Fluorescence Detection of DNA/RNA G‐Quadruplexes (G4s) by Twice‐as‐Smart Ligands

Fluorescence detection of DNA and RNA G-quadruplexes (G4s) is a very efficient strategy to assess not only the existence and prevalence of cellular G4s but also their relevance as targets for therapeutic interventions. Among the fluorophores used to this end, turn-on probes are the most interesting since their fluorescence is triggered only upon interaction with their G4 targets, which ensures a high sensitivity and selectivity of detection. We reported on a series of twice-as-smart G4 probes, which are both smart G4 ligands (whose structure is reorganized upon interaction with G4s) and smart fluorescent probes (whose fluorescence is turned on upon interaction with G4s). The fine mechanistic details behind the excellent properties of the best prototype N-TASQ remain to be deciphered: to investigate this, we report here on the synthesis and studies of two analogues, ^TzN-TASQ and ^AlkN-TASQ, and on a careful analysis of their G4-interacting properties, investigated both in vitro and in silico. Our results show that fine-tuning their constitutive structural elements allows for increasing the efficiency of both their 'off' (i. e., a conformation with a low fluorescence) and 'on' states (i. e., a conformation with a high fluorescence), which opens interesting ways for the design of more efficient fluorogenic G4 probes.