Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells

Abstract

Over the past years, fluorescent proteins (e.g., GFP) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large size and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse–electron–demand Diels–Alder cycloaddition (IEDAC) reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine (2’-aTCOK) robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine-fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Playing with the Molecules of Life

“…208, 237 While the refinement and optimization of labeling and imaging approaches is still on going, early examples of applications that are not possible by other methods have already emerged. 238–240 …”

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

“…The ncAA polyspecificity of both wild-type and evolved aaRSs may be caused by the lack of exposure to non-cognate substrates in the course of their evolution. In some cases, the ability of o-aaRS to use structurally similar ncAAs can be advantageous [36–38]. In other cases it can hamper unambiguous site-specific incorporation of multiple ncAAs into a single protein (see below).…”

Pyrrolysine Amber Stop‐Codon Suppression: Development and Applications

“…An assortment of proteins containing norbornenes and trans ‐cyclooctenes (TCOs) 32 – 35 could be produced by using engineered variants of the M. mazei PylRS. Applications have included fluorescently tagged proteins in vitro, rapid labelling of proteins in vivo, live‐cell labelling, synthesis of glycoconjugates by using glycosyl azides, protein crosslinking and SPIEDAC‐initiated rapid decaging through a novel rearrangement mechanism . Stop‐codon suppression and bioorthogonal chemistry are highly complementary techniques in the field of protein modification and have facilitated innovative developments in the field of chemical biology.…”