Abstract
Super-resolution microscopy allows imaging of cellular structures at nanometer resolution. This comes with a demand for small labels which can be attached directly to the structures of interest. In the context of protein labeling, one way to achieve this is by using genetic code expansion (GCE) and click chemistry. With GCE, small labeling handles in the form of noncanonical amino acids (ncAAs) are site-specifically introduced into a target protein. In a subsequent step, these amino acids can be directly labeled with small organic dyes by click chemistry reactions. Click chemistry labeling can also be combined with other methods, such as DNA-PAINT in which a “clickable” oligonucleotide is first attached to the ncAA-bearing target protein and then labeled with complementary fluorescent oligonucleotides. This protocol will cover both aspects: I describe (1) how to encode ncAAs and perform intracellular click chemistry-based labeling with an improved GCE system for eukaryotic cells and (2) how to combine click chemistry-based labeling with DNA-PAINT super-resolution imaging. As an example, I show click-PAINT imaging of vimentin and low-abundance nuclear protein, nucleoporin 153.
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Acknowledgment
I would like to thank Ivana Milošević for her valuable help with the manuscript proofreading. I would like to thank all the members of the Lemke group for their help and support. I would also like to thank EMBL’s Advanced Light Microscopy Facility, as well as my postdoctoral funding (EMBO Long-Term and Marie Curie IEF fellowships). I am currently supported by the Emmy-Noether programme of the Deutsche Forschungsgemeinschaft (DFG) and the Werner Reichardt Centre for Integrative Neuroscience (CIN) at the Eberhard Karls University of Tuebingen. The CIN is an Excellence Cluster funded by the DFG within the framework of the Excellence Initiative (EXC 307).
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Nikić-Spiegel, I. (2018). Genetic Code Expansion- and Click Chemistry-Based Site-Specific Protein Labeling for Intracellular DNA-PAINT Imaging. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_18
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