Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations.

Quantitative labeling of biomolecules is necessary to advance areas of antibody-drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein.

Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites.

Site-specific dual encoding and labeling of proteins via genetic code expansion.

The ability to selectively modify proteins at two or more defined locations opens new avenues for manipulating, engineering, and studying living systems. As a chemical biology tool for the site-specific encoding of non-canonical amino acids into proteins in vivo, genetic code expansion (GCE) represents a powerful tool to achieve such modifications with minimal disruption to structure and function through a two-step "dual encoding and labeling" (DEAL) process. In this review, we summarize the state of the field of DEAL using GCE.

Rational Design for High Bioorthogonal Fluorogenicity of Tetrazine-Encoded Green Fluorescent Proteins.

The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations.

Nanobody assemblies with fully flexible topology enabled by genetically encoded tetrazine amino acids.

Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups-site-specifically inserted by genetic code expansion at Nb surface sites-are compatible with Nb folding and function, enabling Nb assembly at any desired point.

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

The ability to site-specifically modify proteins at multiple sites will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins followed by mutually orthogonal labeling. This general approach, that we call ual ncoding nd abeling (DEAL), allowed us to efficiently encode tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein.

Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity.

During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus the probability of immobilization with reactive surface sites (i.

Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA.

Immobilization of Proteins with Controlled Load and Orientation.

Biomaterials based on immobilized proteins are key elements of many biomedical and industrial technologies. However, applications are limited by an inability to precisely construct materials of high homogeneity and defined content. We present here a general "protein-limited immobilization" strategy by combining the rapid, bioorthogonal, and biocompatible properties of a tetrazine-strained -cyclooctene reaction with genetic code expansion to site-specifically place the tetrazine into a protein.