PT  - JOURNAL ARTICLE
AU  - Gerti Beliu
AU  - Andreas Kurz
AU  - Alexander Kuhlemann
AU  - Lisa Behringer-Pliess
AU  - Natalia Wolf
AU  - Jürgen Seibel
AU  - Zhen-Dan Shi
AU  - Martin Schnermann
AU  - Jonathan B. Grimm
AU  - Luke D. Lavis
AU  - Sören Doose
AU  - Markus Sauer
TI  - Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy
AID  - 10.1101/503821
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 503821
4099  - http://biorxiv.org/content/early/2018/12/21/503821.short
4100  - http://biorxiv.org/content/early/2018/12/21/503821.full
AB  - 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.