Cellular Detection of G‐Quadruplexes by Optical Imaging Methods

Souheila Amor1, Sunny Y. Yang2, Judy M. Y. Wong2, David Monchaud1

1 Institut de Chimie Moléculaire, UBFC Dijon, 2 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.33
DOI:  10.1002/cpcb.29
Online Posting Date:  September, 2017
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Abstract

G‐quadruplexes (G4s) are higher‐order nucleic acid structures that fold from guanine (G)–rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex‐selective fluorophores, the twice‐as‐smart quadruplex ligands named TASQ (for template‐assembled synthetic G‐quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real‐time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.

Keywords: G‐quadruplex; fluorescence; smart probes; microscopy; live cell imaging

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Detection of Quadruplexes in Cells: Confocal versus Two‐Photon Microscopes, Fixed versus Live‐Cell Imaging
  • Basic Protocol 1: Use of N‐TASQ for Live and Post‐Fixation Staining of Cultured Human Cells
  • Basic Protocol 2: Example of Use of N‐TASQ for Live‐Cell and Post‐Fixation Staining of MCF‐7 Cells
  • Alternate Protocol 1: Example of Use of N‐TASQ for Post‐Fixation Staining on Conventional Coverslips
  • Alternate Protocol 2: Use of N‐TASQ in Combination with Quadruplex‐Specific Antibodies
  • Basic Protocol 3: Detection of Quadruplexes In Cellulo: Real‐Time Monitoring in Live Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Use of N‐TASQ for Live and Post‐Fixation Staining of Cultured Human Cells

  Materials
  • Cell line of interest and appropriate medium (ATCC, or any other cell repository/provider)
  • Fluorophore N‐TASQ in sterile water (see Critical Parameters)
  • Dulbecco's phosphate‐buffered saline (DPBS), pH 7.4 (Gibco, cat no. 1001023)
  • Fixatives: cooled (−20°C) methanol or 4% (w/v) paraformaldehyde (PFA) in 1× DPBS
  • Permeabilizing solution: 0.1% to 0.5% (v/v) Triton X‐100 in PBS
  • Antifade mounting medium (Fluoromount‐G, Southern Biotech)
  • Clear nail polish
  • Removable chambered coverglass specially treated to ensure a consistent growth surface, staining and microscopic examination (8 chamber, Corning Falcon)
  • Humidified 37°C, 5% CO 2 incubator
  • Coverslips suitable for viewing at high magnification on the microscope
  • Additional reagents and equipment for cell culture (unit 1.1; Phelan & May, ) and confocal microscopy (Smith, )
NOTE: All solutions and equipment coming into contact with living cells must be sterile.NOTE: Volume indicated corresponds to the suppliers’ instructions.

Basic Protocol 2: Example of Use of N‐TASQ for Live‐Cell and Post‐Fixation Staining of MCF‐7 Cells

  Materials
  • MCF‐7 cells (ATCC® HTB‐22TM)
  • DMEM medium supplemented with 10% fetal bovine serum (FBS)
  • 0.25% trypsin/EDTA solution (e.g., Gibco)
  • 2.5 µM N‐TASQ in DMEM supplemented with 10% FBS
  • 4% (w/v) paraformaldehyde (PFA) in 1× DPBS
  • Permeabilizing solution: 0.5% (v/v) Triton X‐100 in 1× DPBS
  • Dulbecco's phosphate‐buffered saline (DPBS; Gibco, cat. no. 1001023)
  • Antifade mounting medium: Fluoromount‐G (Southern Biotech)
  • Clear nail polish
  • Methanol, cold (–20°C)
  • 75‐cm2 tissue culture flasks (Nunc)
  • Humidified 37°C, 5% CO 2 incubator
  • Removable chambered coverglass specially treated to ensure a consistent growth surface, staining and microscopic examination (8 chambers, Corning Falcon)
  • Coverslips suitable for viewing at high magnification on the microscope

Alternate Protocol 1: Example of Use of N‐TASQ for Post‐Fixation Staining on Conventional Coverslips

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • DMEM medium supplemented with 5% fetal bovine serum (FBS) and 1× penicillin‐streptomycin
  • 0.25% trypsin/EDTA solution (e.g., Gibco)
  • 70% ethanol
  • Washing solution: 1× DPBS or 1× DPBS/0.1% (v/v) Triton X‐100 (PBS‐T)
  • Blocking solution: 4% (w/v) bovine serum albumin (BSA) in 1× DPBS
  • Nuclear stain DAPI or DRAQ5 dyes
  • Sterilized 12‐mm‐diameter round glass coverslips suitable for imaging at high magnification
  • 24‐well tissue culture plates
  • Forceps and a needle
  • Microscope slides

Alternate Protocol 2: Use of N‐TASQ in Combination with Quadruplex‐Specific Antibodies

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • BG4 ScvF antibody: produced in E. coli BL21(DE3) transfected with pSANG10‐3F (ampicillin selection) (courtesy of Dr. Shankar Balasubramanian (Cambridge University, U.K.). BG4 (His‐tagged) is purified through a single round of Ni‐NTA resin binding. By SDS PAGE and standard Bradford protein assay the eluted fraction is quantified and the purity controlled by western blot (anti‐FLAG monoclonal antibody).
  • PBS/0.1% (v/v) Triton X‐100 (PBS‐T)
  • Blocking solution: 4% (w/v) bovine serum albumin (BSA) in 1× DPBS
  • Primary antibody: 2 μg/ml FLAG M2 monoclonal antibody (Sigma)
  • Secondary antibody: anti‐mouse IgG antibody conjugated with AF594 (Molecular Probes)

Basic Protocol 3: Detection of Quadruplexes In Cellulo: Real‐Time Monitoring in Live Cells

  Materials
  • Cell lines of interest and appropriate medium, according to ATCC or Essen Bioscience for NucLight red lentivirus transduced cells: We used breast cancer MDA‐MB‐231 cells transduced with NucLight Red lentivirus (Essen Bioscience, cat. no. 4487) that directs the production of red fluorescent proteins (RFP) that label cell nuclei. The RFP can be visualized, for quantification of cell growth purposes, through the red channel of the microscope (λ ex = 585 nm, emission filter: 625 to 705 nm), leaving N‐TASQ to be detected via the green channel (λ ex = 460 nm, emission filter: 504‐544 nm) where its fluorescence signal is highest. For comparison, non‐cancerous foreskin fibroblast BJ cells immortalized by transducing in retrovirus‐encoded hTERT (consequently termed BJ‐hTERT; Zeng et al., ) were used. Since these cells do not express fluorescent proteins, we used the phase‐contrast channel to quantify cell confluency, as measure of growth rate.
  • 0.25% trypsin/EDTA solution (e.g., Gibco)
  • Fluorophore N‐TASQ (solubilized in sterile H 2O)
  • Vessels for growth, compatible with real‐time facility (e.g., 96‐well flat bottom plates)
  • Counting device (e.g., cell counter Z2 Beckman Coulter)
  • Sterilized plastic vessels routinely used in cell culture
  • Live cell monitoring and analysis system (e.g., IncuCyte ZOOM from Essen Bioscience, which comprises a microscope housed in a cell incubator and a networked, external‐controller hard drive that gathers and processes image data at up to 2000 images/hr)
Preparation of the plates for real‐time monitoring systems
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Figures

Videos

Literature Cited

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