Factors affecting stability of DNA are H-bonds between the bases. The H-bonds already provide specificity but they also confer stability to the structure. The phosphate groups must be neutralized (by Na+ or Mg2+ ions) to allow the negatively charged phosphates to be in close proximity. The hydrophobic interactions between the planar base pairs stabilize the bases on the inside of the helix, so these provide stability to the structure but do not contribute to the specificity. Let’s discuss each factors one by one:-
Table of Contents
- Hydrogen bonding (2-3 kcal/mol per base pair)
- Stacking interactions (4-15 kcal/mol per base pair)
- Hydrophobic Force
- Stacked Base Pairs
- Charge-Charge Interactions
- Physical Properties of DNA
Hydrogen bonding (2-3 kcal/mol per base pair)
- This is one of the most important factors affecting stability of DNA.
- Hydrogen bonds formed between bases, although weak in energy, but is able to stabilize the helix because of its large number present in DNA molecule.
Stacking interactions (4-15 kcal/mol per base pair)
- Stacking Interactions, or also known as Van der Waals interactions between bases are weak, but the large amounts of these interactions help to stabilize the overall structure of the helix.
- The two strands are also held together via forces generated by the hydrophobic effect and pi-stacking, which are not influenced by the sequence of the DNA
- Double helix is stabilized by hydrophobic effects
- Hydrophobic effects are formed by burying the bases in the interior of the helix, that keeps away the surrounding water, whereas the more polar surfaces. Hence, increases the stability
Stacked Base Pairs
- Stacked base pairs attract to one another through Van der Waals forces . Single, van der Waals interaction generates small energy to the overall DNA structure.
- But many such Van der Waals forces, however, summed to generate large energy over that results in substantial stability.
- refers to the electrostatic (ion-ion) repulsion of the negatively charged phosphate is potentially unstable, however the presence of Mg2+ and cationic proteins with abundant Arginine and Lysine residues that stabilizes the double helix.
- Double-stranded helix structure thus, promoted by having phosphates on outside, interact with H2O and counter ions (K+, Mg2+, etc.).
Solvation also plays a role in stabilizing the double helix that affects base pairing to mediating binding events.
Note* All conformational parameters discussed above, also plays an important role in DNA stability.
Physical Properties of DNA
Physical Properties of DNA constitute mutiple factors affecting stability of DNA
- In the laboratory, the duplex stability can be measured by finding the temperature required to break the hydrogen bonds, their melting temperature (also called Tm value).
- When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules.
- These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others.
- DNA “Melting” is the term given to the separation of the two DNA strands.
There are a number of ways to do this experimentally
- Helical formation can be monitored by observing the optical density of a solution.
- The disruption of base stacking alters the electronic interaction between the bases. As the electronic interaction decreases, it becomes easier for an electron to absorb a photon.
- Hence, denaturation of DNA leads to the “hyperchromic” effect, i.e., the increased absorption of light.
- Thus, an increase in Temperature – when temperature of a DNA solution increases to the melting point (Tm), the strands separate.
- The absorbance of the solution changes as shown in the graph below. The increase in absorbance as the strands separate is due to the irregular orientation of the bases in the SS-DNA compared to the regular planar orientation in the helix.
- In general, helical stability is linearly related to fractional G+C base pair content in DNA.
- As G+C increases so does stability
- An empirical formula for calculating the melting temperature of a particular helix is given as Tm (°C) = 69.3 + 41 * fG/C.
- This expression quantifies the observed result that there is a linear relation between Tm and G+C content.
Helical Stability and Salt
- It has been observed that multiple stranded polynucleotide helices are stabilized by increasing monovalent cation concentration.
- In fact the Tm of a given DNA is linearly dependent on the log of the monovalent cation concentration.
- DNA phosphate backbone is negatively charged. Thus, in salt solutions, cations are associated with it.
- But, when DNA is denatured only fewer cations are associated with the separated DNA strands than with the DNA helix in its native state, because the charge density on double stranded DNA is higher than single strand nucleic acids. This creates a larger electrostatic potential, which more effectively attracts counter ions.
Thus, in the denaturation reaction, the mass action equation can be written.
DNA (Helix). Mx <—-> DNA(Coil) My + M(x-y)
x= no. of ions bound/base pair in a helix
y= no. of ions bound/base in a coil
x-y = net gain in free cations due to denaturation
Therefore, the denaturation reaction equilibrium can be shifted by adjusting the cation concentration.
- The effects of divalent cations are much more complex due to their multiple interactions with the DNA phosphate backbone-each M2+ can potentially bind one or two DNA phosphates and the binding is likely to be cooperative.
- Hence, the Tm dependence on divalent cation concentration is decidedly non-linear.
Effect of Acid
- In strong acid and at elevated temperatures, for example perchloric acid (HClO4) at more than 100°C, nucleic acids are hydrolyzed completely to their constituents: bases, ribose or deoxyribose and phosphate.
- In more dilute mineral acid, for example at pH 3–4, the most easily hydrolyzed bonds which are glycosylic bonds are broken and hence the nucleic acid becomes apurinic.
Effect of Alkali DNA
- Effect of alkali on DNA is to change the tautomeric state of the bases.
- At neutral pH, the compound is predominantly in the keto form, increasing the pH causes a shift to the enolate form.
- This affects the specific hydrogen bonding between the base pairs, with the result that the double stranded structure of the DNA breaks down; that is the DNA becomes denatured.
- Number of chemical agents can cause the denaturation of DNA or RNA at neutral pH. Examples — urea (H2NCONH2) and formamide (HCONH2)
- These agents (several molar) has the effect of disrupting the hydrogen bonding of the bulk water solution.
- Analysis and purification of DNA can be carried out according to its density.
- In solutions containing high concentrations of a high molecular weight salt, for example 8 M cesium chloride (CsCl), DNA has a similar density to the bulk solution, around 1.7 g cm–3.
- If the solution is centrifuged at very high speed, the dense cesium salt tends to migrate down the tube, setting up a density gradient.
- Eventually the DNA sample will migrate to a sharp band at a position in the gradient corresponding to its own buoyant density. This technique is known as equilibrium density gradient centrifugation or isopycnic centrifugation.
- Since, under these conditions, RNA pellets at the bottom of the tube and protein floats, this can be an effective way of purifying DNA away from these two contaminants.
- However, the method is also analytically useful, since the precise buoyant density of the DNA (ρ) is a linear function of its G+C content
- ρ = 1.66 x Frac (G + C)
- Hence, the sedimentation of DNA may be used to determine its average G+C content or, in some cases, DNA fragments with different G+C contents from the bulk sequence can be separated from it.