A genetically encoded cyclobutene probe for labelling of live cells

Abstract

We have identified an aminoacyl-tRNA synthetase/tRNA pair for the efficient and site-specific incorporation of a cyclobutene-containing amino acid into proteins in response to an amber nonsense codon. Fast and fluorescent labeling of purified proteins and intact proteins in live cells was demonstrated using the inverse electron demand Diels-Alder reaction with a tetrazine.

Fig. 1

Fluorescent protein labelling with a genetically encoded unnatural amino acid containing a cyclobutene moiety.

Fig. 2

Genetic incorporation of CbK. (A) Crystal structure of the PylRS in complex with pyrrolysyl-AMP. The structure is derived from PDB 2ZIM; (B) Fluorescence readings of cells expressing CbKRS and sfGFP-Asn149TAG mutant. The expressions were conducted either in the presence or in the absence of 1.0 mM CbK. Fluorescence intensity was normalized to cell growth.

Fig. 3

In vitro protein labelling of sfGFP variants with cyclobutene-tetrazine reaction. Following labeling reactions, protein samples were denatured by heating, then analyzed by SDS-PAGE. The top panel in each figure shows Coomassie blue stained gel and the bottom panel shows the fluorescent image of the same gel before Coomassie blue treatment. (A) Labeling of sfGFP-N149CbK protein with varied concentrations of Fl-Tet for 80 minutes; (B) Reaction progress of sfGFP-N149CbK protein labeling with 50 μM of Fl-Tet. Wild-type sfGFP was included in both experiments as the control. The fluorescence intensities of these images were quantified and presented in Fig. 7 of ESI. The k_obs was estimated as 0.37 M⁻¹ s⁻¹.

Fig. 4

Selective labeling of E. coli cells expressing OmpX-CbK. (A) OmpX-F28CbK mutant that was expressed in the presence of CbK; (B) Wild-type OmpX that was expressed in the presence of CbK. For all images, the left panel shows fluorescent images of E. coli, the middle panel shows bright-field images of the same E. coli cells, and the right panel shows composite images of bright-field and fluorescent images. Scale bars, 5 μm.

Scheme 1

Model of cyclobutene/tetrazine cycloaddition

Scheme 2

Synthesis of unnatural amino acid CbK containing short chain cyclobutene moiety.