Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes

Abstract

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Figure 1

Genetic incorporation of 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine, 1, into proteins. (A) Amino acid 1 reacts with sTCO, 2, then loses nitrogen gas to form in the stable conjugate 3. (B) The evolved MjRS/tRNA_CUA pair in pDule-mtaF allows for site-specific incorporation of 1 in response to an amber codon. Lane 2 shows expression levels of GFP-wt from pBad-GFP-His₆. Production of GFP-1 from pBad-GFP-150TAG-His₆ is dependent on 1 in the growth media, lane 3 without 1 present, lane 4 with 1 mM 1 present. Protein was purified by Co⁺² affinity chromatography, separated by SDS-PAGE and stained with Coomassie. (C) MS/MS fragmentation of tryptic peptides derived from GFP-1 samples demonstrates the efficient high-fidelity incorporation of a single 1 UAA in response to an amber stop codon using the mtaF synthetase. The spectra confirm 1 incorporation at codon 150. The fragmentation sites are illustrated above the spectrum.

Figure 2

Characterization of tetrazine transcyclooctene reaction on GFP. (A) Quenched GFP-1 is “turned on” by coupling with 2, forming fluorescence GFP-3. (B) Excitation at 488 nm produces low flourescene for GFP-1, while the reaction forming GFP-3 produces full fluorescence for GFP. (C) ESI-Q-Tof MS analysis of product from GFP-1 with 2 demonstrates specific and quantitative labeling of GFP-1. (D) MALDI MS analysis of GFP-1 reaction with diacetyl-fluorescein labeled sTCO.

Figure 3

Rate constant determination for in vitro and in vivo reaction of GFP-1 and 2. (A) In vitro kinetics resulted in rate k= 880±10 M⁻¹s⁻¹ (B) In vivo kinetics resulted in k= 330±20 M⁻¹s⁻¹. For both experiments unimolecular rate constants were calculated by fitting the rate of GFP-3 formation to a single exponential at five different concentrations of 2. Inset is bimolecular rate constant determination using unimolecular k_obs=k[2].