Functional Application Areas

Nucleic Acid Stability

Nucleic acid structures are stabilized by non-covalent intramolecular interactions between the bases. All biological processes involving DNA and RNA require these structures to be in the stable and in the appropriate conformation. It is important to know how nucleic acids form their biologically active states and how these active states are stabilized.

There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of nucleic acid structure alone does not ensure accurate prediction of stability, function and biological activity. The complete characterization of any biomolecule requires stability determination and the forces which lead to stability and correct folding.

Differential Scanning Calorimetry (DSC) is a powerful analytical tool which directly measures the stability and unfolding of biomolecules.  In DSC, the sample is heated at a constant rate, and there is a detectable heat change associated with thermal denaturation.

A single DSC experiment can determine:

  • Transition midpoint (Tm)
  • Enthalpy (∆H) and heat capacity change (∆Cp) associated with uncoiling
  • Presence of multiple melting site domains

A nucleic acid in aqueous solution is in equilibrium between the native conformation and its uncoiled conformation.  The stability of the native state is based on the magnitude of the Gibbs free energy (∆G) of the system and the thermodynamic relationships between enthalpy (∆H) and entropy (∆S) changes.  A positive ∆G indicates the native state is more stable than the denatured state – the more positive the ∆G, the greater the stability.  For a DNA molecule to melt, stabilizing forces need to be broken.

The transition midpoint (Tm) is the temperature where 50% of the DNA is in its native confirmation and the other 50% is melted. In general, the higher the Tm, the more stable the DNA.

DSC measures ∆H due to heat denaturation. Nucleic acid unfolding is typically endothermic. During the same experiment, DSC also measures the change in heat capacity (∆Cp) for denaturation.

Many factors are responsible for the stability of nucleic acids, including hydrogen bonding, conformational entropy, and the physical environment (pH, buffer, ionic strength, excipients, etc.).

DSC data, either used alone or in conjunction with stability and structural data, can provide information on:

  • Effects of DNA and RNA sequence
  • Effects of buffer, pH, salt, additives
  • Duplex, triplex and quadruplex structures
  • Formation of RNA and DNA complexes
  • Formation of nucleic acid-protein complexes
  • Effects of small molecule drugs on nucleic acid stability

References

Length and pH-dependent energetics of (CCG)n and (CGG)n trinucleotide repeats
Amrane, S. and Mergny, J. L.
Biochimie 88, 1125-1134 (2006)

Biophysical characterization of Hhe human telomeric (TTAGGG)4 repeat in a potassium solution.
Antonacci, C., Chaires, J. B. and Sheardy, R. D.
Biochemistry 46, 4654-4660 (2007)

Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design.
Bruylants, G., Wouters, J., and Michaux, C.
Curr Med Chem 12, 2011-2020 (2005)

Effects of monofunctional adducts of platinum(II) complexes on thermodynamic stability and energetics of DNA duplexes.
Bursova, V., Kasparkova, J., Hofr, C., and Brabec, V.
Biophys J 88, 1207-1214 (2005)

Structure-based design of a new bisintercalating anthracycline antibiotic.
Chaires, J. B., Leng, F., Przewloka, T., Fokt, I., Ling, Y. H., Perez-Soler, R., and Priebe, W.
J Med Chem 40, 261-266 (1997)

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry.
Chakrabarti, M.C., Schwartz, F.P.
Nucleic Acids Res. 27, 4801-4806 (1999)

Biophysical studies of the c-MYC NHE III1 promoter: model quadruplex interactions with a cationic porphyrin.
Freyer, M. W., Buscaglia, R., Kaplan, K., Cashman, D., Hurley, L. H. and Lewis, E. A.
Biophys J 92, 2007-2015 (2007)

Heat capacity changes associated with nucleic acid folding.
Mikulecky, P. J. and Feig, A. L.
Biopolymers  82, 38-58 (2006)

Energetic and conformational contributions to the stability of Okazaki fragments.
Soto, A.M., Gmeiner, W.H., Marky, L.A. 
Biochemistry 41, 6842-6849 (2002)

Reference Lists

DSC – Nucleic Acid Stability Reference List

DSC – Nucleic Acid Folding and Stability Studies Reference List

DSC – Protein–Nucleic Acid Interactions Reference List

DSC – Nucleic Acid-Small Molecule Interactions Reference List

DSC – Nucleic Acid-Lipid Interactions Reference List

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