究基于他们的核心假设，即由于氧和硒属于同一元素家族，硒可以稳定地取代核酸原子中的氧--特别是不会造成重大扰动。我们已经成功地证明了它们的硒衍生核酸(SENA)可以用来解决相问题。此外，我们还发现，原子特异性的Se衍生化可以在很大程度上促进DNA、RNAs及其与蛋白质的络合物的结晶。因此，SENA战略具有巨大的潜力，可以为这两个长期存在的问题提供新颖合理的解决方案，带来有价值的产品和服务。虽然我们已经合成了几种硒衍生的亚磷酰胺和三磷酸盐(Se构块试剂)，但相对较低的合成规模和低效的提纯方案限制了它们合成少量和数量的Se-RNA和Se-DNA(Se-寡核苷酸/核酸试剂)，用于结晶、物相确定和结构与功能表征。尽管我们已经成功地完成了STTRI期项目，但合成规模在很大程度上限制了新试剂和新技术的应用。在这个二期研究项目中，我们计划将亚磷酸盐的合成提高到几十克的规模(每个50克)，将三磷酸的合成提高到克的规模(每个1克)。这些鳞片比目前的合成鳞片大20-30倍。我们的主要目标是实现Se构筑块和Se-DNA和Se-RNAs的大规模合成，并通过Se衍生化促进核酸及其蛋白质络合物的结晶和相结构的确定。充足的初步结果有力地支持了拟议工作的可行性，该小组在拟议的研究领域拥有广泛的专业知识。我们的长期目标是充分建立标准和方便的策略，通过Se衍生化来合理地促进核酸的结晶和确定其物相结构。我们的新型电子技术将为客户服务于核酸及其与蛋白质和小分子的络合物的结构测定，以发现新的药物和生物。

 DESCRIPTION (provided by applicant): There are growing demands for 3D structure determination of nucleic acids and their protein complexes for understanding disease molecule-level mechanisms and discovering innovative drugs. X-ray crystallography is one of the most direct and powerful tools for structure determination of these macromolecules and complexes. However, the crystallization and phase determination (two long-standing and bottle-neck problems) have largely slowed down structural determination of new structures and folds. Therefore, it is of tremendous value to develop novel technologies that allow the crystallization facilitation and phase determination. Recently, the PI's group has pioneered and successfully demonstrated a novel derivatization strategy via replacement of oxygen of nucleic acids with selenium (Se). Their research is based on their central hypothesis that since oxygen and selenium are in the same elemental family, selenium can be used to stably replace oxygen of nucleic acids atom-specifically without causing significant perturbation. We have successfully demonstrated that their Se-derivatized nucleic acids (SeNA) can be used to solve the phase problem. Furthermore, we have discovered that the atom-specific Se-derivatization can largely facilitate crystallization of DNAs, RNAs and their complexes with proteins. Therefore, the SeNA strategy has great potential to provide novel and rational solutions to these two long-standing problems, leading to valuable products and services. Though we have already synthesized several Se-derivatized phosphoramidites and triphosphates (the Se-building block reagents), the relatively-low synthetic scales and inefficient purification protocols limit them to synthesizea small number and quantity of the Se-RNAs and Se-DNAs (the Se-oligonucleotide/nucleic acid reagents) for crystallization, phase determination, and structure & function characterizations. The synthesis scales largely limit applications of the novel reagents and technologies, though we have successfully completed STTR Phase I project. In this Phase II research project, we plan to increase the Se- phosphoramidite synthesis to tens of grams in scale (50 gram each) and the Se-triphosphate synthesis to gram scale (1 gram each). These are 20-30 times larger than their current synthetic scales. Our primary objectives are to achieve larger-scale synthesis of the Se-building blocks and Se-DNAs & Se-RNAs and to facilitate crystallization and phase & structure determination of nucleic acids and their protein complexes via the Se- derivatizations. Ample preliminary results strongly support feasibility of the proposed work, and the team has extensive expertise in the proposed research areas. Our long-term goal is to fully establish standard and convenient strategies for rational crystallization facilitation and phase & structure determination of nucleic acids via the Se-derivatizations. Our novel Se-technologies will service the customers in structure determination of nucleic acids and their complexes with proteins and small molecules for discovering new drugs and biology.