Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy |
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| Authors: | Dr Ivana Niki? Gemma Estrada?Girona Jun Hee Kang Giulia Paci Sofya Mikhaleva Christine Koehler Dr Nataliia V Shymanska Camilla Ventura?Santos Daniel Spitz Dr Edward A Lemke |
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| Institution: | 1. Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany;2. Present address: Werner Reichardt Centre for Integrative, Neuroscience, University of Tübingen, Tübingen, Germany |
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| 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. |
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| Keywords: | click chemistry genetic code expansion PAINT protein labeling super-resolution microscopy |
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