Directly lighting up RNA G-quadruplexes from test tubes to living human cells

Abstract

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

Scheme 1.

Molecular formula of the cyanine dye CyT.

Figure 1.

Fluorescence emission spectra of 2 μM CyT with various oligonucleotides (4 μM) in 20 mM Tris-HCl (40 mM KCl, pH 7.0) solution.

Figure 2.

Dependence of CyT (2 μM) fluorescence intensity at 595 nm on a variety of RNA sequences (4 μM) in 20 mM Tris-HCl (40 mM KCl, pH 7.0) solution.

Figure 3.

(A) Dependence of CyT (2 μM) fluorescence intensity at 595 nm on Tel22, VEGF, TRF2, BCL-2 and NRAS sequences (4 μM) in K⁺ and Li⁺ solutions. (B) Circular dichroism spectra of RNA G4 sequences (4 μM) in K⁺ and Li⁺ solutions. (C) Dependence of CyT (2 μM) fluorescence intensity at 595 nm on Tel22, VEGF, TRF2, BCL-2 and NRAS sequences (4 μM) at 25°C and 75°C. (D) Circular dichroism spectra of RNA G4 sequences (4 μM) at 25°C and 75°C.

Figure 4.

(A) Absorption spectra and (B) fluorescence emission spectra of CyT (2 μM) with RNA G4 sequence (NRAS) at seven concentrations (μM): (i) 0, (ii) 0.125, (iii) 0.25, (iv) 0.5, (v) 1, (vi) 2, (vii) 4.

Figure 5.

Recognition experiments on PAGE. RNA samples (5 μM) were prepared in 20 mM Tris-HCl buffer with 40 mM K⁺ and loaded on a non-denaturing 12% acrylamide gel. The gels were stained with (A) 20 μM CyT and (B) SYBR Gold, and visualized on GE Typhoon Trio.

Figure 6.

Experiment showing detection limits on native PAGE gels. Oligonucleotide sequences HP-18 (left panels) and BCL-2 (right panels) were tested at nine concentrations (0.125, 0.25, 0.5, 0.7, 1, 2, 2.5, 3 and 5 μM). RNA samples were prepared in 20 mM Tris-HCl buffer with 40 mM K⁺ and loaded on a non-denaturing 12% acrylamide gel. The 12% acrylamide gels (12%) were stained with either (A) 20 μM CyT or (B) SYBR Gold, and were visualized on GE Typhoon Trio.

Figure 7.

(A) Non-canonical G4 sequences. Guanine bases involved in the formation of RNA G4 structures are marked blue. (B) Dependence of CyT (2 μM) fluorescence intensity on noncanonical G4 sequences and nonG4 sequences (4 μM in 20 mM Tris-HCl, pH 7.0, 40 mM K⁺).

Figure 8.

Dependence of TO and CyT fluorescence intensity at 595 nm on G4 and nonG4 sequences: (i) HP-18, (ii) ssAf17, (iii) tRNA, (iv) tRNA-Ala fragment, (iv) tRNA-Cys fragment, (vi) Spinach (vii) Bulges-TB1, (viii) VEGF and (ix) Tel22.

Figure 9.

Confocal laser scanning microscopy showing binding of the fluorescence probe CyT with RNA G4s in the cytoplasm of A549 cells. Nuclei were colored blue by counterstaining with DAPI. Scale bars, 20 μM. (A) After treatment with the CyT probe (5 μM) for 5 min, red fluorescent foci were observed in the cytoplasm. (B) Loss of the red fluorescent foci in the cytoplasm after RNase T1 treatment.

Figure 10.

Confocal laser scanning microscopy showing binding of the CyT probe with RNA G4s in the cytoplasm of A549 cells. Nuclei were colored blue by counterstaining with DAPI. (A) After treatment with the CyT probe (5 μM) for 5 min, red fluorescent foci were observed in the cytoplasm.

Figure 11.

Confocal laser scanning microscopy showing binding of the fluorescence probe CyT with RNA G4s in the cytoplasm of A549 living cells. Nuclei were colored blue by counterstaining with the DNA dye Hoechst 33258, (A) with or (B) without incubation with the CyT probe (1.25 μM) for 24 h. The red channel shows CyT fluorescence in the cytoplasm, and the blue channel shows Hoechst 33258 for nuclear staining (the dotted lines indicate nuclear boundaries). Scale bar, 20 μM.