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Abstract Using genetic code expansion (GCE) to encode bioorthogonal chemistry has emerged as a promising method for protein labeling, both in vitro and within cells. Here, we demonstrate that tetrazine amino acids incorporated into proteins are highly tunable and have extraordinary potential for fast and quantitative bioorthogonal ligations. We describe the synthesis and characterize reaction rates of 29 tetrazine amino acids (20 of which are new) and compare their encoding ability into proteins using evolved Tet ncAA encoding tRNA/RS pairs. For these systems, we characterized on-protein Tet stability, reaction rates, and ligation extents, as the utility of a bioorthogonal labeling group depends on its stability and reactivity when encoded into proteins. By integrating data on encoding efficiency, selectivity, on-protein stability, and in-cell labeling for Tet tRNA/RS pairs, we developed the smallest, fastest, and most stable Tet system to date. This was achieved by introducing fluorine substituents to Tet4, resulting in reaction rates at the 10⁶ M⁻¹s⁻¹ level while minimizing degradation. This study expands the toolbox of bioorthogonal reagents for Tet-sTCO-based, site-specific protein labeling and demonstrates that the Tet-ncAA is a uniquely tunable, highly reactive, and encodable bioorthogonal functional group. These findings provide a foundation to further explore Tet-ncAA encoding and reactivity.
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Rational design for high bioorthogonal fluorogenicity of tetrazine‐encoded green fluorescent proteins
Abstract 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. Förster resonance energy transfer is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150‐Tet with a fluorescence turn‐on ratio of ∼9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet‐v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn‐on ratios to ∼14/31, making the fluorogenicity of GFP150‐Tet‐S202Y the highest among all up‐to‐date tetrazine‐encoded GFPs. In live eukaryotic cells, the GFP150‐Tet‐v3.0‐S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy. Key pointsUltrafast spectroscopy reveals FRET in action and inhomogeneous populations with different transition dipole moment alignments.Rational protein design of two new superfolder GFP mutants with record‐high fluorogenicity.Bioimaging application of the designed bioorthogonal protein mutant in live eukaryotic cells.
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- PAR ID:
- 10373306
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Natural Sciences
- Volume:
- 2
- Issue:
- 4
- ISSN:
- 2698-6248
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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