Chemical Methods for Illuminating Cellular DNA

照亮细胞 DNA 的化学方法

• 批准号: RGPIN-2020-05048
• 负责人: Luedtke, Nathan
• 金额: $ 5.76万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=747984
• 关键词: Chemical; Methods; Illuminating; Cellular; DNA

Fluorescent probes have revolutionized the study of biological macromolecules in their native environments. However, as compared to the extensive methodologies developed for proteins, relatively few imaging strategies are currently available for cellular nucleic acids. These methods are limited by their negative impact on native systems and/or inability to be applied in live cells and animals. My program aims to develop non-toxic fluorescent probes for characterizing nucleic acid structures and dynamics in living cells. We design and synthesize new fluorophores and suitable metabolic precursors for their introduction into cells. In addition to the study of DNA folding and ligand binding interactions, a number of probes developed in my lab have been commercialized and used by many other research groups working in the fields of regenerative medicine, genome architecture, stem cell biology, and virology. My group is currently moving to McGill University. An award from NSERC will be critical for re-establishing my rigorous research and training program in Canada, where we aim to: 1) synthesize fluorescent nucleobase analogues with sufficient brightness to probe DNA dynamics in single molecules and living cells; 2) develop fluorogenic nucleoside triphosphate analogues that report DNA or RNA synthesis in real time; 3) conduct bioorthogonal synthesis of fluorescent nucleobase analogues in genomic DNA; 4) develop new fluorogenic probes that selectively react with bioorthogonal functional groups present in cellular G-quadruplex structures. Our new probes and techniques for studying biopolymer synthesis, structure, and dynamics will provide unprecedented insights into the behavior of cellular nucleic acids. The visualization of DNA/RNA dynamics in living cells will require the development of much brighter fluorescent nucleobase analogues than are currently available. The C-nucleosides proposed here constitute a novel family of intensely bright nucleobase analogues containing a single C-H----O or C-H----N interaction in the Watson-Crick interface that supports their faithful enzymatic incorporation into cellular nucleic acids. We will also develop copper-free bioorthogonal chemical reactions between two small precursors that generate a fluorescent nucleobase as the product in the chromatin of live cells. The fluorescence properties of these probes will provide specific information about structural context (ex. duplex, single-stranded, i-motif, or G-quadruplex), and can also be used to track real-time DNA-drug and DNA-metal binding reactions in live cells. Future applications of our research are very broad and include the development of theranostic agents for cancer analysis and treatment. In addition to excellent student training opportunities that build a highly qualified workforce, this program has the potential to benefit Canadian society by generating new business and health care opportunities in pharmaceuticals, diagnostics, and biotechnology.

荧光探针已经彻底改变了生物大分子在其原生环境中的研究。然而，与为蛋白质开发的广泛方法相比，目前可用于细胞核酸的成像策略相对较少。这些方法因其对天然系统的负面影响和/或无法应用于活细胞和动物而受到限制。我的项目旨在开发无毒荧光探针，用于表征活细胞中的核酸结构和动力学。我们设计并合成了新的荧光团和合适的代谢前体，将它们引入细胞。除了DNA折叠和配体结合相互作用的研究外，我的实验室开发的许多探针已经商业化，并被许多其他研究小组在再生医学、基因组结构、干细胞生物学和病毒学领域使用。我的团队现在要搬到麦吉尔大学。NSERC的奖励对于我在加拿大重新建立严格的研究和培训计划至关重要，在那里我们的目标是：1)合成具有足够亮度的荧光核碱基类似物，以探测单分子和活细胞中的DNA动力学；2)开发实时报告DNA或RNA合成的荧光三磷酸核苷类似物；3)对基因组DNA荧光核碱基类似物进行生物正交合成；4)开发新的荧光探针，选择性地与细胞g -四联结构中的生物正交官能团反应。我们用于研究生物聚合物合成、结构和动力学的新探针和技术将为细胞核酸的行为提供前所未有的见解。活细胞中DNA/RNA动力学的可视化将需要开发比目前可用的更明亮的荧光核碱基类似物。这里提出的c -核苷构成了一个新的强烈明亮的核碱基类似物家族，在沃森-克里克界面中含有单个C-H----O或C-H----N相互作用，支持它们忠实的酶结合到细胞核酸中。我们还将开发两个小前体之间的无铜生物正交化学反应，在活细胞的染色质中产生荧光核碱基作为产物。这些探针的荧光特性将提供有关结构背景的特定信息（如双链、单链、i-motif或g -四链），也可用于实时跟踪活细胞中的dna -药物和dna -金属结合反应。我们研究的未来应用是非常广泛的，包括开发用于癌症分析和治疗的治疗剂。除了优秀的学生培训机会，建立高素质的劳动力，这个项目有潜力通过在制药、诊断和生物技术领域创造新的商业和医疗保健机会，造福加拿大社会。