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Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy
Author(s) -
Nikić Ivana,
Estrada Girona Gemma,
Kang Jun Hee,
Paci Giulia,
Mikhaleva Sofya,
Koehler Christine,
Shymanska Nataliia V.,
Ventura Santos Camilla,
Spitz Daniel,
Lemke Edward A.
Publication year - 2016
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201608284
Subject(s) - fluorescence microscope , click chemistry , microscopy , genetic code , subcellular localization , fluorescence , chemistry , debugging , residue (chemistry) , biophysics , nanotechnology , biochemistry , computational biology , dna , biology , combinatorial chemistry , materials science , computer science , cytoplasm , programming language , physics , optics
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.