Chemical Methods for Illuminating Cellular DNA

荧光探针彻底改变了生物大分子在其自然环境中的研究。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。这些方法受到其对天然系统的负面影响和/或不能应用于活细胞和动物的限制。 我的项目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。我们设计和合成新的荧光团和合适的代谢前体，将其引入细胞。除了DNA折叠和配体结合相互作用的研究之外，我实验室开发的许多探针已经商业化，并被再生医学，基因组结构，干细胞生物学和病毒学领域的许多其他研究小组使用。 我的团队目前正在转移到麦吉尔大学。来自NSERC的奖励将对我在加拿大重建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碱基类似物，以探测单分子和活细胞中的DNA动力学; 2）开发荧光核苷三磷酸类似物，以真实的时间报告DNA或RNA合成; 3）在基因组DNA中进行荧光核碱基类似物的生物正交合成; 4）开发新的荧光探针，其选择性地与细胞G-四链体结构中存在的生物正交官能团反应。我们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家庭强烈明亮的核碱基类似物含有一个单一的C-H-O或C-H-N在沃森-克里克接口，支持他们忠实的酶掺入细胞核酸的相互作用。我们还将开发两个小前体之间的无铜生物正交化学反应，该反应产生荧光核碱基作为活细胞染色质中的产物。这些探针的荧光性质将提供有关结构背景的特定信息（例如，双链体、单链、i-基序或G-四链体），并且还可用于跟踪活细胞中的实时DNA-药物和DNA-金属结合反应。我们研究的未来应用非常广泛，包括开发用于癌症分析和治疗的治疗诊断剂。除了优秀的学生培训机会，建立一个高素质的劳动力，该计划有可能通过在制药，诊断和生物技术产生新的商业和医疗保健机会，使加拿大社会受益。

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.