Functional Application Areas

Nucleic Acid-Nucleic Acid Interactions

Nucleic acids are able to exist in several different forms, which are involved in biological processes like gene replication and cell growth, and these may play a role in disease states. These include:

  • Single stranded RNA
  • Double helix DNA
  • Triple helix (triplex) DNA
  • Quadruplex DNA
  • Telomeres
  • DNA junctions
  • Okazaki fragments
  • Multi-branched (looped) RNA
  • DNA/RNA complexes

Knowledge of how nucleic acids form such complexes is important to understand how nucleic acids function in biological systems. 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 function and biological activity. The complete characterization of any binding interaction requires a quantification of the affinity, number of binding sites, and the thermodynamics. 

Thermodynamic data, specifically enthalpy (ΔH) and entropy (ΔS), reveal the forces that drive complex formation and mechanism of action. Thermodynamics provide information on conformational changes, hydrogen bonding, hydrophobic interactions, and charge-charge interactions.  This information is used to describe the function and mechanism at a molecular level. 

Isothermal Titration Calorimetry (ITC) is a powerful analytical tool which measures the binding affinity and thermodynamics between any two biomolecules. ITC is considered the “gold standard” assay for binding

ITC is vital in the study of multi-probe structure activity relationships (SAR) since it can detect contributions that affinity-only methods may miss.  For example, the affinity measured by these methods may be similar for formation of triplex DNA from different double-stranded and single-stranded DNA sequences, but ITC can reveal differences in ΔH and ΔS that can describe the mechanism of action of binding.  This information can validate in-silico modeling. ITC is also commonly used to validate other binding assays.

Since ITC is done in-solution, it can utilize any biological buffer. For a full characterization of a biomolecular interaction, it is important to observe how salt, pH, temperature, etc affects binding affinity and thermodynamics.

References

Thermodynamics of three-way multibranch loops in RNA.
Diamond J. M., Turner D. H., and Mathews D. H.  
Biochemistry 40, 6971-6981 (2001)

Applications of isothermal titration calorimetry in RNA biochemistry and biophysics.
Feig A. L.
Biopolymers 87, 293-301 (2007)

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength.
Lang B. E. and Schwarz F. P.
Biophys Chem 131, 96-104 (2007)

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation.
Mikulecky P. J. and Feig A. L.
Nucleic Acids Res 32, 3967-3976 (2004).

Duplex dissociation of telomere DNAs induced by molecular crowding.
Miyoshi D., Matsumura S., Nakano S., and Sugimoto N.  
J Am Chem Soc 126, 165-169 (2004)

ITC – Nucleic Acid-Nucleic Acid Interactions Reference List

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