Novel Fluorescent Nucleotide Analogues to Probe Nucleic Acid Metabolism

说明(申请人提供)：荧光核苷酸是DNA和RNA的合成构件，除了是人造结构成分外，当受到更长波长的光照射时，还会发出彩色光。由于这种发射的性质对化学环境很敏感，荧光核苷酸可以作为DNA加工酶活性的高度敏感的视觉探针。一类特殊的合成荧光核苷酸，三环胞苷类似物，具有在双链DNA(双螺旋)中保持荧光的独特性质，与自然界合成DNA和RNA的机制(DNA和RNA聚合酶)高度兼容，并且一旦加入，对核酸的扰动最小。然而，它们的局限性在于，它们只能在很小的可见光谱范围内使用颜色，而且它们的亮度比许多常见的商业氟低一个数量级。在这个项目中，将通过系统地改变先导化合物的化学结构来开发新的荧光核苷酸来克服这些限制。初步结果表明，三环胞苷类似物的荧光波长和强度对取代基和其他结构修饰具有很强的敏感性。利用化学合成，将系统地研究一系列新的修饰，并将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反复使用，以完善明亮的核苷酸荧光的战略开发，这种荧光在更广泛的可见光谱窗口中提供吸收和发射波长。在这些荧光测量的同时，新的核苷酸将被用作DNA和RNA聚合酶的测试底物，其结果将被用来建立它们与核酸结合的方案，并提供关于聚合酶用于区分正确和错误传入核苷酸的机制的新知识。随着吸收和发射窗口的成功扩大和染料的增亮，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和重要的医学过程。例如，大多数癌症和抗病毒治疗药物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，这将导致新疗法的开发和使用现有疗法的新方法，以及对细胞如何代谢核酸的基本见解。 与公共卫生相关：DNA复制在医疗保健和疾病中扮演着多种关键角色--细胞和病毒DNA复制是癌症化疗和抗病毒药物的主要靶点，不正确的复制可能导致多种疾病状态，包括癌症。拟议的研究将提供工具，以更好地了解这一过程，然后可能导致更有效的治疗剂。

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.