What stabilizes the N.A. double helix? < *

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What stabilizes the N.A. double helix?
1 Hydrogen bonding
G•C 3 hydrogen bonds
A•T 2 hydrogen bonds
2 Stacking interactions
Stacking of aromatic rings stabilized by hydrophobic forces and
induced dipole.
Different sequences will have different stacking interactions, i.e. aromatic
rings will stack better or worse.
Thermodynamic parameters have been calculated for all DNA & RNA dinucleotides.
e.g.
5’d(GAGAGAGA)3’
3’d(CTCTCTCT)5’
1 & 2 are about equally important
Important for specificity and stability.
< d(ACGTACGT)
(TGCATGCA)
T m
~20° different
3 Ionic interactions
Bp hydrophobic; P hydrophilic -- but electrostatic repulsion of - P
destabilizes DNA. Stabilized by cation binding.
Since divalents bind specifically to P , are much more effective shielding
agents than monovalent cations
4 Ordered waters (and ions) in grooves, especially minor groove
(spine of hydration)

DNA denaturation
DNA bases absorb UV light due to aromatic rings.
λ max
at A 260
(average for the four bases at pH 7.0)
Recall: A = εcl
path length, usually 1 cm
1 mg/ml DNA has A ~
260 = 20
50 µg/ml DNA has A ~
260 = 1
absorbance
extinction
coefficient
concentration
Hyperchromic effect: stacking of nucleotide bases decreases ε.
∴ bases in ss absorb more than bases in ds.
⇒ Absorbance increases as DNA denatures.
ds → ss, A 260 ↑ ~37%
transition breadth
T m
(melting temperature) is transition midpoint
[wc] = [w] = [c] half melted
Approximate inflection point on melting curve

DNA melting is a cooperative process
Increasing Conformational Entropy
Increasing translational
Entropy

What makes a process cooperative?
First, it must occur in multiple steps. For example:
K
A
1
K
B
2
K
C
3
D
Second, the equilibrium constants of the successive steps must
satisfy the following relationship:
K 1 < K 2 < K 3

Stability of DNA & ∴ T m
are affected by:
1) ionic strength of solution
2) GC content of DNA
3) pH
4) solvent
5) binding of molecules
(e.g. drugs, proteins, poly cations)
1) Ionic strength:
µ = (1/2)c i
z i
2
charge of each species
concentration of each species
for a given ion, as [cation] ↑, T m
↑
2) GC content:
@ same ionic strength, DNAs with
higher GC content will melt higher
[Exception: short DNA oligonucleotides ≤ 50 bp]
Combine 1) & 2) to get empirical equation for Na + :
T m
= 41.1 (X G+C
) + 16.6 log[Na + ] + 81.5
mole fraction
(know how to use,
but don’t need to memorize)

Relationship between GC content and T m
Original Marmur/Doty equation:
T m
= 0.411 (%GC) + 69
%GC = 100χ GC
T m
= 41.1 χ GC
+ 69
Marmur and Doty
(1962) JMB 5, 119-131
Experimental observation: 10-fold
increase in [Na + ] increases T m
by 16.6˚.
This leads to the salt corrected
Marmur/Doty equation:
T m
= 41.1 χ GC
+ 16.6log [Na + ] + 81.5

Marmur/Doty relationship breaks down
for small and/or non-complex duplexes
Sequence GC Exp T m M/D T m
GGATCC
CCTAGG
CAAGCTTG
GTTCGAAC
GGTATACC
CCATATGG
0.67 30.8 C 109 C
0.50 44.6 C 103
0.50 36.0 C 103
C
C
Discrepancies with Marmur/Doty equation are due to length
effects and nearest neighbor effects.

Renaturation of DNA
Melted DNA can be reannealed
to an extent which depends on
sequence complexity & rate of reannealing.
e.g.
Rate: If cools too fast, strands don’t have time to find neighbors
Sequence Complexity: For random DNA (e.g. calf thymus, historical source)
don’t get much reannealing. For poly d(AT) n
, can get complete reannealing
Historically important for finding highly repetitive DNA sequences in chromatin,
e.g. satellite DNA (before DNA sequencing).
Important today in molecular biology experiments,
e.g. DNA or RNA hybridization (Northern blots, Southern blots, PCR)