Visualization of Parallel G-Quadruplexes in Cells with a Series of New Developed Bis(4-aminobenzylidene)acetone Derivatives

Abstract

G-quadruplexes (G4s) are unique four-stranded nucleic acid secondary structures formed by G-rich nucleic acid sequences which are prevalent in gene promoter and telomere regions and deemed to play essential roles in many biological and pathological processes. Although attentions to G4s have been paid for nearly 40 years, G4 selectivity and its topology discrimination in cells is still pending. Small fluorescence molecules are emerging as a versatile tool of interrogation of cellular features in vivo. Herein, a new class of bis(4-aminobenzylidene)acetone derivatives GD1, GD2, and GD3 with excellent environment-sensitive emission properties were developed and used for fluorescent detection of G4s. Among them, compound GD3 owning four methoxy groups presented preferable capability of lighting up parallel G4s with a strong red-emission enhancement. The photophysical property of GD3 was systematically investigated to elucidate the turn-on mechanism of GD3 toward parallel G4 structures, which reveal that the binding-induced polarity change of the microenvironment around GD3 together with the fluorophore conformational confinement affected the molecular intramolecular charge-transfer state and resulted the enhanced emission. G4s staining with GD3 in fixed cells was further applied, demonstrating GD3 a promising probe with the ability to visualize the distribution of G4 structures in biological processes. In general, this study provides a new potential scaffold-bis(4-aminobenzylidene)acetone-for design of G4-selective fluorescence probes.

Scheme 1. Structures of the Three Bis(4-aminobenzylidene)acetone Derivatives GD1, GD2, and GD3

Figure 1

(A) Emission responses of GD1, GD2, and GD3 at 600 nm and (B) emission spectra of GD3 toward 0.5 equiv oligonucleotides of 10 different sequences (1 μM) in Tris-HCl buffer (10 mM, pH = 7.4) containing 50 mM KCl. GD1, GD2, and GD3 (2 μM) were severally incubated with DNA at room temperature for 10 min, and the solutions were excited at 450 nm.

Figure 2

(A) Fluorescence titration of GD3 (2 μM) with the 10 preannealed oligonucleotides; (B) CD spectra of the four different parallel G4s (5 μM) and their corresponding complexes with GD3 (5 μM), in Tris-HCl buffer with 50 mM KCl (10 mM, pH = 7.4).

Figure 3

(A) Normalized fluorescence spectra of GD3 (2 μM) in six kinds of solvents with different polarities; (B) fluorescence spectra of GD3 (2 μM) in the mixtures of methanol and Tris-HCl with different ratios (Inset: the emission maximum wavelength of GD3 at different methanol fractions).

Figure 4

Fluorescence images of ARPE-19 cells costained with 1 μM GD3 and 1 μg/mL Hoechst 33258. (A) GD3 red channel (580–620 nm, λ_ex = 488 nm); (B) Hoechst 33258 blue channel (420–450 nm, λ_ex = 405 nm); (C) merged image of (A,B); (D) cell lines pretreated with DNase, (E) cell lines pretreated with RNase, and (F) pretreated with both.