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

开发邻近诱导的荧光反应，通过响应四联体密码子和重新编码信号的非规范氨基酸诱变来成像生物分子相互作用

• 批准号: 10259702
• 负责人: Jiantao Guo
• 金额: $ 30.67万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259702
• 关键词: Alkenes; Amber; Amino Acids; Biological; Biological Process; Biology; Cell Line; Cell physiology; Cells; Chemicals; Chemistry; Codon Nucleotides; Development; Diels Alder reaction; Disease; Electrons; Event; Fluorescence; Goals; Health; Image; Investigation; Kinetics; Knowledge; Label; Lead; Literature; Messenger RNA; Mission; Mutagenesis; Normal Cell; Nucleotides; Peptide Signal Sequences; Property; Proteins; Quadruplet Multiple Birth; Reaction; Reagent; Reporting; Research; Signal Transduction; System; Terminator Codon; Time; United States National Institutes of Health; base; combinatorial; fluorophore; guided inquiry; high throughput screening; human disease; macromolecule; novel strategies; novel therapeutic intervention; protein protein interaction; reaction rate; response; theories; tool

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.

先前在生物方面化学方面的研究工作主要集中在适合蛋白质标记的化学上，这需要高速率常数以适应快速的细胞过程和/或靶标生物分子的丰度。从不同的角度来看，该提案探讨了生物正交化学的有趣而重要的应用，这些化学具有缓慢的动力学，适用于生物正交（例如蛋白质蛋白质）相互作用的研究。在具体而言，我们试图开发一类基于烯烃四嗪化学的接近性诱导的荧光团形成生物缀合反应。当分别接枝在两个分子（例如蛋白质）上的两个反应物通过特定的生物分子相互作用事件将两个分子（例如蛋白质）定位为紧密接近时，这种反应只会产生荧光信号。荧光团形成的机制与先前报道的基于四嗪的荧光反应从根本上有所不同，这些反应依赖于四嗪作为淬火基团的丧失来揭开现有的荧光团。由于反应产物是整个反应系统中唯一的荧光物种，因此这种形成荧光团的反应会产生最小的背景信号。提出的反应对生物学研究的应用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。虽然琥珀抑制是将NCAA引入活细胞中蛋白质中的最流行的方法，但它导致读取内源性停止密码子并干扰正常的细胞生理。在此提案中，我们试图通过使用四倍密码子（uagn; n = a，g，c，u）来开发一种新的方法来减少对琥珀终止密码子的不可抑制，该密码子仅取决于mRNA中含有的合成重新编码信号。没有接近重新编码的信号，内源性UAGN密码子（即常规的琥珀色终止密码子加上以下核OTIDE）并未在很大程度上解码。该策略可以应用于任何可转染的细胞系，因此对活细胞研究的NCAA诱变的一般领域产生了广泛的影响。总体而言，预计拟议的化学生物学工具的开发可以极大地增强人们探测与疾病相关的生物学过程的能力。