Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy. Issue 52 (2nd November 2016)

• Record Type: Journal Article
• Authors: 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.
• Is Part Of: Angewandte Chemie international edition. Volume 55:Issue 52(2016)
• Page Start: 16172
• Page End: 16176
• Publication Date: 2016-11-02
• Subjects: click chemistry -- genetic code expansion -- PAINT -- protein labeling -- super-resolution microscopy; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201608284
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: 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. Abstract : On target : Cytoplasmic re‐targeting of the most widely used eukaryotic genetic code expansion machinery substantially increases the residue‐specific incorporation of noncanonical amino acids into proteins in mammalian cells. This approach can be combined with ultrafast Diels–Alder click reactions for DNA‐PAINT‐based super‐resolution microscopy. The "Click‐PAINT" system enables high‐contrast, residue‐specific imaging of even low‐abundance proteins.