scholarly journals Cover Picture: Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy (Angew. Chem. Int. Ed. 52/2016)

2016 ◽  
Vol 55 (52) ◽  
pp. 15931-15931
Author(s):  
Ivana Nikić ◽  
Gemma Estrada Girona ◽  
Jun Hee Kang ◽  
Giulia Paci ◽  
Sofya Mikhaleva ◽  
...  
Keyword(s):  
Genetic Code ◽  
Super Resolution ◽  
Site Specific ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

scholarly journals Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy

2016 ◽  
Vol 55 (52) ◽  
pp. 16172-16176 ◽  
Author(s):  
Ivana Nikić ◽  
Gemma Estrada Girona ◽  
Jun Hee Kang ◽  
Giulia Paci ◽  
Sofya Mikhaleva ◽  
...  
Keyword(s):  
Genetic Code ◽  
Super Resolution ◽  
Site Specific ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

scholarly journals Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy

2021 ◽  
Vol 13 ◽  
Author(s):  
Alexander Kuhlemann ◽  
Gerti Beliu ◽  
Dieter Janzen ◽  
Enrica Maria Petrini ◽  
Danush Taban ◽  
...  
Keyword(s):  
Genetic Code ◽  
Click Chemistry ◽  
Unnatural Amino Acids ◽  
Super Resolution ◽  
Receptor Expression ◽  
Fluorescence Labeling ◽  
Site Specific ◽  
Gaba A ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

scholarly journals Bioorthogonal double-fluorogenic siliconrhodamine probes for intracellular super-resolution microscopy

2017 ◽  
Vol 53 (50) ◽  
pp. 6696-6699 ◽  
Author(s):  
E. Kozma ◽  
G. Estrada Girona ◽  
G. Paci ◽  
E. A. Lemke ◽  
P. Kele
Keyword(s):  
Super Resolution ◽  
Site Specific ◽  
Intracellular Proteins ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

A series of double-fluorogenic siliconrhodamine-tetrazines were synthesized. One of these tetrazines is a membrane-permeant label allowing site-specific bioorthogonal tagging of intracellular proteins and super-resolution imaging.

scholarly journals Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Gerti Beliu ◽  
Andreas Kurz ◽  
Alexander Kuhlemann ◽  
Lisa Behringer-Pliess ◽  
Natalia Wolf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Alder Reaction ◽  
Cell Permeability ◽  
High Signal ◽  
Noncanonical Amino Acids ◽  
Single Molecule Level ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

scholarly journals Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

2021 ◽  
Author(s):  
Diogo Bessa-Neto ◽  
Alexander Kuhlemann ◽  
Gerti Beliu ◽  
Valeria Pecoraro ◽  
Sören Doose ◽  
...  
Keyword(s):  
Amino Acids ◽  
Brain Slices ◽  
Transmembrane Proteins ◽  
Super Resolution ◽  
Biological Imaging ◽  
Bioorthogonal Labeling ◽  
Resolution Imaging ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy ◽  
Organotypic Brain Slices

ABSTRACTProgress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allowed us to image the differential localization of two glutamate receptor auxiliary proteins in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

scholarly journals Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

2015 ◽  
Vol 137 (14) ◽  
pp. 4602-4605 ◽  
Author(s):  
Chayasith Uttamapinant ◽  
Jonathan D. Howe ◽  
Kathrin Lang ◽  
Václav Beránek ◽  
Lloyd Davis ◽  
...  
Keyword(s):  
Genetic Code ◽  
Super Resolution ◽  
Live Cell ◽  
Cellular Proteins ◽  
Resolution Imaging ◽  
Genetic Code Expansion

scholarly journals Correction to “Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins”

2018 ◽  
Vol 140 (42) ◽  
pp. 13986-13986
Author(s):  
Chayasith Uttamapinant ◽  
Jonathan D. Howe ◽  
Kathrin Lang ◽  
Václav Beránek ◽  
Lloyd Davis ◽  
...  
Keyword(s):  
Genetic Code ◽  
Super Resolution ◽  
Live Cell ◽  
Cellular Proteins ◽  
Resolution Imaging ◽  
Genetic Code Expansion

scholarly journals Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 578 ◽  
Author(s):  
Jan-Erik Hoffmann
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Click Chemistry ◽  
Protein Interactions ◽  
Site Specific ◽  
Genetic Code Expansion

Genetic code expansion is a powerful tool for the study of protein interactions, as it allows for the site-specific incorporation of a photoreactive group via non-canonical amino acids. Recently, several groups have published bifunctional amino acids that carry a handle for click chemistry in addition to the photo-crosslinker. This allows for the specific labeling of crosslinked proteins and therefore the pulldown of peptides for further analysis. This review describes the properties and advantages of different bifunctional amino acids, and gives an overview about current and future applications.

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