Nucleic Acid Stability : Stabilization in biological systems

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.

Importance of Nucleic Acid Stability :

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.

Nucleic Acid Stability Experiment :

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 Nucleic acid 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.

Nucleic Acid Stability : Stabilization in biological systems

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 Nucleic Acid Stability, 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