Development of proximity-induced fluorogenic reactions for imaging biomolecular interaction through noncanonical amino acid mutagenesis in response to quadruplet codon and recoding signal

生物正交化学先前的研究工作主要集中在适合蛋白质标记的化学上，这需要高速率常数来适应快速的细胞过程和/或低丰度的目标生物分子。从不同的角度出发，本提案探索了慢动力学生物正交化学的一个有趣而重要的应用，适用于生物分子（例如蛋白质-蛋白质）相互作用的研究。具体而言，我们寻求开发一类基于烯-四嗪化学的邻近诱导的荧光团形成生物偶联反应。这类反应只有当分别接枝在两个分子（如蛋白质）上的两种反应物通过特定的生物分子相互作用事件定位到近距离时才会产生荧光信号。荧光团的形成机制与先前报道的基于四氮的荧光反应有着根本的不同，后者依赖于四氮作为猝灭基团的损失来揭开预先存在的荧光团。由于反应产物是整个反应体系中唯一的荧光物质，这种形成荧光团的反应产生最小的背景信号。将所提出的反应应用于生物学研究需要将生物正交试剂附着在活细胞中的蛋白质上，这将通过非规范氨基酸（ncAA）诱变来实现。琥珀色抑制是将ncAAs引入活细胞中蛋白质的最常用方法，但它会导致内源性终止密码子的读取并干扰正常的细胞生理。在本文中，我们试图开发一种新的方法，通过使用四联体密码子（UAGN； N= a， G， C， U）解码来减少琥珀色停止密码子的不良抑制，该四联体密码子解码完全依赖于嵌入mRNA中的合成编码信号。如果没有附近的重新编码信号，内源性UAGN密码子（即规则的琥珀色停止密码子加上后面的核苷酸）在很大程度上不会被解码。该策略可应用于任何可转染的细胞系，因此对ncAA诱变活细胞研究的一般领域具有广泛的影响。总的来说，化学生物学工具的发展有望大大提高人们探测疾病相关生物过程的能力。

Prior research efforts in bioorthogonal chemistry have mainly focused on chemistry that is suitable for protein labeling, which requires high rate constants to accommodate rapid cellular processes and/or low abundance of target biomolecules. Taking from a different perspective, this proposal explores an intriguing and important application of bioorthogonal chemistry with slow kinetics that are suitable for the study of biomolecular (e.g., protein-protein) interactions. In specific, we seek to develop a class of proximity-induced fluorophore-forming bioconjugation reaction that is based on alkene-tetrazine chemistry. This class of reaction only produces fluorescence signal when the two reactants that are separately grafted on two molecules (e.g., proteins) are localized to a close proximity through specific biomolecular interaction events. The fluorophore-forming mechanism is fundamentally different from previously reported tetrazine-based fluorogenic reactions that rely on the loss of tetrazine as a quenching group to unmask a pre-existing fluorophore. Since the reaction product is the only fluorescence species within the entire reaction system, this fluorophore-forming reaction produces minimal background signal. The application of the proposed reaction to biological studies requires the attachment of bioorthogonal reagents to proteins in live cells, which will be achieved through noncanonical amino acid (ncAA) mutagenesis. While amber suppression is the most popular approach to introduce ncAAs into proteins in live cells, it leads to read-through of endogenous stop codons and interferes with normal cell physiology. In this proposal, we seek to develop a new approach to reduce undesirable suppression of amber stop codons by using quadruplet codon (UAGN; N= A, G, C, U) decoding that is exclusively dependent on synthetic recoding signals imbedded in mRNA. Without nearby recoding signal, endogenous UAGN codons (i.e. regular amber stop codon plus a following nucleotide) are not decoded to a significant extent. This strategy can be applied to any transfectable cell lines, thus has broad impacts on the general field of ncAA mutagenesis for live-cell studies. Overall, the development of proposed chemical biology tools is expected to greatly enhance one’s ability to probe disease-relevant biological processes.