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

  • 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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Literature Cited

Literature Cited
   Anderson, C.F. and Record, M.T. Jr. 1990. Ion distributions around DNA and other cylindrical polyions: Theoretical descriptions and physical implications. Annu. Rev. Biophys. Biophys. Chem. 19:423‐465.
   Anderson, C.F. and Record, M.T. Jr. 1995. Salt‐nucleic acid interactions. Annu. Rev. Phys. Chem. 46:657‐700.
   Auffinger, P. and Westhof, E. 2001. Water and ion binding around r(UpA)12 and d(TpA)12 oligomers—Comparison with RNA and DNA (CpG)12 duplexes. J. Mol. Biol. 305:1057‐1072.
   Bloomfield, V.A., Crothers, D.M., and Tinoco, I. Jr. 2000. Nucleic Acids: Structures, Properties, and Functions. University Science Books, Sausalito, California.
   Breslauer, K.J., Frank, R., Blocker, H., and Marky, L.A. 1986. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83:3746‐3750.
   Ellis, R.J. and Minton, A.P. 2003. Cell biology: Join the crowd. Nature 425:27‐28.
   Feig, M. and Pettitt, B.M. 1998. A molecular simulation picture of DNA hydration around A‐ and B‐DNA. Biopolymers 48:199‐209.
   Fulton, A.B. 1982. How crowded is the cytoplasm? Cell 30:345‐347.
   Goobes, R., Kahana, N., Cohen, O., and Minsky, A. 2003. Metabolic buffering exerted by macromolecular crowding on DNA‐DNA interactions: Origin and physiological significance. Biochemistry 42:2431‐2440.
   Guttman, H.J., Anderson, C.F., and Record, M.T. Jr. 1995. Analyses of thermodynamic data for concentrated hemoglobin solutions using scaled particle theory: Implications for a simple two‐state model of water in thermodynamic analyses of crowding in vitro and in vivo. Biophys. J. 68:835‐846.
   Hall, D. and Minton, A.P. 2003. Macromolecular crowding: Qualitative and semiquantitative successes, quantitative challenges. Biochim. Biophys. Acta 1649:127‐139.
   Karimata, H., Nakano, S., and Sugimoto, N. 2007. Effects of polyethylene glycol on DNA duplex stability at different NaCl concentrations. Bull. Chem. Soc. Jpn. 80:1987‐1994.
   Kilburn, D., Roh, J.H., Guo, L., Briber, R.M., and Woodson, S.A. 2010. Molecular crowding stabilizes folded RNA structure by the excluded volume effect. J. Am. Chem. Soc. 132:8690‐8696.
   Manning, G.S. 1972. On the application of polyelectrolyte “limiting laws” to the helix‐coil transition of DNA. I. Excess univalent cations. Biopolymers 11:937‐949.
   Miyoshi, D. and Sugimoto, N. 2008. Molecular crowding effects on structure and stability of DNA. Biochimie 90:1040‐1051.
   Miyoshi, D., Karimata, H., and Sugimoto, N. 2006. Hydration regulates thermodynamics of G‐quadruplex formation under molecular crowding conditions. J. Am. Chem. Soc. 128:7957‐7963.
   Miyoshi, D., Nakamura, K., Tateishi‐Karimata, H., Ohmichi, T., and Sugimoto, N. 2009. Hydration of Watson‐Crick base pairs and dehydration of Hoogsteen base pairs inducing structural polymorphism under molecular crowding conditions. J. Am. Chem. Soc. 131:3522‐3531.
   Nagatoishi, S., Isono, N., Tsumoto, K., and Sugimoto, N. 2011. Hydration is required in DNA G‐quadruplex‐protein binding. Chembiochem 12:1822‐1826.
   Nakano, S., Fujimoto, M., Hara, H., and Sugimoto, N. 1999. Nucleic acid duplex stability: Influence of base composition on cation effects. Nucleic Acids Res. 27:2957‐2965.
   Nakano, S., Karimata, H., Ohmichi, T., Kawakami, J., and Sugimoto, N. 2004. The effect of molecular crowding with nucleotide length and cosolute structure on DNA duplex stability. J. Am. Chem. Soc. 126:14330‐14331.
