Functional Application Areas

Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions are important to all biological systems, as well as protein engineering.   These can include:

  • Proteins involved in gene expression, DNA replication, transcription and translation
  • Histones
  • Binding of repressors and promoters to operators
  • Viral assemblies
  • Zinc fingers
  • Protein-single stranded DNA interaction
  • Protein-RNA interactions
  • Formation of quadruplex structures
  • Transport and delivery of DNA into cells
  • Transformation
  • Enzymes involved in cloning and PCR

Knowledge of these interactions is important to understand how proteins and 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 protein and 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 a wild-type and mutant protein binding to an oligonucleotide, 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.

ITC is also used to characterize ligand specificity (i.e. a series of peptides binding a nucleic acid), binding of inhibitors of protein-nucleic acid interactions, and allosteric effects of ligands to the protein-nucleic acid interactions.

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.

Most drug targets are proteins, and drug discovery involves identifying compounds which can either inhibit or activate the target protein. Aptamers are RNA molecules which are a new class of therapeutics, currently under investigation by pharmaceutical and biotechnology companies. ITC is also becoming an important tool in characterizing drug-target interactions, and can be used in many different stages of Drug Discovery and Development.  For example, many antibiotics interfere with protein-nucleic acid interactions.

References

Thermodynamics of the binding of Thermus aquaticus DNA polymerase to primed-template DNA.
Datta K. and LiCata V. J.
Nucleic Acids Res 31, 5590-5597 (2003)

Effect of mutation of the Sac7d intercalating residues on the temperature dependence of DNA distortion and binding thermodynamics.
Peters W. B., Edmondson S. P., and Shriver J. W. 
Biochemistry 44, 4794-4804 (2005)

Calorimetry of protein-DNA complexes and their components.
Read C. M. and Jelesarov I.
Methods Mol Biol 148, 511-533 (2001)

ITC – Protein-Nucleic Acid Interactions Reference List

ITC – Antibiotics Reference List

ITC – Drug Discovery and Development Reference List

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