Functional Application Areas

Nucleic Acid-Small Molecule Interactions

Nucleic acids bind a variety of small molecules and this binding can affect their function. These small molecules include:

  • Drugs
  • Dyes
  • Metals
  • Surfactants

There are a variety of compounds that bind to DNA as “intercalators” while another set of compounds are DNA ‘’groove-binders.” Since DNA is a target of anticancer treatments as well as other diseases, there is a large body of research on the use of ITC to characterize the binding affinity and mechanism of DNA-drug interactions.

Knowledge of these interactions 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 different oligonucleotide sequences binding to a drug, 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 ligands binding to the nucleic acid) and studying allosteric effects.

Many drug targets are nucleic acids, and drug discovery involves identifying compounds which can either inhibit or activate the target protein. Aptamers are RNA molecules which are under investigation as a potential drug. 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.

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.

DSC and DNA Drug Binding Studies

Differential Scanning Calorimetry (DSC) can also be used to study nucleic acid – small molecule interactions, including: the correlation between thermodynamic parameters and solutions structure for the interaction of minor groove ligands with an AAATT/AATTT sequence in DNA (Renzeperis et al, 1995), thermodynamics of DNA-cisplatin and DNA-transplatin (Hofr and Brabec, 2001), study of a bifuntional polymer platinum compound crosslinked to DNA (Hofr et al, 2001), and formation of drug-DNA complexes (Leng, et al, 1999)

Effect of adding ethidium bromide to herring DNA.  DNA (1.1 mM) in 6 mM Na2HPO4, 2mM NaH2PO4, 1mM EDTA, pH 7.0, in the absence (scan 1) and presence (scan 2) of 0.5 mM ethidium bromide.  Scans were performed in the VP-Capillary DSC at 200° C/hour.  DSC data shown are after buffer-buffer reference scan subtraction and concentration normalization.  Binding constant estimated to be 1.6 x 104 M-1.  DNA samples prepared by Dr. J.B. Chaires, University of Mississippi (currently at University of Louisville)

References

Thermodynamic characterization of the multivalent binding of chartreusin to DNA.
Barcelo F., Capo D., and Portugal J.
Nucleic Acids Res 30, 4567-4573 (2002)

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine.
Bhadra K., Maiti M. and Kumar G. S.
Biochim Biophys Acta 1770, 1071-1080 (2007)

Energetic basis of molecular recognition in a DNA aptamer.
Bishop G. R., Ren J., Polander B. C., Jeanfreau B. D., Trent J. O., and Chaires J. B.
Biophys Chem 126, 165-75 (2007)

Parsing free energies of drug-DNA interactions.
Haq I., Jenkins T. C., Chowdhry B. Z., Ren J., and Chaires J. B.
Methods Enzymol 323, 373-405  (2000)

Calorimetric techniques in the study of high-order DNA-drug interactions.
Haq I., Chowdhry B. Z., and Jenkins T. C.
Methods Enzymol 340, 109-149 (2001)

Thermal and thermodynamic properties of duplex DNA containing site-specific interstrand cross-link of antitumor cisplatin or its clinically ineffective trans isomer.
Hofr, C., Brabec, V.
J. Biol. Chem. 276, 9655-9661 (2001)

Thermodynamic properties of duplex DNA containing a site-specific d(GpP) intrastrand crosslink formed by an antitumor dinuclear platinum complex.
Hofr, B., Ferrell, N., Brabec, V.
Nucleic Acids Res. 29, 2034-2040 (2001)

Ultratight DNA binding of a new bisintercalating anthrocycline antibiotic.
Leng, F., Priebe, W., Chaires, J.B.
Biochemistry 37, 1743-1753 (1999)

Interaction of minor groove ligands to an AAATT/AATTT site: Correlation of thermodynamic characterization and solution structure.
Renzeperis, D., Marky, L.A., Dryer, T., J., Geierstanger, B.H., Pelton, J.G., Wemmer, D.E.
Biochemistry 34, 2937-2945 (1995)

ITC – Nucleic Acid-Small Molecule Interactions Reference List

ITC – Drug Discovery and Design Reference List

DSC – Nucleic Acid-Small Molecule Interactions Reference List

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