Figure 1.

Bioorthogonal reactions. (a) The biarsenical fluorescent dye FlAsH specifically reacts with target proteins genetically tagged with the tetracysteine motif⁴. (b) Small, genetically encoded peptide tags (red rectangle) can be specifically modified in or on live cells using engineered enzymes, such as the biotin ligase BirA^6,7. Biotin-tagged target proteins can then be visualized by streptavidin-conjugated probes, such as quantum dots (QD605). (c) In an example of carbonyl condensations as a bioorthogonal method, unnatural, ketone-functionalized amino acids can be metabolically incorporated into proteins and then detected by reaction with a hydrazide-functionalized fluorescent probe (yellow star)¹¹. (d) In an example of the Staudinger ligation, an azidosugar analog of N-acetylmannosamine (termed ManNAz) is used to metabolically label sialic acid residues on cell-surface glycoproteins. The azidosugar is detected by reaction with a tagged phosphine probe (orange rectangle) and visualized with an antibody to the tag^2,21. (e) In an example of CuAAC chemistry, an alkynyl analog of myristic acid is metabolically processed to an acetyl coenzyme-A metabolite and then attached to protein N termini via N-myristoyltransferase¹⁷. The tagged protein can then be labeled via CuAAC using an azide probe (yellow star). (f) In an example of copper-free click chemistry, an azidosugar analog of fucose is metabolically incorporated into cell-surface glycans in live zebrafish embryos and detected by reaction with a fluorescent difluorinated cyclooctyne (yellow star)^39,49. (g) DNA labeled with metabolically incorporated 5′-ethynyl-2′-deoxyuridine is visualized via direct Raman spectroscopic detection of the intact alkyne⁴⁸.