Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.
Super-resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine-based GCE system for click chemistry, combined with DNA-PAINT microscopy, enables the visualization of even low-abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue-specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.|Present address: Werner Reichardt Centre for Integrative, Neuroscience, University of Tübingen, Tübingen, 72076, Germany.
Ivana
0000-0002-6666-3486
I
Nikić
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Gemma
0000-0003-1533-5041
G
Estrada Girona
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Jun Hee
JH
Kang
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Giulia
G
Paci
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Sofya
S
Mikhaleva
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Christine
C
Koehler
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Nataliia V
NV
Shymanska
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Camilla
C
Ventura Santos
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Daniel
D
Spitz
Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
Edward A
0000-0002-0634-0503
EA
Lemke
D057930:Click Chemistry; D004247:DNA / Q000235:genetics*; D005057:Eukaryotic Cells / Q000166:cytology*; D005815:Genetic Code; D006801:Humans; D008856:Microscopy, Fluorescence; D015202:Protein Engineering
D004247:DNA
PAINT; click chemistry; genetic code expansion; protein labeling; super-resolution microscopy