Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

Abstract

The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.

The use of unnatural amino acids to study and engineer protein function

“…This is especially important for super-resolution microscopy, which is presently limited by labelling quality. Optimization of incorporation efficiency, coupling chemistry and labelling protocols recently paved the way to the application of UAA technology in super-resolution microscopy [75][76][77].…”

Diels-Alder reaction–triggered bioorthogonal protein decaging in living cells

“…This Pyl-based system has emerged in recent years as a 'one-stop shop' for encoding unnatural amino acids (UAAs) in diverse living systems, including prokaryotic and eukaryotic cells, and even in multicellular organisms [11][12][13][14] . Recently, a Y306A Y384F double mutant derived from Methanosarcina mazei PylRS (MmPylRS) has been shown to recognize a broad range of aromatic and/or cyclic Pyl analogs including the cyclooctene-containing TCOK (TCOK-a and TCOK-e mixture) [15][16][17] . We used this mutant MmPylRS to site-specifically incorporate isomers TCOK-a and TCOK-e into the model protein GFP at residue N149 (a previously used residue site with high incorporation efficiency) to generate GFP-N149-TCOK-a and GFP-N149-TCOK-e, respectively (Supplementary Fig.…”

Single-molecule FRET and crosslinking studies in structural biology enabled by noncanonical amino acids

“…Some proteins are sensitive to the copper catalyst and require extremely mild labeling conditions. In this respect, other 'clickable' ncAAs such as those containing strained cyclooctyne (SCO) [25], trans-cyclooctene (TCO) [26,27], and bicyclononyne (BCN) [28,29] groups provide potential solutions for the site-specific labeling of proteins under fully physiological and catalyst free conditions [19]. Whereas the potential has been nicely demonstrated for various model systems, the high cost of ring-strained ncAAs remains a limiting factor for engineering proteins that are difficult to express.…”