Bioorthogonal Ligation‐Activated Fluorogenic FRET Dyads

Abstract

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. Suchr elay 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.S ubdiffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopyis also presented.

The Application of Bio-orthogonality for In Vivo Animal Imaging

“…In 2022, Albitz et al demonstrated that bio-orthogonal ligation could be harnessed to activate FRET dyads via manipulating the quenching effect of tetrazine in the FRET pair . In the preactivated FRET pair, coumarin–tetrazine and Cy5 were conjugated into one molecule.…”

Conjugated polymers for biomedical applications

“…Besides the good biocompatibility of CPs modified with tetrazine groups, it can also be designed as turn-on fluorescence probe. 71 The tetrazine groups as energy acceptors can receive the energy given by the CPs so that the fluorescence of the CPs is quenched. However, when tetrazine groups react with TCO, the fluorescence signals of conjugated systems were recovered.…”

Supramolecular ‘catch-and-release’ strategy for bioorthogonal fluorogenic imaging across the visible spectrum

“…Arguably, the most important example of this concept is tetrazine (Tz) probes, which gained popularity due to their fast reactivity via inverseelectron-demand Diels-Alder cycloaddition reaction. [28][29][30][31][32][33][34][35][36][37][38] Tz has been shown to quench fluorescence via various photophysical mechanisms, including Forster resonance energy transfer (FRET), 39,40 through-bond energy transfer (TBET), 26,27 Dexter-type electron exchange, 41 and photoinduced electron transfer (PET). 42 Over the last few years, a range of design strategies has been investigated in collaboration with different fluorophore scaffolds to harness various Tz-mediated quenching mechanisms and achieve fluorogenic tetrazine probes extending up to the far-red/NIR range.…”

“…42 Over the last few years, a range of design strategies has been investigated in collaboration with different fluorophore scaffolds to harness various Tz-mediated quenching mechanisms and achieve fluorogenic tetrazine probes extending up to the far-red/NIR range. 26,27,30,[33][34][35][36][37][38][39][40][41][42] While such fluorogenic probes have been promising, the quenching strategies are not generalizable to diverse libraries of fluorophore families, requiring extensive design efforts to customize quenching mechanisms for each fluorophore scaffold. In addition, access to these fluorogenic probes often requires direct alteration of the core fluorophore skeleton, demanding re-optimization of the laborious fluorophore synthesis scheme and tackling critical synthetic challenges.…”