Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids

Abstract

Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 104 M–1 s–1 with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for substoichiometric in vivo eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.

Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

“…Due to the success of these applications, we hypothesize that transplanting the ncAAs and reactions developed here into eukaryotic cells using currently available systems 13,41 will have a similar level of mutual orthogonality and provided efficient DEAL for a broad range of applications. For example, while we illustrated dual in vivo labeling with two fluorophores, one could also install virtually any combination of probes or moieties, such as protein ligands and inhibitors to modulate protein function, photosensitive probes for spatiotemporal control, or secondary probes with shifted spectral properties, all of which could enable simultaneous monitoring and control of protein functions in real-time in their native context [42][43][44] .…”

“…The IEDDA reaction is a [4+2] cycloaddition that occurs between an electron-deficient diene such as a 1,2,4,5-tetrazine and an electron-rich dienophile such as a strained alkene 1 . Bioorthogonal IEDDA reactions, such as those first described by Fox and colleagues, can reach rates upwards of 10 6 M -1 s -1 , allowing complete reaction within minutes at sub-micromolar concentrations 1,9,[11][12][13][14] . Amino acid derivatives containing azide, cyclopropene, alkyne, trans-cyclooctene (TCO), and tetrazine functionalities have all been genetically encoded into proteins 15 , albeit not yet in a manner conducive to dual encoding and subsequent intracellular labeling in vivo.…”

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

“…To identify a Mb PylRS/tRNA CUA pair capable of site-specically incorporating Acd into proteins in response to an amber stop codon (TAG) we screened a library of Mb PylRS variants in which ve active-site residues were randomized to all 20 amino acids (N 311 , C 313 , V 366 , W 382 , G 387 ) using a standard life/death selection in E. coli. 32,33 Following a round of positive selection in the presence of Acd and a round of negative selection against canonical amino acids, 96 colonies were assessed for their ability to suppress a TAG codon interrupted sfGFP gene (sfGFP-TAG 150 ) in the presence of Acd and suppressor tRNA CUA . The top 25 performing clones were sequenced and 13 unique RS clones were identied with highly similar active site sequences (Fig.…”

Genetic Encoding of a Highly Photostable, Long Lifetime Fluorescent Amino Acid for Imaging in Mammalian Cells

“…The most active and selective variants (32,41,82) had similar mutations in the active site (N311S and V366A, as well as W382T or W382V). Mb AcdRS 32 and 82 also had an additional active site mutation of C313A or C313G, respectively, and Mb AcdRS 82 also contained a non-active site mutation, L155V.…”

“…To identify a Mb PylRS/tRNACUA pair capable of site-specifically incorporating Acd into proteins in response to an amber stop codon (TAG) we screened a library of Mb PylRS variants in which five active-site residues were randomized to all 20 amino acids (N311, C313, V366, W382, G387) using a standard life/death selection in E. coli. 32,33 Following a round of positive selection in the presence of Acd and a round of negative selection against canonical amino acids, 96 colonies were assessed for their ability to suppress a TAG codon interrupted sfGFP gene (sfGFP-TAG150) in the presence of Acd and suppressor tRNACUA. The top 25 performing clones were sequenced and 13 unique RS clones were identified with highly similar active site sequences (Fig.…”