22.5. Nucleic acid stability
Many forces stabilizing the DNA structure
- Base pairing : Base pairing holds both of the strands of DNA together like ‘glue’. Nitrogenous base of a strand form the pairs with another strand. Watson crick base pairing is the more stable of all base pairing in the double helix. It plays a crucial role roll in stability of DNA.
- Hydrophobic force : It is a tendency of water molecules to form a bigger cage around the hydrophobic molecules. In the DNA, pyrimidines and purines are hydrophobic, so water molecules forms the hydrophopic interaction with nitrogenous bases. Hydrophobic force plays a central role in determining nucleic acid structure.
- Base stacking interaction : Interaction between two adjacent nitrogenous bases which are out in plane, in the parallel manner. Different sets of base pairs in a stack have different stacking energies. Stacked G and C bases have greater interaction compare to stacked A and T bases. This force helps to minimize the contact of water with nitrogenous bases.
- Hydrogen bond : When a hydrogen atom present between two electronegative molecules than it forms an extra bond. This extra bond is known as hydrogen bond. Adenine is always hydrogen-bonded to thymine and guanine is always hydrogen bonded to cytosine by watson-crick pairing. The surface atoms in the sugar-fourth and phosphate form hydrogen bond to water molecules.
- Ionic interaction : Phosphate group of back bone of DNA play an important role in DNA stability due to their one charge. If we add the positive ions (Na+) in the DNA solution, the melting temperature of duplex DNA increase means stability of DNA increase because these ion shield the anionic phosphate group and decrease the repulsion.
22.6. Thermal Stability
The temperature at which 50% DNA in given sample become denatured, known as melting temperature (Tm). The double stranded DNA show the less absorbance at 260 nm. While the denature DNA show the more absorbance at 260 nm this phenomena known as hyperchromatic effect.
Factors affecting Tm
- Ionic strength : When the lower the ionic strength is low it can not completely shield the anionic phosphate. So repulsion makes it energetically more favorable to separate the strands thus Tm temperature become lower.
- AT and GC Content : GC content has higher number of Hydrogen bond and base stacking interaction compare to AT content. So the high GC content has higher Tm than DNA with high AT content.
- pH : If the pH is greater than 10, the hydrogen bonds of Nitrogenous bases are destroyed and DNA become denatured. If the pH is less than 3, it also disrupt, the base pairing due to extensive protonation. So very high or very low pH lowers the Tm.