Chemical Synthesis and Applications of Nucleic Acid Analogues

核酸是生命的基本分子，携带着地球上所有生物的基因蓝图。经过数十亿年的进化，它们实现了复杂和优雅之间的理想平衡，形式和功能的完美结合。因此，合成的DNA和RNA寡核苷酸及其类似物是分子生物学、功能基因组学和生物化学研究的非常有用的工具。*我们将在这项拟议的NSERC发现拨款计划中追求三个具体目标。首先，我们将合成和表征分枝RNA类似物，以研究人类套索RNA去分支酶(HDbr1)的结构和性质，hDbr1是已知的唯一能降解套索RNA内含子2‘，5’-磷酸二酯键的酶；通过引入糖和碱基修饰来进行非水解性RNA分支点类似物的结构指导合成，以创建模拟自然界中观察到的套索RNA分支点的构象；确定DBR1·抑制剂复合体的结构，并测试DBR1抑制剂化合物对神经细胞系的毒性。其次，我们将设计人工修饰的DNA和RNA结构，这些结构能够通过截取mRNA来“关闭”基因(例如，致病基因)，这是因为它们与RNA的结合亲和力预期会增加，并且在生物介质中具有稳定性。第三，我们计划研究折叠的基因组DNA结构，即G-四链和I-基序。虽然以前的结构已经知道控制基因的表达，但I基序的生物学作用仍然难以捉摸。我们首次提出端粒酶活性与双链DNA中G-四链体和假定的I-基序的瞬时形成相关。通过我们提出的方法在生理条件下稳定I-基序结构，将为I-基序和G-四链体开辟新的研究方向。*我们建议的研究计划将利用前一次授权期内获得的专业知识和研究进展，进一步推动最新技术的发展，并阐明生物有机化学中几个临床相关的问题。这项拟议的计划通过在以前成果的基础上将风险降至最低，同时仍然延续了我们实验室大胆推动核酸研究前沿的主题。我们的科学方法结合了溶液和固相合成方法、药物发现、光谱分析和分子生物学技术。这些研究让我的学生接触到核酸化学和生物学研究的广泛主题，以及用于识别候选分子并将其开发成有用的探针和/或治疗药物的策略。

Nucleic acids are the fundamental molecules of life, carrying the genetic blueprints for all living things on Earth. Refined through billions of years of evolution, they achieve an ideal balance between complexity and elegance, a perfect marriage of form and function. As such, synthetic DNA and RNA oligonucleotides and their analogues are incredibly useful tools for molecular biology, functional genomics, and biochemistry research.******We will pursue three specific aims within this proposed NSERC Discovery Grant program. First, we will synthesize and characterize branched RNA analogues in order to study the structure and properties of the human lariat RNA debranching enzyme (hDbr1), the only enzyme known to hydrolyze 2',5'-phosphodiester bonds of lariat RNA introns; carry out structure-guided synthesis of non-hydrolyzable RNA branchpoint analogues through the introduction of sugar and base modifications to create conformations that mimic that of the lariat RNA branchpoint observed in nature; determine the structures of Dbr1•inhibitor complexes, and test the toxicity of Dbr1 inhibitor compounds in neuronal cell lines. Second, we will design synthetically modified DNA and RNA structures that are able to "switch off" genes (e.g., disease-causing genes) by intercepting mRNA, by virtue of their expected increased binding affinity for RNA and stability in biological media. Third, we plan to study folded genomic DNA structures, i.e., the G-quadruplexes and i-motifs. While the former structures are already known to control gene expression, the biological role of i-motifs remains elusive. We propose to correlate, for the first time, telomerase activity with transient formation of G-quadruplexes and putative i-motifs within duplex DNA. The stabilization of i-motif structures at physiological conditions by our proposed approach will open new research directions on i-motifs and G-quadruplexes.******Our proposed research program will capitalize on the expertise and research advances obtained during the previous granting period to further advance the state of the art and shed light on several clinically relevant questions in bioorganic chemistry. This proposed program minimizes risk through building on previous achievements, while still continuing our lab's theme of boldly pushing the leading edge of nucleic acids research. Our scientific approach combines solution and solid-phase synthetic methods, drug discovery, spectroscopic analysis, and molecular biology techniques. These studies expose my students to a wide range of topics in nucleic acid chemistry and biology research, and to strategies used for identifying candidate molecules and developing them into useful probes and/or therapeutic drugs.**