Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy.
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.
Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany).
Ivana
I
Nikić
Tilman
T
Plass
Oliver
O
Schraidt
Jędrzej
J
Szymański
John A G
JA
Briggs
Carsten
C
Schultz
Edward A
EA
Lemke
D000596:Amino Acids / Q000737:chemistry; D061565:Cycloaddition Reaction; D004583:Electrons; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D009975:Orthomyxoviridae / Q000378:metabolism; D015202:Protein Engineering; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D005456:Fluorescent Dyes; D006388:Hemagglutinins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D011972:Receptor, Insulin
amino acids; click chemistry; cycloaddition; protein engineering; super-resolution microscopy