Determination of Nucleic Acid Hydration Using Osmotic Stress

Eriks Rozners1

1 Binghamton University, The State University of New York, Binghamton, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.14
DOI:  10.1002/0471142700.nc0714s43
Online Posting Date:  December, 2010
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Abstract

Water plays an important role in structure and molecular recognition of biopolymers. Understanding hydration of biopolymers is a significant problem in structural chemistry and biology. However, hydration is a dynamic process that is difficult to study. While X‐ray crystallography, NMR, and molecular modeling have provided structural detail on nucleic acid hydration and valuable insights into water dynamics, the thermodynamic contribution of water molecules to conformational equilibria and recognition of nucleic acids remains poorly understood. This unit describes a thermodynamic analysis of nucleic acid hydration using osmotic stress. Osmotic stress monitors the depression of melting temperature upon decreasing water activity, and calculates the number of thermodynamically unique water molecules associated with the double helix and released from single strands upon melting. Comparison of the number of water molecules released upon melting of nucleic acids with different sequences and chemical modifications provides insights that complement and enhance information obtained by other methods. Curr. Protoc. Nucleic Acid Chem. 43:7.14.1‐7.14.13. © 2010 by John Wiley & Sons, Inc.

Keywords: hydration; nucleic acids; thermodynamics; osmotic stress

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: UV Thermal Melting for Osmotic Stress Studies
  • Basic Protocol 2: Determination of Melting Enthalpy for Osmotic Stress Calculation
  • Basic Protocol 3: Calculation of the Number of Water Molecules Released Upon Melting
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: UV Thermal Melting for Osmotic Stress Studies

  Materials
  • Aqueous buffer (CNE 0.3) containing 10 mM sodium cacodylate (pH 7.4), 300 mM NaCl, and 0.1 mM EDTA
  • Solutions of ethylene glycol, glycerol, and acetamide in cacodylate buffer (see Table 7.14.1)
  • 240 µM oligonucleotide stock solution(s): either a single self‐complementary oligonucleotide or two stock solutions of complementary strands
  • UV‐visible spectrometer with multi‐position Peltier temperature controller (see )
  • Microsoft Excel software for statistical data analysis
    Table 7.4.1   Materials   Water Activity (lna W) of Co‐Solute Buffers a   Water Activity (lna W) of Co‐Solute Buffers

    Buffer Co‐solute concentration (g/mL) lna W
    CNE 0.3 alone b 0.0104
    5% ethylene glycol in CNE 0.3 0.0516 0.0248
    10% ethylene glycol in CNE 0.3 0.1002 0.0392
    15% ethylene glycol in CNE 0.3 0.1510 0.0541
    20% ethylene glycol in CNE 0.3 0.2078 0.0665
    5% glycerol in CNE 0.3 0.0508 0.0204
    10% glycerol in CNE 0.3 0.0992 0.0311
    15% glycerol in CNE 0.3 0.1499 0.0433
    20% glycerol in CNE 0.3 0.1998 0.0569
    5% acetamide in CNE 0.3 0.0500 0.0250
    10% acetamide in CNE 0.3 0.1006 0.0399
    15% acetamide in CNE 0.3 0.1510 0.0551
    20% acetamide in CNE 0.3 0.2026 0.0686

     aWater activities were determined experimentally by Spink and Chaires ( ).
     bSolutions are prepared by weighing the precise amount of co‐solute directly into a volumetric flask and diluting to desired volume with CNE 0.3 buffer (10 mM sodium cacodylate, pH 7.4, 300 mM NaCl, 0.1 mM EDTA).

