Visualization of ligand-induced c-MYC duplex-quadruplex transition and direct exploration of the altered c-MYC DNA-protein interactions in cells

Abstract

Ligand-Induced duplex-quadruplex transition within the c-MYC promoter region is one of the most studied and advanced ideas for c-MYC regulation. Despite its importance, there is a lack of methods for monitoring such process in cells, hindering a better understanding of the essence of c-MYC G-quadruplex as a drug target. Here we developed a new fluorescent probe ISCH-MYC for specific c-MYC G-quadruplex recognition based on GTFH (G-quadruplex-Triggered Fluorogenic Hybridization) strategy. We validated that ISCH-MYC displayed distinct fluorescence enhancement upon binding to c-MYC G-quadruplex, which allowed the duplex-quadruplex transition detection of c-MYC G-rich DNA in cells. Using ISCH-MYC, we successfully characterized the induction of duplex to G-quadruplex transition in the presence of G-quadruplex stabilizing ligand PDS and further monitored and evaluated the altered interactions of relevant transcription factors Sp1 and CNBP with c-MYC G-rich DNA. Thus, our study provides a visualization strategy to explore the mechanism of G-quadruplex stabilizing ligand action on c-MYC G-rich DNA and relevant proteins, thereby empowering future drug discovery efforts targeting G-quadruplexes.

Figure 1.

Illustration of GTFH probe ISCH-MYC for c-MYC G-quadruplex detection. (A) c-MYC G-rich DNA sequences, complementary sequences and GTFH probes used in this study. (B) Synthesis of ISCH-MYC. (C) Design principle of ISCH-MYC.

Figure 2.

Fluorescence spectra of ISCH-MYC with DNAs. (A) Fluorescence spectra of 1 μM ISCH-MYC with or without 2 μM DNAs in 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.2. (B) Fluorescence spectra of 1 μM ISCH-MYC with Pu27T of different concentrations in 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.2. (C) Fluorescence spectra of 1 μM ISCH-MYC with 2 μM Pu27T in 10 mM Tris-HCl buffer, pH 7.2 containing 100 mM KCl or LiCl. (D) Fluorescence spectra of 1 μM ISCH-MYC with or without 2 μM DNA G-quadruplexes in 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.2.

Figure 3.

Confocal imaging of FAM-labeled DNA-transfected cells stained by ISCH-MYC. (A) Schematic diagram of cell imaging process using Lipofectamine 3000 (LIPO) or Streptolysin O (SLO) as transfection reagent. (B) Imaging of FAM-labeled DNA-transfected cells stained by ISCH-MYC. The DNAs are delivered into cytoplasm by Lipofectamine 3000. (C) Imaging of FAM-labeled DNA-transfected cells stained by ISCH-MYC. The DNAs are delivered into nucleus by Streptolysin O.

Figure 4.

Detection of ligand-induced duplex-quadruplex transition in cells. (A) Schematic diagram of cell imaging process using Lipofectamine 3000 (LIPO) or Streptolysin O (SLO) as transfection reagent. (B) Imaging of c-MYC G-rich DNA-transfected cells treated with PDS or PDS-A then stained by ISCH-MYC. The DNAs are delivered into cytoplasm by Lipofectamine 3000. (C) Imaging of c-MYC G-rich DNA-transfected cells treated with PDS or PDS-A then stained by ISCH-MYC. The DNAs are delivered into nucleus by Streptolysin O. Quantification of fluorescence was shown on the right. Quantification data are expressed as the mean ± SEM (standard error of mean). For each sample, about 100 cells were measured. The standard error was calculated from a set of three replicate experiments. Statistical significance was determined by the t test as (ns) not significant, (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

Figure 5.

Visualization of the altered c-MYC DNA-Sp1 interactions. (A) Schematic diagram of cell imaging and analysis process using Lipofectamine 3000 (LIPO) or Streptolysin O (SLO) as transfection reagent. (B) Immunofluorescence imaging of Sp1 in Pu27T or Pu27T/Py27 transfected cells treated with or without PDS then stained by ISCH-MYC. The DNAs are delivered into cytoplasm by Lipofectamine 3000. (C) Immunofluorescence imaging of Sp1 in Pu27T or Pu27T/Py27 transfected cells treated with or without PDS then stained by ISCH-MYC. The DNAs are delivered into nucleus by Streptolysin O. White arrows indicate the distinct colocalization of FAM foci with ‘turn-on’ ISCH-MYC signals; while red arrows indicate the distinct colocalization of FAM foci with Sp1 signals (ISCH-MYC signals were ‘turn-off’ in this case).

Figure 6.

Visualization of the altered c-MYC DNA-CNBP interactions. (A) Schematic diagram of cell imaging and analysis process using Lipofectamine 3000 (LIPO) or Streptolysin O (SLO) as transfection reagent. (B) Immunofluorescence imaging of CNBP in Pu27T or Pu27T/Py27 transfected cells treated with or without PDS then stained by ISCH-MYC. The DNAs are delivered into cytoplasm by Lipofectamine 3000. (C) Immunofluorescence imaging of CNBP in Pu27T or Pu27T/Py27 transfected cells treated with or without PDS then stained by ISCH-MYC. The DNAs are delivered into nucleus by Streptolysin O. White arrows indicate the distinct colocalization of FAM foci, CNBP foci and ‘turn-on’ ISCH-MYC signals.