Systematic Derivatization of Nucleic Acids with Selenium for X-ray Crystallography

用于 X 射线晶体学的硒系统核酸衍生化

• 批准号: 0517092
• 负责人: Zhen Huang
• 金额: --
• 依托单位: Georgia State University Research Foundation, Inc.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517092&HistoricalAwards=false
• 关键词: Systematic; Derivatization; Nucleic; Acids; Selenium

X-ray crystallography is the most powerful approach for determining the structures of RNAs, DNAs, and nucleic acid-protein complexes to reveal their functional insights. However, the difficulties related to crystallization and to heavy atom derivatization for phase determination are the major limiting factors in nucleic acid X-ray crystallography. To address the latter problem, this project will develop methodologies for selective replacement of nucleotide oxygen with selenium. This derivatization is expected to maintain functional and structural properties of nucleic acids, although the size of Se atom (radius: 1.16 angstroms) is much larger than that of O atom (radius: 0.73 angstroms). Se and O elements are in the same family in periodic table. The project will test if this derivatization can create stable Se-labeled nucleic acids that are structurally and chemically isomorphous to the native molecules. Unlike conventional halogen derivatization (Br or I), where halogens are primarily introduced to the 5-position of deoxyuridine (a mimic of thymidine) and the 5-position of uridine, Se can be introduced to a variety of positions via oxygen replacement (e.g., 2'- or 3'-ribose oxygen, non-bridging phosphate oxygen, or oxygen on nucleobases). Selective incorporation of Se can avoid disruption of structure and function. Because the selenomethionine replacement has revolutionized protein phase and structure determination using Multiwavelength (or Single-Wavelength) Anomalous Dispersion (MAD or SAD), Se should also be able to serve as an ideal anomalous scatterer in nucleic acid crystallography. In addition, Br derivatives are light sensitive, and long-time exposure to X-ray sources may cause decomposition. Therefore, this novel Se derivatization of nucleic acids should be a much better alternative to the conventional Br derivatization. The intellectual merit of this project is to develop general methods for systematic and site-specific replacement of O with Se in RNAs and DNAs to facilitate phase and structure determination by X-ray crystallography via MAD or SAD. The project involves development of routes to synthesize a variety of the Se-containing nucleoside phosphoramidites and triphosphates, and develop chemical and enzymatic procedures to prepare Se-RNAs and Se-DNAs on large scales (10 mg). Chemical and thermodynamic stabilities of Se-derivatized nucleotides will be studied, and the Se-derivatization strategy in X-ray crystal structure study will be evaluated. This Se strategy provides a novel approach to derivatize nucleic acid-protein complexes, where the nucleic acids instead of the protein counterparts will be derivatized. Broader Impacts: Development of methods and reagents for structural analysis of nucleic acids will be a major contribution of this project to the research community. In addition, the project will involve substantial research training of students. In 2003, Georgia State University (GSU) was ranked #1 among non-historically black institutions in the nation and #7 overall in awarding Bachelor's degrees in the Chemistry and other Physical Sciences to African-American students. Therefore, the project will provide an excellent opportunity to train underrepresented undergraduate and graduate students in macromolecular structural research. The students will obtain hands-on skills and experimental research, especially in organic chemistry and biochemistry skills. As a long tradition of the Chemistry Department in educating and serving undergraduate and graduate students at GSU, the applicant and his department offer various extensive training programs. The research project will enhance the environment for these training activities.

X 射线晶体学是确定 RNA、DNA 和核酸-蛋白质复合物结构以揭示其功能的最强大方法。然而，与结晶和重原子衍生化相测定相关的困难是核酸 X 射线晶体学的主要限制因素。为了解决后一个问题，该项目将开发用硒选择性取代核苷酸氧的方法。尽管Se原子（半径：1.16埃）的尺寸远大于O原子（半径：0.73埃），但这种衍生化有望保持核酸的功能和结构特性。 Se和O元素在元素周期表中属于同一族。该项目将测试这种衍生化是否可以产生稳定的硒标记核酸，这些核酸在结构和化学上与天然分子同构。与传统的卤素衍生化（Br 或 I）不同，其中卤素主要被引入到脱氧尿苷（胸苷的模拟物）的 5 位和尿苷的 5 位，Se 可以通过氧取代引入到多个位置（例如、2'-或3'-核糖氧、非桥联磷酸氧或核碱基上的氧）。选择性掺入 Se 可以避免结构和功能的破坏。由于硒代蛋氨酸的替代彻底改变了使用多波长（或单波长）反常色散（MAD 或 SAD）的蛋白质相和结构测定，因此 Se 也应该能够作为核酸晶体学中理想的反常散射体。另外，Br衍生物对光敏感，长时间暴露在X射线源下可能会引起分解。因此，这种新型核酸 Se 衍生化应该是传统 Br 衍生化更好的替代方案。该项目的智力价值是开发在 RNA 和 DNA 中系统地、位点特异性地用 Se 取代 O 的通用方法，以促进 X 射线晶体学通过 MAD 或 SAD 确定物相和结构。该项目涉及开发合成各种含硒核苷亚磷酰胺和三磷酸的路线，并开发化学和酶促程序来大规模制备 Se-RNA 和 Se-DNA（10 毫克）。研究硒衍生核苷酸的化学和热力学稳定性，并对X射线晶体结构研究中的硒衍生策略进行评估。这种硒策略提供了一种衍生化核酸-蛋白质复合物的新方法，其中核酸而不是蛋白质对应物将被衍生化。更广泛的影响：开发核酸结构分析方法和试剂将是该项目对研究界的重大贡献。此外，该项目还将涉及对学生的大量研究培训。 2003 年，佐治亚州立大学 (GSU) 在全国非历史黑人院校中排名第一，在向非裔美国学生授予化学和其他物理科学学士学位方面总体排名第七。因此，该项目将为培养大分子结构研究领域代表性不足的本科生和研究生提供绝佳的机会。学生将获得实践技能和实验研究，特别是有机化学和生物化学技能。作为 GSU 化学系教育和服务本科生和研究生的悠久传统，申请人及其所在部门提供各种广泛的培训项目。该研究项目将改善这些培训活动的环境。