Modulation of the Properties of Nucleic Acids through Structural Modifications

核酸对许多基本的生物过程是至关重要的。它们携带遗传信息，并通过复制、转录和翻译传递这些信息。这些分子也因其在许多细胞过程中的调节作用而被公认。这一提议旨在将独特的结构部分引入核酸，从而赋予这些分子性质，从而进一步加深我们对基本生物学过程的了解。在这方面，将研究三种化学方法。首先，将探索一种代谢性标记核酸的方法。这一化学是基于四嗪和应变烯烃之间的反电子需求Diels-Alder反应。有人提出，应变的烯烃部分将被结合到核苷中，然后以酶的方式结合到核酸中。在这种结合后，将加入共价连接到BODIPY荧光团(以四嗪-BODIPY共轭形式的非荧光)的四嗪，其中发生反向电子需求Diels-Alder反应以恢复荧光。由于这种反应通常非常快，这种化学反应实际上将允许实时跟踪涉及核酸的细胞过程，而且重要的是，具有最少的荧光背景。第二，提出了一种限制酶的化学模拟物，它将以序列特异性的方式切割核酸。这种方法是基于烯二炔部分的Bergman环化反应，在环化过程中会产生反应性的双自由基。通过将烯二炔部分共价连接到合成的寡核苷酸上，由于寡核苷酸与目标核酸的互补性，烯二炔的Bergman环化可以指向所需的切割位置。在细胞和分子生物学研究中，这种方法在裂解大的核酸，如基因组DNA方面将特别有用。最后，将探索通过在核酸中掺入环偶氮苯来用光调节核酸性质的方法。由于外界光刺激引起的环状偶氮苯的结构变化，带有这种修饰的核酸将对特定波长的光做出响应。这种化学将被探索用于杂交和转录等过程的时空调节，并从长远来看，研究生命系统中的基因功能。拟议的研究将为十几名急需的高素质人员提供培训基础。这项拟议研究的工作将进一步加深我们对一些基本生物过程的了解，这些过程将造福加拿大人，并为加拿大的知识驱动型经济做出贡献。

Nucleic acids are crucial for many fundamental biological processes. They carry genetic information, and transmit it through replication, transcription and translation. These molecules are also recognized for their regulatory roles in many cellular processes. This proposal aims at approaches to introduce unique structural moieties into nucleic acids and consequently, confer properties to these molecules that will further our knowledge on the fundamental biological processes. In this regard, three chemical methods will be investigated. First, a methodology to metabolically label nucleic acids will be explored. This chemistry is based on the inverse Electron Demand Diels-Alder reaction between tetrazine and strained alkenes. It is proposed that a strained alkene moiety will be incorporated into nucleosides and subsequently, incorporated into nucleic acids enzymatically. Upon this incorporation, tetrazine that is covalently linked to a BODIPY fluorophore (non-fluorescent in the form of tetrazine-BODIPY conjugate) will be added, where an inverse Electron Demand Diels-Alder reaction takes place to restore the fluorescence. As such reactions are typically very fast, this chemistry will virtually allow for real-time tracking of cellular processes involving nucleic acids, and importantly, with minimal fluorescent background. Second, a chemical mimetic of restriction enzymes that will cleave nucleic acids in a sequence-specific manner is proposed. This approach is based on the Bergman cyclization of enediyne moieties that generate reactive biradicals during the cyclization. By covalently linking enediyne moieties to synthetic oligonucleotides, Bergman cyclization of enediyne can be directed towards the desired cleavage site due to the complementarity of the oligonucleotides with the target nucleic acids. This approach will be particularly useful in fragmenting large nucleic acids, such as genomic DNAs, in cell and molecular biology studies. Last, a methodology to regulate nucleic acid properties by light will be explored through the incorporation of cyclic azobenzene into nucleic acids. Due to the structural changes in cyclic azobenzene as a result of external light stimuli, nucleic acids bearing this modification will respond to light of specific wavelengths. This chemistry will be explored for spatiotemporal regulation of processes such as hybridization and transcription, and in the long term gene functions in living systems. The proposed research will provide a training ground for over a dozen much-needed highly qualified personnel. Work from the proposed research will further our knowledge on some fundamental biological processes that will benefit the wellbeing of Canadians, and contribute to Canada's knowledge-driven economy.**