   Nakano, S., Kirihata, T., and Sugimoto, N. 2008a. Capture of cationic ligands bound diffusely to base pairs during DNA refolding. Chem. Commun. 6:700‐702.
   Nakano, S., Wu, L., Oka, H., Karimata, H.T., Kirihata, T., Sato, Y., Fujii, S., Sakai, H., Kuwahara, M., Sawai, H., and Sugimoto, N. 2008b. Conformation and the sodium ion condensation on DNA and RNA structures in the presence of a neutral cosolute as a mimic of the intracellular media. Mol. Biosyst. 4:579‐588.
   Nakano, S., Karimata‐Tateishi, H., Kitagawa, Y., and Sugimoto, N. 2009. Facilitation of RNA enzyme activity in the molecular crowding media of cosolutes. J. Am. Chem. Soc. 131:16881‐16888.
   Nakano, S., Yamaguchi, D., Tateishi‐Karimata, H., Miyoshi, D., and Sugimoto, N. 2012. Hydration changes upon DNA folding studied by osmotic stress experiments. Biophys. J. 102:2808‐2817.
   Nordstrom, L.J., Clark, C.A., Andersen, B., Champlin, S.M., and Schwinefus, J.J. 2006. Effect of ethylene glycol, urea, and N‐methylated glycines on DNA thermal stability: The role of DNA base pair composition and hydration. Biochemistry 45:9604‐9614.
   Pramanik, S., Nakamura, K., Usui, K., Nakano, S., Saxena, S., Matsui, J., Miyoshi, D., and Sugimoto, N. 2011. Thermodynamic stability of Hoogsteen and Watson‐Crick base pairs in the presence of histone H3‐mimicking peptide. Chem. Commun. 47:2790‐2792.
   Qu, X. and Chaires, J.B. 2001. Hydration changes for DNA intercalation reactions. J. Am. Chem. Soc. 123:1‐7.
   Record, M.T. Jr., Anderson, C.F., and Lohman, T.M. 1978. Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: The roles of ion association or release, screening, and ion effects on water activity. Q. Rev. Biophys. 11:103‐178.
   Richards, E.G. 1975. Nucleic acids and polynucleotides. In Handbook of Biochemistry and Molecular Biology: Nucleic Acids, 3rd ed., Vol. 1. (G.D. Fasman, ed.) pp. 596‐598. CRC Press, Cleveland, Ohio.
   Rozners, E. and Moulder, J. 2004. Hydration of short DNA, RNA and 2′‐OMe oligonucleotides determined by osmotic stressing. Nucleic Acids Res. 32:248‐254.
   Spink, C.H. and Chaires, J.B. 1999. Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA. Biochemistry 38:496‐508.
   Spink, C.H., Garbett, N., and Chaires, J.B. 2007 Enthalpies of DNA melting in the presence of osmolytes. Biophys. Chem. 126:176‐185.
   Sugimoto, N., Kierzek, R., Freier, S.M., and Turner, D.H. 1986. Energetics of internal GU mismatches in ribooligonucleotide helixes. Biochemistry 25:5755‐5759.
   Tateishi‐Karimata, H. and Sugimoto, N. 2012. A‐T base pairs are more stable than G‐C base pairs in a hydrated ionic liquid. Angew Chem. Int. Ed. 51:1416‐1419.
   Tobe, S., Heams, T., Vergne, J., Herve, G., and Maurel, M.C. 2005. The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure. Nucleic Acids Res. 33:2557‐2564.
   Wenner, J.R. and Bloomfield, V.A. 1999. Crowding effects on EcoRV kinetics and binding. Biophys. J. 77:3234‐3241.
   Xia, T. SantaLucia, J. Jr., Burkard, M.E., Kierzek, R., Schroeder, S.J., Jiao, X., Cox, C., and Turner, D.H. 1998. Thermodynamic parameters for an expanded nearest‐neighbor model for formation of RNA duplexes with Watson‐Crick base pairs. Biochemistry 37:14719‐14735.
   Zhou, H.X., Rivas, G., and Minton, A.P. 2008. Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 37:375‐397.
   Zimmerman, S.B. and Minton, A.P. 1993. Macromolecular crowding: Biochemical, biophysical, and physiological consequences. Annu. Rev. Biophys. Biomol. Struct. 22:27‐65.
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