Functional Application Areas

Interactions with Nucleic Acids

Nucleic acids, including DNA and RNA, are involved in gene replication and expression, transcription, and protein expression.  Modern molecular biology and protein expression requires cloning, mutagenesis and amplification of nucleic acids.  As part of their function, nucleic acids specifically bind a biomolecule such as a lipid, protein, or another nucleic acid, and nucleic acids can also bind drugs, surfactants or other ligands. Understanding how nucleic acids function and interact with other molecules is important in biochemistry and drug discovery and development.

Accompanying the rapid advances in structural biology is the definition of biochemical function and mechanism in terms of molecular forces.  However, knowledge of structure alone does not ensure accurate prediction of function and biological activity. The complete characterization of any binding interaction requires a quantification of the binding affinity, number of binding sites, and the thermodynamics. 

Thermodynamics provides information on the energetic forces that drive biomolecular interactions. Enthalpy (ΔH) and entropy (ΔS), reveals the forces that drive complex formation and mechanism of action, and provides 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 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 commonly used to validate other binding assays.

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 binding of a biomolecular interaction, it is important to observe how salt, pH, temperature, etc affects binding affinity and thermodynamics.

ITC is used to characterize any binding event with a nucleic acid, including:

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