in this pdf, we will discover: - the nucleic acid structure (DNA & RNA) - their properties -How to deal with them in the lab

1. NUCLEIC ACID
STRUCTURE

2. Function:
– store & transmit
genetic information
• Structure:
– monomers = nucleotides
– Polymers = polynucleotides (DNA, RNA)
• Examples:
– RNA (ribonucleic acid) (rRNA, mRNA, tRNA)
– DNA (deoxyribonucleic acid)
– ATP (adenosine triphosphate) which:
• Supplies energy for synthetic reactions and for other
• energy-requiring processes in cells

3. sugar
Nitrogenous
base
Phosphate
group
Nucleoside

4. The sugar

9. Forces that
contribute to
stability of helix
formation
•Hydrogen bonding in
base-pairing
•Hydrophobic
interactions in base
stacking

10. Forces That Stabilize Nucleic Acid Double
Helix
• There are THREE major forces that contribute to stability of
helix formation
• Hydrogen bonding in base-pairing
• Hydrophobic interactions in base stacking

11. Chemical forces that stabilize the DNA double
helix:
❖The helical structure of nucleic acids is determine by stacking between
adjacent bases in the same strand.
❖
The double-stranded helical structure of DNA is maintained by hydrogen-
bonding between the bases in the base pairs.
❖
Hydrogen bonding and hydrophobic interaction work cooperatively to form
a very stable structure of ddDNA
❖
If one of the interactions is eliminated , the other is weakened; this
explains why Tm drops so markedly after the addition of a reagant that
destroys either type of interaction

12. Stacking
• A hydrophobic interaction is an interaction between two molecules (or portions of
molecules) that are somewhat insoluble in water. In response to their repulsion in
water they tend to associate.
•
This is true for nucleic acids the bases of nucleic acids are planar molecules
carrying localized weak charges. The localized charges will maintain solubility but
the large poorly soluble organic rings of the bases tend to cluster. In a nucleic acid
this produces an array known as base-stacking

13. Denaturation AND Renaturation of
DNA
• When duplex DNA molecules are subjected to conditions of pH, temperature or ionic
strength that disrupt hydrogen bonds, the strands are no longer held together. The
double helix is denatured.
• If the temperature is the denaturing agent, the double helix is said to melt;
ENZYMES DRIVE THIS IN THE CELL.
• The phenomenon that the relative absorbance of the DNA solution at 260 nm
increases as the bases unstack is called hyperchromic shift;
• If one follows the absorbance as a function of temperature, the midpoint
temperature of the absorbance curve is termed melting temperature, Tm.

14. Denaturation AND Renaturation of
DNA
• Denaturation can be detected by observing the increase in the ability of a DNA
solution to absorb UV light at a wavelength of 260 nm.
•
When bases are highly ordered they absorb less light than when they are in a less
ordered state
•
If the A260 of dsDNA solution is 1.00, the denatured (ssDNA) solution will be A260
1.37.
• If a DNA solution is slowly heated and the A260 is measured at various temperatures
a melting curve is obtained

15. Denaturation AND Renaturation of
DNA

16. DenaturationAND
Renaturation of
DNA

17. Properties affecs
DNA structure
•pH
•Temperature
•Ionic strength

19. Effects of pH on the structure of DNA
• Hydrogen bonding between the complimentary strands is stable
between pH 4 and 10
• Phosphodiester linkages in the DNA backbone are stable between pH
3 and 12.
• N-glycosidic bonds to purine bases are hydrolysed at pH value of 3
and less.
• Another very effective denaturant is NaOH
• At a pH greater than 11.3 all hydrogen bonds are eliminated and DNA
is completely denatured

20. Temperature
• There is considerable variation in the temperature stability of the
hydrogen bonds in the double helix, but most DNA begins to unwind
in the range of 80-90 C.
• Phosphodiester linkages and N—glycosidic bonds are stable up to
100C.
• Heat is often used to denature DNA and as result this process is
referred to as melting.
• During melting all covalent bonds including phosphodiester bonds
remain intact. Only hydrogen bonds and stacking interactions are
disrupted.

21. Ionic strength
• DNA is most stable and soluble in salt solutions. Salt concentrations
of less than 0.05M weaken the hydrogen bonding between
complementary strands.