Novel Fluorescent Nucleotide Analogues to Probe Nucleic Acid Metabolism

用于探测核酸代谢的新型荧光核苷酸类似物

• 批准号: 7939946
• 负责人: Byron Willis Purse
• 金额: $ 34.79万
• 依托单位: UNIVERSITY OF DENVER (COLORADO SEMINARY)
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7939946
• 关键词: 2-Aminopurine; Address; Affect; Antiviral Agents; Biochemical; Cells; Chemical Structure; Chemicals; Collaborations; Color; Colorado; Complex; Cytidine; DNA; DNA Polymerase I; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Disease; Dyes; Electrons; Environment; Enzymes; Flour; Fluorescein; Fluoresceins; Fluorescence; Fluorescence Resonance Energy Transfer; Future; Gene Mutation; Genetic Recombination; Goals; Healthcare; Human; Image; Knowledge; Label; Lead; Letters; Life; Light; Literature; Malignant Neoplasms; Measurement; Measures; Methods; Mitochondrial DNA; Modification; Motion; Movement; Nature; Nuclear; Nucleic Acid Probes; Nucleic Acids; Nucleosides; Nucleotides; Outcome; Performance; Play; Polymerase; Positioning Attribute; Process; Property; Proteins; Protocols documentation; RNA; RNA chemical synthesis; Research; Role; Route; Simplexvirus; Structure; Testing; Therapeutic; Therapeutic Agents; Time; Universities; Vent; Visual; Xenopus oocyte; absorption; analog; base; chemical synthesis; design; direct application; ds-DNA; fluorescence imaging; fluorophore; in vivo; insight; knowledge base; novel; novel therapeutics; nucleic acid metabolism; nucleobase; nucleotide analog; polymerization; public health relevance; quantum; synthetic nucleotide; tool; viral DNA

DESCRIPTION (provided by applicant): Fluorescent nucleotides are synthetic building blocks of DNA and RNA that, in addition to being artificial structural components, emit colored light when irradiated with light of a longer wavelength. Because the character of this emission is sensitive to the chemical environment, fluorescent nucleotides can be used as exquisitely sensitive visual probes of the activity of DNA-processing enzymes. A special emerging class of synthetic fluorescent nucleotides, tricyclic cytidine analogues, has the unique properties of maintaining fluorescence in double-stranded DNA (the double helix), being highly compatible with Nature's machinery for DNA and RNA synthesis (DNA and RNA polymerases), and minimally perturbing the nucleic acids, once incorporated. They are limited, however, in that they are available with colors in only a small range of the visible spectrum and they are an order of magnitude less bright than many common commercial fluors. In this project, new fluorescent nucleotides will be developed to overcome these limitations by systematically modifying the chemical structure of the lead compounds. Preliminary results indicate that the fluorescence wavelengths and intensities are strongly sensitive to substituent and other structural modifications to the tricyclic cytidine analogues. Using chemical synthesis, a new set of modifications will be systematically examined and a number of novel tri- and tetracyclic cytidine analogues frameworks will be prepared. The results of systematic substituent testing will be used iteratively to refine the strategic development of bright nucleotide fluors that offer absorption and emission wavelengths across a broader window of the visible spectrum. Concurrently with these fluorescence measurements, the new nucleotides will be used as test substrates for DNA and RNA polymerases, the results of which will be used to establish protocols for their incorporation into nucleic acids and to provide new knowledge of the mechanisms used by polymerases to discriminate between correct and incorrect incoming nucleotides. With the successful expansion of the absorption and emission window and the brightening of the dyes, new FRET (Fluorescence Resonance Energy Transfer) applications and direct in vivo fluorescence imaging of DNA synthesis are likely to be possible. Nucleic acid metabolism is an extremely complex and medically important process. For example, most cancer and antiviral therapeutics attack some aspect of this process. The proposed fluorescent nucleotides are expected to offer new methods for analyzing nucleic acid metabolism, which can lead to the development of novel therapeutics and new ways of using existing therapeutics, in addition to fundamental insights into the mechanisms of how cells metabolize nucleic acids. PUBLIC HEALTH RELEVANCE: DNA replication plays multiple critical roles in health care and disease - cellular and viral DNA replication are primary targets for cancer chemotherapeutics and antiviral agents, and incorrect replication can lead to multiple disease states, including cancer. The proposed studies will provide tools to better understand this process, which may then lead to more effective therapeutic agents.

描述（由申请人提供）：荧光核苷酸是DNA和RNA的合成构件，除了是人工结构成分外，当用较长波长的光照射时，会发出有色光。由于这种发射特性对化学环境敏感，荧光核苷酸可以作为dna加工酶活性的灵敏的视觉探针。一种特殊的新兴合成荧光核苷酸，三环胞苷类似物，具有保持双链DNA（双螺旋）荧光的独特特性，与自然的DNA和RNA合成机制（DNA和RNA聚合酶）高度兼容，并且一旦结合对核酸的干扰最小。然而，它们是有限的，因为它们只能在可见光谱的一小部分范围内提供颜色，而且它们的亮度比许多常见的商业荧光低一个数量级。在这个项目中，新的荧光核苷酸将通过系统地修饰先导化合物的化学结构来克服这些限制。初步结果表明，荧光波长和强度对三环胞苷类似物的取代基和其他结构修饰非常敏感。利用化学合成，将系统地研究一组新的修饰，并制备一些新的三环和四环胞苷类似物框架。系统取代基测试的结果将被迭代地用于改进明亮核苷酸荧光的战略开发，这些荧光可以在更宽的可见光谱窗口内提供吸收和发射波长。与这些荧光测量同时，新的核苷酸将被用作DNA和RNA聚合酶的测试底物，其结果将用于建立将其纳入核酸的方案，并提供聚合酶用于区分正确和不正确进入核苷酸的机制的新知识。随着吸收和发射窗口的成功扩展以及染料的增亮，新的FRET（荧光共振能量转移）应用和DNA合成的直接体内荧光成像可能成为可能。核酸代谢是一个极其复杂且具有重要医学意义的过程。例如，大多数癌症和抗病毒疗法会攻击这一过程的某些方面。所提出的荧光核苷酸有望为分析核酸代谢提供新的方法，这可以导致新的治疗方法和使用现有治疗方法的新方法的发展，以及对细胞如何代谢核酸的机制的基本见解。