G‐Quartets, 4‐Way Junctions and Triple Helices but Not DNA Duplexes: Planarization of Twisted Push–Pull Flipper Probes by Surface Recognition Rather Than Physical Compression

Abstract

Planarizable push-pull flipper probes have been introduced to image order and tension of membranes in living cells. In this report, we show that fluorescent flippers can also be planarized by chemical interactions on DNA architectures rather than by physical forces in biomembranes. Compared to fluorescence in water, the planarization on parallel cMyc G-quartets is characterized by a 100 nm red shift of the excitation maximum and a strong increase in fluorescence intensity. A coinciding 100 nm red shift of the emission maximum compared to planarized flippers in ordered membranes reveals that planarization occurs on the water-accessible surface rather than within the G-quartets. Less relevant for physical compression in the ground state in hydrophobic membranes, this functional relevance of flipper solvatochromism in the excited state, reflected by red shifts in emission rather than excitation, is unprecedented and introduces attractive dual sensing perspectives. With decreasing efficiency, flippers are also planarized by antiparallel hTel22 G-quartets, 4-way junctions and triple helices, while the aromatic surface of B-DNA duplexes is too small for strong enough π stacking. These remarkably consistent results expand the understanding of planarizable push-pull flipper probes and enable applications in new directions.

Hydrophobic Interfacing of Fluorescent Membrane Probes

“…Minimal hydration, in turn, should minimize solvatochromism. [1,54] The small blue shift of the emission thus supported that maximized hydrophobic interfacing also minimizes hydration around the probe, which is reflected by a small blue shift of the emission maximum. [54] Compared to the impact of mechanical compression in equilibrium in the ground state on the excitation spectra (Figure 2a, b), the impact of membrane dehydration on solvatochromism in the emission spectra is minor and without importance for use in practice (Figure 2d).…”

“…In aqueous media, the fully twisted flippers absorb in the blue, emit red shifted, and have a short fluorescence lifetime. [54] In lipid bilayers, flipper planarization with increasing order from liquid-disordered (L d ) to liquid-ordered (L o ) and solid-ordered (S o ) membranes causes massive red shifts in excitation and equally distinct increases in fluorescence lifetime and intensity (Figure 1b). Membrane tension applied to single-component membranes (+ σ) decreases flipper lifetimes, which is consistent with probe deplanarization by mechanical lipid decompression (Figure 1c).…”

Fluorescent Membrane Probes Obey the Israelachvili Rules

“…Today, a rich flipper collection is available to target essentially any membrane of interest within cells, [19] using either empirical trackers [19,29,30] or cellular engineering [31,32] together with controlled release by chemical stimulation [31] or with light. [32] Studies to improve on the performance of flippers covered topics as diverse as turn-on donors, [33] alternative targets, [34] dynamiccovalent acceptors, [35] chalcogen-bonding cascade switching [35,36] and fluorophore architectures different from the twisted push-pull dithienothiophene dimers, from trimers [37] up to molecular papillons for excitedstate unbending rather than ground-state untwisting, or dynamers. [19] These studies have contributed to an advanced understanding of the original Flipper-TR® 1, fluorescent membrane probes in general and membrane tension imaging, including access to superresolution and dual sensing.…”

Fluorogenic In Situ Thioacetalization: Expanding the Chemical Space of Fluorescent Probes, Including Unorthodox, Bifurcated, and Mechanosensitive Chalcogen Bonds

“…Located at the membrane surface, variable targeting units R have to be attached to this terminus (Figure ). ,− Moreover, donors D are needed in planarized flippers II to generate a strong push–pull system accounting for large red-shifts, but they are incompatible with twisted flippers I because the decoupled donor side oxidizes . Flippers such as 1 with simple alkoxy donors are not stable for this reason (Figure d).…”