Efficient Synthesis of Se-DNAs and Se-RNAs for Structure and Function Studies

描述（由申请人提供）：对核酸和蛋白质-核酸复合物的3D结构测定的需求日益增长，以了解疾病分子水平的机制和发现新药。X射线晶体学是测定这些大分子和配合物结构的最直接、最有力的工具之一。然而，用于相测定的结晶和重原子衍生化在很大程度上减慢了新结构和折叠的结构测定。因此，开发允许相确定和结晶促进的新技术具有巨大的价值。最近，PI和他的研究小组开创并成功地证明了一种新的衍生化策略，通过用硒取代核酸的氧。他们的研究基于他们的中心假设，即由于氧和硒属于同一元素家族，因此硒可以用于稳定地取代核酸原子中的氧-特别是没有显著的扰动。他们已经成功地证明，他们的硒衍生化的核酸可以用来解决相位问题。此外，他们已经发现Se衍生化可以极大地促进DNA的结晶。因此，他们的硒衍生化策略具有很大的潜力，可以为这两个长期存在的问题提供新颖合理的解决方案，从而产生新的有价值的产品和服务。 虽然他们已经合成了许多硒衍生的亚磷酰胺和三磷酸盐（结构单元），但其相对低的合成规模和低效的纯化方案限制了他们合成少量和少量的Se-DNA和Se-RNA用于结晶，结构和功能表征。这些合成和纯化问题在很大程度上限制了其新技术的应用。在这个第一阶段的研究项目中，他们计划将亚磷酰胺的合成增加到克级（从目前的规模增加10倍），将三磷酸硒的合成增加到100毫克级（从目前的规模增加50-100倍）。他们在第一阶段项目中的主要目标是为硒结构单元和硒修饰的DNA和RNA开发有效的合成程序和纯化方案。此外，他们还将探索硒衍生化核酸的结晶。大量的初步结果有力地支持了拟议工作的可行性，研究团队在拟议的研究领域拥有广泛的专业知识。他们的长期目标是通过硒衍生化充分建立核酸的合理定相和结晶的标准和方便策略，以服务客户并满足这些新材料和核酸及其蛋白质复合物的3D结构测定的需求。 公共卫生相关性：硒衍生核酸在合理的相态确定和结晶促进方面具有巨大的潜力，可用于核酸及其蛋白质复合物的三维晶体结构测定，从而提供分子水平的疾病机制，并导致新的药物发现和疾病治疗。

DESCRIPTION (provided by applicant): There are growing demands for 3D structure determination of nucleic acids and protein-nucleic acid complexes for understanding disease molecule-level mechanisms and discovering new 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 heavy atom derivatization for phase determination have largely slowed down structural determination of new structures and folds. Therefore, developing novel technologies that allow the phase determination and crystallization facilitation is of tremendous value. Recently the PI and his research group have pioneered and successfully demonstrated a novel derivatization strategy via replacement of oxygen of nucleic acids with selenium. 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 significant perturbation. They have successfully demonstrated that their selenium derivatization of nucleic acids can be used to solve the phase problem. Furthermore, they have discovered that the Se-derivatization can largely facilitate crystallization of DNAs. Therefore, their Se-derivatization strategy has great potential to provide novel and rational solutions to these two long- standing problems, leading to new and valuable products and services. Though they have already synthesized many Se-derivatized phosphoramidites and triphosphates (the building blocks), their relatively-low synthetic scales and inefficient purification protocols limit them to synthesis of a small number and quantity of Se-DNAs and Se-RNAs for crystallization, structure and function characterizations. These synthesis and purification problems largely limit the applications of their novel technologies. In this Phase I research project, they plan to increase the phosphoramidite synthesis to the gram scale (10 fold increase from their current scale), and the Se-triphosphate synthesis to the 100-miligram scale (50-100 fold increase from their current scale). Their primary objectives in this Phase I project are to develop efficient synthesis procedures and purification protocols for the Se-building blocks and DNAs & RNAs modified with selenium. In addition, they will also explore crystallization of nucleic acids with selenium derivatization. Ample preliminary results strongly support the feasibility of the proposed work, and the research team has extensive expertise in the proposed research areas. Their long-term goal is to fully establish the standard and convenient strategies for the rational phasing and crystallization of nucleic acids via the selenium derivatization, in order to service the customers and meet the needs on these novel materials and the 3D structure determination of nucleic acids and their protein complexes. PUBLIC HEALTH RELEVANCE: Selenium-derivatized nucleic acids have great potentials in rational phase determination and crystallization facilitation for 3D crystal structure determination of nucleic acids and their protein complexes, which provide molecule-level disease mechanisms and lead to new drug discoveries and disease treatments.