Basic Protocol 2: Determination of Melting Enthalpy for Osmotic Stress Calculation

  Materials
  • 240 µM oligonucleotide stock solution (same as in UV melting studies)
  • Aqueous buffer containing 10 mM sodium phosphate (pH 7.4) and 300 mM NaCl
  • Round‐bottom cryovials
  • Speedvac evaporator
  • Dialysis bag (e.g., Flot‐A‐Lyzer G2, 100‐500 D, Spectrum Laboratories)
  • Differential scanning microcalorimeter suitable for biopolymer studies (This protocol is described for the TA Instruments NanoDSC equipped with 0.3‐mL capillary cells)
  • Scintillation vial
  • Oil‐free vacuum pump, vacuum desiccator, and stirring plate for degassing
  • Thermal analysis software for calculation of van't Hoff enthalpy from melting curves
  • KaleidaGraph software for graphical fitting of data
  • Microsoft Excel software for statistical data analysis

Basic Protocol 3: Calculation of the Number of Water Molecules Released Upon Melting

  Materials
  • KaleidaGraph software for graphical fitting of data
  • Microsoft Excel software for statistical data analysis
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Figures

Videos

Literature Cited

Literature Cited
   Bewington, P.R. and Robinson, D.K. 1992. Data reduction and error analysis for the physical sciences. McGraw Hill, New York.
   Breslauer, K.J. 1995. Extracting thermodynamic data from equilibrium melting curves for oligonucleotide order‐disorder transitions. Methods Enzymol. 259:221‐242.
   Courtenay, E.S., Capp, M.W., Anderson, C.F., and Record, M.T. Jr. 2000. Vapor pressure osmometry studies of osmolyte‐protein interactions: Implications for the action of osmoprotectants in vivo and for the interpretation of “Osmotic Stress” experiments in vitro. Biochemistry 39:4455‐4471.
   Hwang, G.T., Hari, Y., and Romesberg, F.E. 2009. The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation. Nucleic Acids Res. 37:4757‐4763.
   Kolarovic, A., Schweizer, E., Greene, E., Gironda, M., Pallan, P.S., Egli, M., and Rozners, E. 2009. Interplay of structure, hydration and thermal stability in formacetal modified oligonucleotides: RNA may tolerate nonionic modifications better than DNA. J. Am. Chem. Soc. 131:14932‐14937.
   Li, F., Pallan, P.S., Maier, M.A., Rajeev, K.G., Mathieu, S.L., Kreutz, C., Fan, Y., Sanghvi, J., Micura, R., Rozners, E., Manoharan, M., and Egli, M. 2007. Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo‐difluorotoluyl nucleotide: Insights into substrate requirements by the human RISC Ago2 enzyme. Nucleic Acids Res. 35:6424‐6438.
   Parsegian, V.A., Rand, R.P., and Rau, D.C. 1995. Macromolecules and water: Probing with osmotic stress. Methods Enzymol. 259:43‐94.
   Parsegian, V.A., Rand, R.P., and Rau, D.C. 2000. Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives. Proc. Natl. Acad. Sci. U.S.A. 97:3987‐3992.
   Pilch, D.S. 2000. Calorimetry of nucleic acids. Curr. Protoc. Nucleic Acid Chem. 0:7.4.1‐7.4.9.
   Qu, X. and Chaires, J.B. 1999. Contrasting hydration changes for ethidium and daunomycin binding to DNA. J. Am. Chem. Soc. 121:2649‐2650.
   Qu, X. and Chaires, J.B. 2001. Hydration changes for DNA intercalation reactions. J. Am. Chem. Soc. 123:1‐7.
   Robinson, C.R. and Sligar, S.G. 1995. Hydrostatic and osmotic pressure as tools to study macromolecular recognition. Methods Enzymol. 259:395‐427.
   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.
   Rozners, E., Smicius, R., and Uchiyama, C. 2005. Expanding functionality of RNA: Synthesis and properties of RNA containing imidazole modified tandem G‐U wobble base pairs. Chem. Commun. 46:5778‐5780.
   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.
   Timasheff, S.N. 1998. In disperse solution, “osmotic stress” is a restricted case of preferential interactions. Proc. Natl. Acad. Sci. U.S.A. 95:7363‐7367.
   Watts, J.K., Martin‐Pintado, N., Gomez‐Pinto, I., Schwartzentruber, J., Portella, G., Orozco, M., Gonzalez, C., and Damha, M.J. 2010. Differential stability of 2′F‐ANA‐RNA and ANA‐RNA hybrid duplexes: Roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility. Nucleic Acids Res. 38:2498‐2511.
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