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-蛋白复合物结构以揭示其功能见解的最强大方法。然而，与结晶和重原子衍生化相测定有关的困难是限制核酸x射线晶体学的主要因素。为了解决后一个问题，本项目将开发用硒选择性替代核苷酸氧的方法。尽管Se原子（半径为1.16埃）比O原子（半径为0.73埃）大得多，但这种衍生化有望保持核酸的功能和结构特性。元素周期表中的硒元素和氧元素属于同一族。该项目将测试这种衍生化是否能产生稳定的硒标记核酸，在结构和化学上与天然分子同构。与传统卤素衍生化（Br或I）不同，卤素主要被引入到脱氧尿嘧啶（胸苷的模拟物）的5位和尿嘧啶的5位，硒可以通过氧取代（例如，2‘或3’-核糖氧，非桥接磷酸氧或核碱基上的氧）被引入到各种位置。硒的选择性掺入可以避免结构和功能的破坏。由于硒代蛋氨酸已经彻底改变了使用多波长（或单波长）异常色散（MAD或SAD）测定蛋白质的相和结构，硒也应该能够作为核酸晶体学中理想的异常散射体。此外，Br衍生物对光敏感，长时间暴露于x射线源可能导致分解。因此，这种新型的核酸硒衍生化应该是一种比传统的溴衍生化更好的替代品。该项目的智力优势在于开发了一种通用的方法，系统地和特定位点地将rna和dna中的O替换为Se，以便通过MAD或SAD通过x射线晶体学确定相和结构。该项目涉及开发合成各种含硒核苷磷酰胺和三磷酸盐的途径，并开发大规模（10毫克）制备硒rna和硒dna的化学和酶程序。研究了硒衍生核苷酸的化学和热力学稳定性，并对硒衍生化策略在x射线晶体结构研究中的应用进行了评价。这种Se策略提供了一种衍生化核酸-蛋白复合物的新方法，其中核酸而不是相应的蛋白质将被衍生化。更广泛的影响：开发核酸结构分析的方法和试剂将是本项目对研究界的主要贡献。此外，该项目将涉及大量的研究训练的学生。2003年，佐治亚州立大学（GSU）在全美非黑人院校中排名第一，在授予非裔美国学生化学和其他物理科学学士学位方面排名第七。因此，该项目将为培养大分子结构研究方面代表性不足的本科生和研究生提供一个极好的机会。学生将获得动手技能和实验研究，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，申请人和他的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。