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

用于核酸 X 射线晶体学的硒衍生新试剂

• 批准号: 8907532
• 负责人: Zhen Huang
• 金额: $ 30.79万
• 依托单位: SENA RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8907532
• 关键词: Address; Antisense DNA; Area; Award; Biology; Complex; Crystallization; DNA; Disease; Drug Targeting; Family; Gene Silencing; Genetic Transcription; Goals; Hereditary Disease; Lead; Length; Letters; Marketing; Neck; Nucleic Acids; Nucleosides; Oligonucleotides; Oxygen; Pharmaceutical Preparations; Phase; Preparation; Proteins; Protocols documentation; RNA; Reagent; Research; Research Project Grants; Resolution; Role; S Phase; Selenium; Series; Services; Small Business Technology Transfer Research; Small Interfering RNA; Solid; Solutions; Structure; Technology; Thymidine; Time; Uridine; Work; X-Ray Crystallography; aptamer; base; chemical synthesis; commercialization; drug discovery; innovation; macromolecule; meetings; new technology; novel; nucleic acid structure; nucleobase; phosphoramidite; polymerization; protein complex; protein structure; public health relevance; scale up; small molecule; three dimensional structure; tool; tripolyphosphate

 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.

 描述（由申请人提供）：对核酸及其蛋白质复合物的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创新药物。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，结晶和相确定（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，允许结晶促进和相确定是具有巨大价值的。最近，PI的小组已经开创并成功地证明了一种新的衍生化策略，通过用硒（Se）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，因此硒可以用于原子特异性地稳定取代核酸中的氧，而不会引起显著的扰动。我们已经成功地证明，他们的硒衍生核酸（SeNA）可以用来解决相位问题。此外，我们还发现原子特异性硒衍生化可以极大地促进DNA、RNA及其与蛋白质的复合物的结晶。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而合理的解决方案，从而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（硒构件试剂），但相对较低的合成规模和低效的纯化方案限制了它们合成用于结晶、相确定和结构与功能表征的少量Se-RNA和Se-DNA（硒寡核苷酸/核酸试剂）。尽管我们已经成功完成了STTR一期项目，但合成规模在很大程度上限制了新试剂和新技术的应用。在这个第二阶段研究项目中，我们计划将Se-亚磷酰胺的合成增加到数十克规模（每个50克），并将Se-三磷酸盐的合成增加到克规模（每个1克）。这些比目前的合成规模大20-30倍。我们的主要目标是实现硒结构单元和Se-DNA和Se-RNA的大规模合成，并通过硒衍生化促进核酸及其蛋白质复合物的结晶和相和结构测定。大量的初步结果有力地支持了拟议工作的可行性，该团队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战略，合理的结晶促进和相和结构的核酸通过硒衍生化的测定。我们的新型硒技术将为客户提供核酸及其与蛋白质和小分子复合物的结构测定服务，以发现新药和生物学。