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.
The goal of this proposal is to develop enabling chemical biology tools for biomedical investigations. The development of a class of proximity-induced fluorophore-forming bioconjugation reaction will advance one?s ability to probe disease-relevant biological processes, which will accelerate and guide the discovery of new therapeutic interventions for the treatment of human diseases. Therefore, the proposed research is relevant to the NIH's mission in uncovering new fundamental knowledge that will lead to better health for everyone.
