Abstract
Precise temporally regulated protein function directs the highly complex processes that make up embryo development. The zebrafish embryo is an excellent model organism to study development, and conditional control over enzymatic activity is desirable to target chemical intervention to specific developmental events and to investigate biological mechanisms. Surprisingly few, generally applicable small molecule switches of protein function exist in zebrafish. Genetic code expansion allows for site-specific incorporation of unnatural amino acids into proteins that contain caging groups that are removed through addition of small molecule triggers such as phosphines or tetrazines. This broadly applicable control of protein function was applied to activate several enzymes, including a GTPase and a protease, with temporal precision in zebrafish embryos. Simple addition of the small molecule to the media produces robust and tunable protein activation, which was used to gain insight into the development of a congenital heart defect from a RASopathy mutant of NRAS and to control DNA and protein cleavage events catalyzed by a viral recombinase and a viral protease, respectively.
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