UV Melting of G‐Quadruplexes

Jean‐Louis Mergny1, Laurent Lacroix1

1 Laboratoire de Biophysique, Inserm U565, Muséum National d'Histoire Naturelle, Paris, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 17.1
DOI:  10.1002/0471142700.nc1701s37
Online Posting Date:  June, 2009
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Absorbance versus temperature curves can provide information on the thermal stability of DNA or RNA quadruplexes. Quadruplex denaturation or renaturation can be followed by recording absorbance at 295 nm rather than 260 nm, the wavelength used to monitor duplex denaturation. This unit describes the use of absorbance versus temperature curves to determine melting temperatures (Tm values) and model‐dependent thermodynamic parameters. Curr. Protoc. Nucleic Acid Chem. 37:17.1.1‐17.1.15. © 2009 by John Wiley & Sons, Inc.

Keywords: quartets; thermodynamics; van't Hoff analysis; thermal melting

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Table of Contents

  • Introduction
  • Basic Protocol 1: Melting G‐Quadruplexes and Measuring UV Absorbance
  • Basic Protocol 2: Analysis of Reversible Melting Profiles
  • Alternate Protocol 1: Using Other Approaches in Conjuction with UV Melting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Melting G‐Quadruplexes and Measuring UV Absorbance

  • Oligonucleotide stock solutions (store at −20°C between experiments)
  • 2× stock buffer, e.g., 20 mM lithium cacodylate (see recipe)
  • 10× salt solution, e.g., 1 M NaCl (filter sterilize using a 0.2‐µm filter and store up to several weeks at 4°C)
  • MgCl 2 or other ligand solution, optional
  • Mineral oil, optional
  • Pure ethanol
  • Quartz cuvettes and caps
  • UV‐visible spectrophotometer equipped with a cell changer device
  • Temperature control device (e.g., water bath or Peltier effect heater)
  • 1.5‐mL microcentrifuge tubes, optional
  • Hair dryer, optional
  • External temperature probe
  • Computer attached to spectrophotometer
  • Additional reagents and equipment for determining molar extinction coefficients (e.g., see unit 7.3)
NOTE: It is recommended that oligonucleotide purity and concentration be determined before any thermal analysis begins. Do not depend on indicated quantities provided by the oligonucleotide manufacturer. Ensure that all cuvettes are clean and samples are transparent. Even weak diffusion (resulting from aggregation or precipitation) will ruin the experiment. Use UV‐grade reagents of highest purity for all experiments.CAUTION: Cacodylate buffer contains arsenic and should not be discarded into the drain system; rather the solution should be disposed of as toxic waste.
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Literature Cited

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   Amrane, S., Saccà, B., Mills, M., Chauhan, M., Klump, H.H., and Mergny, J.L. 2005. Length‐dependent energetics of (CTG)n and (CAG)n trinucleotide repeats. Nucleic Acids Res. 33:4065‐4077.
   Bishop, G.R., Ren, J., Polander, B.C., Jeanfreau, B.D., Trent, J.O., and Chaires, J.B. 2007. Energetic basis of molecular recognition in a DNA aptamer. Biophys. Chem. 126:165‐175.
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   Gray, R.D., and Chaires, J.B. 2008. Kinetics and mechanism of K+‐ and Na+‐induced folding of models of human telomeric DNA into G‐quadruplex structures. Nucleic Acids Res. 36:4191‐4203.
   Haq, I., Chowdhry, B.Z., and Chaires, J.B. 1997. Singular value decomposition of 3‐D DNA melting curves reveals complexity in the melting process. Eur. Biophys. J. 26:419‐426.
   Haq, I., Chowdhry, B.Z., and Jenkins, T.C. 2001. Calorimetric techniques in the study of high‐order DNA‐drug interactions. In Drug Nucleic Acid Interaction, Vol. 350 (J.B. Chaires and M.J. Waring, eds.) pp. 109‐149. Academic Press Inc, San Diego, Calif.
   Mergny, J.L. and Lacroix, L. 2003. Analysis of thermal melting curves. Oligonucleotides 13:515‐537.
   Mergny, J.L. and Maurizot, J.C. 2001. Fluorescence resonance energy transfer as a probe for G‐quartet formation by a telomeric repeat. ChemBioChem 2:124‐132.
   Mergny, J.L., Phan, A.T., and Lacroix, L. 1998. Following G‐quartet formation by UV‐spectroscopy. FEBS Lett. 435:74‐78.
   Mergny, J.L., De Cian, A., Ghelab, A., Saccà, B., and Lacroix, L. 2005a. Kinetics of tetramolecular quadruplexes. Nucleic Acids Res. 33:81‐94.
   Mergny, J.L., Li, J., Lacroix, L., Amrane, S., and Chaires, J.B. 2005b. Thermal difference spectra: A specific signature for nucleic acid structures. Nucleic Acids Res. 33:e138.
   Merkina, E.E. and Fox, K.R. 2005. Kinetic stability of intermolecular DNA quadruplexes. Biophys. J. 89:365‐373.
   Mills, M., Arimondo, P., Lacroix, L., Garestier, T., Hélène, C., Klump, H.H., and Mergny, J.L. 1999. Energetics of strand displacement reactions in triple helices: A spectroscopic study. J. Mol. Biol. 291:1035‐1054.
   Petersheim, M. and Turner, D. 1983. Base‐stacking and base‐pairing contributions to helix stability: Thermodynamics of double‐helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp. Biochemistry 22:256‐263.
   Puglisi, J.D. and Tinoco, I. Jr. 1989. Absorbance melting curves of RNA. Methods Enzymol. 180:304‐325.
   Rachwal, P.A. and Fox, K.R. 2007. Quadruplex melting. Methods 43:291‐301.
   Saccà, B., Lacroix, L., and Mergny, J.L. 2005. The effect of chemical modifications on the thermal stability of different G‐quadruplexes‐forming oligonucleotides. Nucleic Acids Res. 33:1182‐1192.
   Testa, S.M. and Gilham, P.T. 1993. Analysis of oligonucleotide structure using hyperchromism measurements at long wavelengths. Nucleic Acids Res. 21:3907‐3908.
   Völker, J., Makube, N., Plum, G.E., Klump, H.H., and Breslauer, K.J. 2002. Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: Implications for triplet expansion diseases. Proc. Natl. Acad. Sci. U.S.A. 99:14700‐14705.
Internet Resources
  A Website to serve the quadruplex community.
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