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

生物正交化学以前的研究主要集中在适合蛋白质标记的化学上，这需要高的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。从不同的角度来看，这一建议探索了生物正交化学的一个有趣和重要的应用，该化学具有缓慢的动力学，适用于研究生物分子(例如蛋白质-蛋白质)相互作用。具体地说，我们试图开发一类基于烯烃-四嗪化学的邻位诱导荧光团形成生物偶联反应。这类反应只有在两个分子(例如蛋白质)上分别嫁接的两个反应物通过特定的生物分子相互作用事件定位到非常接近的位置时才会产生荧光信号。荧光团的形成机制与以前报道的基于四嗪的荧光反应有根本的不同，后者依赖于四嗪的损失作为猝灭基团来揭示预先存在的荧光团。由于反应产物是整个反应体系中唯一的荧光物种，这种形成荧光团的反应产生的本底信号最小。将所提出的反应应用于生物学研究需要将生物正交试剂连接到活细胞中的蛋白质上，这将通过非规范氨基酸(NCAA)突变实现。虽然琥珀抑制是将NCAA引入活细胞中蛋白质的最常用的方法，但它会导致内源终止密码子的通读，并干扰正常的细胞生理。在这个建议中，我们试图开发一种新的方法，通过使用四重密码子(UAGN；N=A，G，C，U)解码来减少对琥珀终止密码子的不良抑制，这种解码完全依赖于嵌入在mRNA中的合成重新编码信号。如果没有附近的重新编码信号，内源UAGN密码子(即正常的琥珀终止密码子加上随后的核苷酸)在很大程度上不会被解码。这一策略可以应用于任何可转基因的细胞系，因此对活细胞研究的NCAA诱变的一般领域具有广泛的影响。总体而言，拟议的化学生物学工具的开发有望极大地提高人们探索与疾病相关的生物过程的能力。

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.