Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method

Abstract

Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities.

Figure 1. Bioinspired recognition of quadruplexes by synthetic G-quartets.

Structure of a template-assembled synthetic G-quartet (or TASQ, here N-TASQ, left) and of a G-quartet (right); schematic representation of a quadruplex-forming sequence in its unfolded and folded states. The bioinspired interaction between N-TASQ and quadruplexes takes place through quartet-quartet recognition, stabilized by π-stacking interactions (grey arrows) and cation chelation (yellow dashed lines), along with electrostatic interactions (pink arrows). The ordered hydration shell of the TASQ/quadruplex assembly is schematically represented as a pale blue rectangle.

Figure 2. Demonstration and exploitation of the red-edge effect.

(A) Dependence of the emission maximum (λ_em^max) on the excitation wavelength (λ_ex): fluorescence emission spectra recorded for an experiment carried out with N-TASQ (10 μM) and a quadruplex (22AG, 5 μM) with λ_ex between 488 and 588 nm (every 10 nm). (B) Dependence of the excitation maximum (λ_ex^max) on the emission wavelength (λ_em): fluorescence excitation spectra recorded for an experiment carried out with N-TASQ (10 μM) and a quadruplex (22AG, 5 μM) with λ_em between 734 and 634 nm (every 10 nm). (C) Fluorescence titrations performed without (black lines) or with N-TASQ (10 μM) and increasing amounts of 22AG (from 0 to 5 μM) with λ_ex at 408 (left), 488 (center) and 555 nm (right). Black stars indicate the Raman signals of the buffer. All experiments are performed in 10 mM lithium cacodylate buffer (pH 7.2) + 10 mM KCl/90 mM LiCl.

Figure 3. Assessing the properties of N-TASQ as quadruplex-specific REE probe.

(A–D) Fluorescence titrations performed without (black lines) or with N-TASQ (10 μM) and increasing amounts of oligonucleotides (from 0 to 5 μM, from grey to red lines), either quadruplex-DNA (22AG, (A) or c-Myc, (B)), quadruplex-RNA (TERRA, (C)) or duplex-DNA (ds17, (D)). (E,F) control experiments carried out without (black lines) or with 10 μM of ^PNADOTASQ (E) or N-NH₂ (F) and increasing amounts of 22AG (from 0 to 5 μM, from grey to red lines). All experiments are performed under excitation (λ_ex) at 555 nm. (G) REE experiments performed with N-TASQ (colored lines), ^PNADOTASQ (black lines) or N-NH₂ (grey lines) and 22AG (5 μM) with λ_ex at 618, 628 and 638 nm. All experiments are performed in 10 mM lithium cacodylate buffer (pH 7.2) + 10 mM KCl/90 mM LiCl. (H) structures of N-TASQ, ^PNADOTASQ and N-NH₂.

Figure 4. Implementation of the red-edge effect for fluorescence cell imaging.

(A) Images of MCF7 cells live-treated with N-TASQ (2.5 μM) for 24 h before fixation (PFA-triton). (B) Images of fixed MCF7 cells (PFA-triton) post-labelled with N-TASQ (100 μM). (C) Images of fixed MCF7 cells (MeOH) post-labelled with N-TASQ (100 μM). (D) Images of MCF7 cells live-treated with BRACO-19 (2.5 μM), followed by incubation with N-TASQ (100 μM) for 1 h after cell fixation (MeOH). Fixed cells are mounted (Fluoromount-G^TM) for confocal analyses carried out with lasers at 408, 488 and 555 nm and visualized through DAPI (blue), FITC (green) and Alexa channels (red). White arrows in insets indicate representative clusters of RNA (A,B) and DNA (C,D) quadruplexes.

Figure 5. Co-incubation of BG4 and N-TASQ.

(A,B) MCF7 cells are fixed either with PFA-triton (A) or MeOH (B) and labelled with BG4 (10 μg/mL) for 3 h (followed by secondary and tertiary antibodies, see the Experimental Section) and then N-TASQ (100 μM) for 3 h before mounting steps (Fluoromount-G^TM) and confocal analyses carried out with lasers at 408, 488 and 555 nm and visualized through DAPI (blue), FITC (green) and Alexa channels (red). White arrows indicate some of the most representative N-TASQ/BG4 co-staining sites, i.e., clusters of RNA (A) or DNA (B) quadruplexes.