DNA structure (with a side of RNA)

The sugar HOCH 2 OH H H H H HOCH 2 OH H H H

HOCH 2 OH H H H 1’ 2’ 3’ 4’ 5’

Nucleosides Ribose + nitrogenous base Deoxyribose + nitrogenous base RNA bases- Adenine, Guanine, Cytosine and Uracil DNA bases- Adenine, Guanine, Cytosine and Thymine

Purine name is adenine while the nucleoside name is adenosine The base is attached to the 1’ carbon via a glycosidic bond

Nucleotides aka nucleoside phosphates The phosphate is attached to the 5’ carbon by a phosphodiester linkage

aka dTTP

Native DNA is a double helix of complementary antiparallel chains

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What governs basepairing rules? –Size –Charge

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Circular and superhelical DNA

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 –Dipole-dipole interactions (responsible for twist) 5’ 3’ Same strand stacking cross-strand stacking

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 or perturbed, the other is weakened; this explains why Tm drops so markedly after the addition of a reaganet that destroys either type of interaction

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

RNA is single stranded or is it?

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, T m.

Structural Changes in DNA Melting

Denaturation AND Renaturation of DNA TmTm DNA of different sequences have different T m. T m is higher for DNA that contain more GC pairs; T m is also directly proportional to the ionic strength of the solution. (salts can shield repulsions of negatively charged phosphate groups)

1.The rise occurs over a narrow range 2.The maximum A260 is about 37% higher than the starting value 3.The temperature where the rise in A260 is half complete is the melting temperature or Tm.

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 A 260 of dsDNA solution is 1.00, the denatured (ssDNA) solution will be A If a DNA solution is slowly heated and the A 260 is measured at various temperatures a melting curve is obtained.

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.

Another very effective denaturant is NaOH At a pH greater than 11.3 all hydrogen bonds are eliminated and DNA is completely denatured

A solution of denatured DNA can be treated in such a way that native DNA structure reforms this is called renaturation of reannealing. Two requirements must be meet –The salt concentration must be high enough that electrostatic repulsion between the two strands is eliminated NaCl –Temperature must be high enough to eliminate intrastrand hydrogen bonds but not so high that interstrand base-pairing will not occur. The opitimal temperature is C below the Tm.

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.

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 C. Phosphodiester linkages and N— glycosidic bonds are stable up to 100C.

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. At low salt concentrations the phosphate groups destabilize the DNA.