Chemical Methods for Illuminating Cellular DNA

荧光探针使天然环境中生物大分子的研究发生了革命性的变化。然而，与为蛋白质开发的广泛方法学相比，目前可用于细胞核酸的成像策略相对较少。这些方法因其对本地系统的负面影响和/或无法应用于活细胞和动物而受到限制。我的计划旨在开发无毒的荧光探针，用于表征活细胞中的核酸结构和动力学。我们设计和合成了新的荧光团和合适的代谢前体，以将它们引入细胞。除了研究DNA折叠和配体结合相互作用外，我实验室开发的一些探针已经商业化，并被许多在再生医学、基因组结构、干细胞生物学和病毒学领域工作的其他研究小组使用。我的团队目前正迁往麦吉尔大学。NSERC的获奖将对我在加拿大重新建立严格的研究和培训计划至关重要，我们的目标是：1)合成具有足够亮度的荧光核酸基类似物，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合成的荧光核苷三磷酸类似物；3)进行基因组DNA中荧光核苷酸碱基类似物的生物正交合；4)开发新的荧光探针，它能选择性地与细胞G-四链结构中的生物正交功能基团发生反应。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。在活细胞中显示DNA/RNA动态将需要开发比目前可用的更明亮的荧光核酸基类似物。这里提出的C-核苷构成了一类新型的非常明亮的碱基类似物，它们在Watson-Crick界面上含有单一的C-H-O或C-H-N相互作用，支持它们忠实地被酶结合到细胞核酸中。我们还将在两个小的前体之间进行无铜生物正交化学反应，产生一个荧光核苷酸作为活细胞染色质中的产物。这些探针的荧光性质将提供关于结构上下文的特定信息(例如。该技术可用于跟踪活细胞中DNA-药物和DNA-金属的实时结合反应。我们未来的研究应用非常广泛，包括开发用于癌症分析和治疗的治疗试剂。除了培养高素质劳动力的优秀学生培训机会外，该项目还有可能通过在制药、诊断和生物技术领域创造新的商业和医疗保健机会而造福加拿大社会。

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.