DOI: 10.1101/503821Dec 21, 2018Paper

Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

BioRxiv : the Preprint Server for Biology
Gerti BeliuMarkus Sauer

Abstract

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.

Related Concepts

Cell Growth
Cell Membrane Permeability
Dyes
Iprindole
Microscopy
Electron Microscopy
Oxazines
Rhodamines
Fluorescence Imaging
Extracellular

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