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

Abstract

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.

Keywords: Click chemistry; Fluorescent probes; G-quadruplex; Molecular dynamics.

Figure 1

A. Schematic representation of the folding of a guanine (G)‐rich sequence into a G‐quadruplex (G4) structure and of its fluorescence detection using N‐TASQ. B. Chemical structures of N‐TASQ, ^TzN‐TASQ and ^AlkN‐TASQ (dark spheres indicate that the four arms are strictly identical within each molecule).

Figure 2

Chemical synthesis of N‐TASQ, ^TzN‐TASQ and ^AlkN‐TASQ (dark spheres indicate that the four arms are strictly identical within each molecule).

Figure 3

A. Example of competitive FRET‐melting curves (n=1) obtained with F‐21‐T (0.2 μM) with no ligand (black) or with 1.0 μM N‐TASQ (yellow) or ^TzN‐TASQ (red), in absence or presence of an excess (3 and 10 μM) of competitive calf thymus DNA (ctDNA); right panel: competitive FRET‐melting results for experiments performed with both N‐TASQ and ^TzN‐TASQ (1.0 μM) against F‐21‐T, F‐Myc‐T, F‐TERRA‐T and F‐VEGF‐T (0.2 μM) in the presence of increasing amounts (0, 3 and 10 μM) of competitive calf thymus DNA (ctDNA). B. Water box (50×50×100 Å) at the bottom of which the TASQ/G‐quartet complex (on the right) is placed. C. Averaged free energy profiles for pulling N‐TASQ (blue), ^TzN‐TASQ (green) and ^AlkN‐TASQ (orange) apart from the G‐quartet (a zoom is provided in the inset).

Figure 4

A. Fluorescence titration of N‐TASQ, ^TzN‐TASQ and ^AlkN‐TASQ (2.0 μM) in presence of increasing amounts (0 to 5 μM) of G4 (SRC) upon excitation at 286 nm (left panel, upward and downward arrows indicate an increase and decrease in fluorescence, respectively) and of both N‐TASQ (yellow) and ^TzN‐TASQ (red) (2.0 μM) versus increasing concentrations (up to 5.0 μM) of DNA G4 (SRC, rhombus), RNA G4 (VEGF, square) and a duplex‐DNA (ds17, star) (right panel). B. Snapshot of TASQ conformations using Replica Exchange Molecular Dynamical (REMD) calculation and quantification of the occurrence of G stacking atop the naphthalene template, defined as both proximity (3–5 Å) and coplanarity (0–10°) between the two units during the simulation.