Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy |
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| Authors: | Dr Ivana Niki? Dr Tilman Plass Dr Oliver Schraidt Dr J?drzej Szymański Dr John A G Briggs Priv‐Doz?Dr Carsten Schultz Dr Edward A Lemke |
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| Affiliation: | Structural and Computational Biology Unit and Cell Biology and Biophysics Unit, EMBL, Meyerhofstrasse 1, 69117 Heidelberg (Germany) |
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| Abstract: | The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy. |
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| Keywords: | amino acids click chemistry cycloaddition protein engineering super‐resolution microscopy |
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