Evaluation of bioorthogonally applicable tetrazine-Cy3 probes for fluorogenic labeling schemes.

The fluorogenic features of three sets of tetrazine-Cy3 probes were evaluated in bioorthogonal tetrazine-cyclooctyne ligation schemes. These studies revealed that the more efficient, internal conversion-based quenching of fluorescence by the tetrazine modul is translated to improved fluorogenicity compared to the more conventional, energy transfer-enabled design. Furthermore, a comparison of directly conjugated probes and vinylene-linked tetrazine-Cy3 probes revealed that more intimate conjugation of the tetrazine and the chromophore results in more efficient IC-based quenching even in spectral ranges where tetrazine exhibits diminished modulation efficiency. The applicability of these tetrazine-quenched fluorogenic Cy3 probes was demonstrated in the fluorogenic labeling schemes of the extra- and intracellular proteins of live cells.

Bioorthogonal Ligation-Activated Fluorogenic FRET Dyads.

An energy transfer-based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue-excitable coumarins to yellow/red emitting cyanine frames is presented. Such relay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes-shifts allowing lower background fluorescence even in no-wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells. These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three-color imaging of intracellular targets using one single excitation source with separate emission windows. Sub-diffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopy is also presented.