Secondary Structure continued

Hydrogen bonds:

A-T base pair has 2: 10 kJ/mol

G-C base pair has 3: 15 kJ/mol

In addition to hydrogen bonding, DNA, in its double helical configuration, is stabilized by base stacking.

The bases of DNA are aromatic organic ring compounds (remember what the pi electron cloud looks like) and interact via van der Waals forces.

Types of helical structures:

Three types of DNA helix => A, B, Z

A-DNA = more compact (flattened = 11 residues/turn vs. 10 for B, see below) form of DNA produced by dehydration of B-DNA

B-DNA = standard Watson-Crick DNA (10 resiudues/turn); a right handed helix (requires high humidity - like the inside of the cell)

Z-DNA = 12 residues/turn; observed in x-ray patterns of oligonucleotides [d(CG)3 - a left-handed double helix], discovered in 1979. Z-DNA requires alternating sequence of pyrimidine and purine.

Tertiary Structure of Nucleic Acids

In addition to the a-helix of DNA there is an additional level of twisting (supercoiling).

Discovered by Jerome Vinograd (1963) at Caltech; noticed multiple bands of DNA in preparations of circular DNA (plasmids).

Circular DNA with no superhelical turns = relaxed (takes energy to supertwist)

Superhelical turns serve two purposes:

1. yield a more compact shape
2. can result in unwinding of DNA helix (aids in replication)

Can express the degree of twisting of DNA by the following equation:

L = T + W

L = linking number (# time each strand crosses, normally = once per 10.5 bases)
T = twist number
W = writhe = number of superhelical turns (coiling)

Another example of nucleic acid tertiary structure is transfer RNA.

Nucleic Acid Denaturation

(once used for estimation of base composition and for hybridization studies)

Hyperchromiscity = DNA has a higher absorbance (at 260 nm) when in the denatured state (or as hydrolyzed bases) than in the native double-stranded form. Why? Look at how bases lie on top of each other in the double strand configuration.

Disrupting agents: acid, base, heat, hydrogen-bond breaker (formamide = H-C-NH2)

Heat is the most commonly used agent (easy to monitor and control).

Lower ionic strength lowers T_m (melting temperature). Why? Hydrophobic forces involved in base stacking are minimized (i.e. high salt increases stability of hydrophobic forces).

Under fixed conditions of ionic strength and pH, the melting temperature of a segment of DNA is related to its GC content. Why? GC has three hydrogen bonds/base pair vs. two for AT

Annealing: lower the temperature slowly and DNA spontaneously re-associates. Renaturation is used in locating a gene; oligonucleotide (labeled) hybridizes to a fragment of DNA contained in a plasmid. A clone has been identified

The speed with which this occurs relates to the degree of repetitiveness of a particular DNA sequence (some of which are highly repeated). A display of this data in what is called a C_ot curve (C_ot) C = concentration of DNA bases and t = time allowed the discovery of highly repetitive DNA sequences in eukaryotes.