ON–OFF Fluorescent Cyanine Dye Based on a Benzothiophenyl Rotor Enables Selective Illumination of G-Quadruplexes in Mitochondria

Abstract

Conventional cyanine dyes exist as "always-on" fluorescent probes leading to inevitable background signals which often limit their performance and scope of applications. To develop specific fluorescent probes with high sensitivity and robust OFF/ON switching for targeting G4s, we introduced aromatic heterocycles through conjugation with polymethine chains to construct a rotor-π system. Here, a universal strategy is presented to synthesize pentamethine cyanines with different aromatic heterocycle substituents on the meso-polymethine chain. In these probes, SN-Cy5-S is self-quenched in aqueous solution due to H-aggregation. The structure indicates that SN-Cy5-S with a flexible meso-benzothiophenyl rotor conjugated to the cyanine backbone matches adaptively with G-tetrad planes, enhancing π−π stacking and resulting in triggered fluorescence. This allows recognition of G-quadruplexes due to the synergy of disaggregation-induced emission (DIE) and inhibited twisted intramolecular charge-transfer effects. This combination leads to a robust lighting-up fluorescence response for c-myc G4 with superior fluorescence enhancement (98-fold), allowing for a low detection limit of 1.51 nM, which is much more sensitive than the previously reported DIE-based G4 probes (22−83.5 nM). In addition, the superior imaging properties and rapid internalization time (5 min) in mitochondria allow SN-Cy5-S to also have a high potential for mitochondrially targeting anti-cancer therapy.

Red-Emitting Pyrrolyl Squaraine Molecular Rotor Reports Variations of Plasma Membrane and Vesicular Viscosity in Fluorescence Lifetime Imaging

“…To study the response of pSQs toward variations of viscosity, we did not use the golden standard glycerol/water or glycerol/methanol mixtures, as they are not adapted to hydrophobic molecular rotors. Indeed, in these very polar solvents the probes tend to form aggregates, thus leading to underestimated fluorescence quantum yield and fluorescence lifetime values due to aggregation-caused quenching. , In light of these considerations, the viscosity sensitivity of the pSQs was assessed using mixtures of model nonpolar lipid oils, namely, medium-chain triglyceride oil (MCT) and the highly viscous vitamin E acetate (VEA). The viscosity of these mixtures was measured by rheology (see the Materials and Methods section) and ranged from 30 and 4890 mPa·s (or cP).…”

“…Despite notable efforts, the development of red-shifted BODIPY rotors appeared tedious as slight variation of the BODIPY rotor structure led to a dramatic decrease of sensitivity toward the viscosity. , Styryl dyes − and carbocyanines − were shown to be efficient viscosity probes; however, their cationic nature and broad excitation and emission spectra limit their applications in cell imaging. The group of Kuimova developed an elegant red-shifted ratiometric dual emissive system based on a conjugated porphyrin dimer, but its cationic nature did not enable to selectively target it to subcellular compartments .…”

Recent advances in FRET probes for mitochondrial imaging and sensing

“…Most of the reported fluorogenic probes that can selectively target mitochondria are based on the mitochondrial phospholipid bilayer with a membrane potential of approximately −180 mV. 22,28,29 These probes are designed with positively charged moieties 22,30–33 (such as triphenylphosphonium cations, 34–36 pyridinium cations, 11,37,38 cyanine cations, 39–41 etc. ) and commercialized mitochondrial dyes 22,42 (such as rhodamine 123 (Rh123), tetramethylrhodamine methyl ester (TMRM), cyanine dye series, etc. )…”