Quantitative Analyses of Nucleic Acid Stability Under the Molecular Crowding Condition Induced by Cosolutes

Hisae Tateishi‐Karimata1, Shu‐ichi Nakano2, Naoki Sugimoto2

1 Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan, 2 Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.19
DOI:  10.1002/0471142700.nc0719s53
Online Posting Date:  June, 2013
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Abstract

A variety of biomolecules, including nucleic acids, proteins, polysaccharides, and other soluble and insoluble low‐molecular weight components, are present in living cells. These molecules occupy a significant fraction of the cellular volume (up to 40%), resulting in a highly crowded intracellular environment. This situation is referred to as molecular crowding. Although the thermodynamic stabilities of DNA structures are known to be altered in a crowded environment, less is known about the behavior of nucleic acids and their interactions with cations and water molecules under such conditions. This unit describes methods that can be used to quantitatively analyze the molecular crowding effects caused by cosolutes on the thermodynamic stability, hydration, and cation binding of nucleic acid structures. Curr. Protoc. Nucleic Acid Chem. 53:7.19.1‐7.19.17. © 2013 by John Wiley & Sons, Inc.

Keywords: molecular crowding; hydration; cation binding; thermodynamics; nucleic acids

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Calculation of Thermodynamic Parameters for Formation of Nucleic Acid Structures Based on UV Melting Curves
  • Basic Protocol 2: Determination of the Number of Water Molecules and the Number of Cations Bound During DNA Structure Formation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Calculation of Thermodynamic Parameters for Formation of Nucleic Acid Structures Based on UV Melting Curves

  Materials
  • 50% (v/v) nitric acid, optional
  • Crowding solution: phosphate buffer solution at pH 7.0 containing 1.0 M NaCl (1 M NaCl, 10 mM Na 2HPO 4, pH 7.0 at 25°C, and 1 mM Na 2EDTA)
  • ODNs (0.2‐µmol scale synthesis; see Strategic Planning)
  • UV spectrophotometer equipped with a temperature controller
  • Quartz cuvette cells with 1‐mm and 1‐cm path–lengths
  • Hot plate and magnetic stir bar
  • Heating block
  • Personal computer (PC) for computational data analysis
NOTE: The synthesis scale indicates the amount of starting material present, not the amount of final product produced.NOTE: The authors use a Shimadzu 1700 spectrophotometer (Shimadzu) connected to a thermoprogrammer to measure the ODN absorbance at 260 nm. This machine can hold eight micro‐multicells to allow for simultaneous measurement of eight samples.NOTE: A PC with installed curve fitting software, such as IGOR Pro (WaveMetrics), KaleidaGraph (HULINKS), and ORIGIN (Light Stone), is convenient for thermodynamic analysis.
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Figures

Videos

Literature Cited

Literature Cited
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