Quantitative Analysis of Nucleic Acid Stability with Ligands Under High Pressure to Design Novel Drugs Targeting G‐Quadruplexes

Shuntaro Takahashi1, Naoki Sugimoto2

1 Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, 2 Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 17.9
DOI:  10.1002/cpnc.39
Online Posting Date:  September, 2017
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Abstract

Nucleic acids (DNA and RNA) can form various non‐canonical structures. Because some serious diseases are caused by the conformational change of G‐quadruplex DNA structures, the development of ligands that bind and stabilize G‐quadruplex DNA is of interest to the field of nucleic acid chemistry. Volumetric changes (ΔV) in the biomolecular reaction include the structural change of biomolecules and hydration behaviors, which provide information about the tertiary interaction between G‐quadruplex DNA and ligands. Thus, it is valuable to investigate ΔV values to understand the mechanism of interaction between non‐canonical structures and their ligands. This unit describes methods that can be used to quantitatively analyze the interaction between G‐quadruplex DNA and ligands by using high‐pressure UV melting. The combination of thermodynamic parameters (ΔG, ΔH, ΔS, and ΔV) is a powerful tool to elucidate the mechanism of ligand binding to G‐quadruplex without real structural analysis by NMR and X‐ray spectroscopy, and gives useful information to design novel drugs. © 2017 by John Wiley & Sons, Inc.

Keywords: DNA; drug; G‐quadruplex ligand; pressure; UV melting

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Setup of High‐Pressure Apparatus for UV Melting Analysis of Nucleic Acid Stability
  • Basic Protocol 2: UV Melting Measurement of G‐Quadruplex DNA Under High Pressure
  • Basic Protocol 3: Analysis of Thermodynamic Parameters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Setup of High‐Pressure Apparatus for UV Melting Analysis of Nucleic Acid Stability

  Materials
  • Grease
  • Concentrated nitric acid
  • Methanol or ethanol
  • Buffer solution (see recipe)
  • High‐pressure cell
  • UV spectrophotometer
  • Refrigerated and heating circulators
  • Pressure gauge
  • Pump
  • Pressure reservoir system
  • Quartz cuvette cells for use under high pressure
  • Computer for controlling temperature and recording absorbance
NOTE: The authors used a high‐pressure system obtained from Syn‐Corporation Ltd (Kyoto, Japan). As for UV measurement, temperature was controlled by using refrigerated and heating circulators from Julabo ME‐25 (Seelbach, Germany). This protocol is entirely based on usage of these apparatuses and equipped software. Similar systems may be available from other suppliers.NOTE: The authors use water for the circulator. If heating above 100°C is necessary, oil as a temperature control liquid can be used if your pressure cell allows it.

Basic Protocol 2: UV Melting Measurement of G‐Quadruplex DNA Under High Pressure

  Materials
  • ODN solution (0.2 μM scale synthesis; see Strategic Planning)
  • PEG200
  • Ligand molecule (e.g., N‐methylmesoporphyrin IX [NMM] and iron(III)‐protoporphyrin IX [hemin])
  • Quartz cuvette cells for use under high pressure
  • High‐pressure system (see protocol 1)
  • Pressure reservoir system
  • Refrigerated and heating circulators
  • Computer for controlling temperature and recording absorbance
  • UV spectrophotometer
  • Pressure gauge
  • Pump
NOTE: The synthesis scale indicates the amount of starting material present, not the amount of final product produced.

Basic Protocol 3: Analysis of Thermodynamic Parameters

  Materials
  • UV melting data (see protocol 2)
  • Computer running curve‐fitting software
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Figures

Videos

Literature Cited

Literature Cited
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