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COURSE: MBLG1001
Lecturer: Dale Hancock
Lecture 8
MBLG1001 Lecture 8
Nucleic Acid Structure
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Properties of Nucleic Acids
What is wrong with these pictures?
•
•
•
•
•
Stability of DNA
UV absorbance
Negative charge
Detection by fluorescence
Manipulation of the weak forces
experimentally
• Proteins that interact with DNA
Stability of DNA
Why can we isolate the DNA
from this animal after
thousands of years in the
tundra?
• Why is RNA so much less stable? Some
RNA molecules only last a few minutes inside
the cell.
• How can the small structural differences
between RNA and DNA make such a
difference?
1
The sugar phosphate backbone of
DNA is very stable to chemical
attack.
The RNA sugar phosphate
backbone is vulnerable to base
attack
• The absence of the –OH at position 2 in the
sugar makes DNA very resistant to alkali (unlike
RNA).
• The –OH at position 2 in ribose enables the
phosphate nearby to form a cyclic compound
when the proton is removed (at high pH).
• This cyclic compound rapidly breaks the
phosphodiester bond resulting in a 2’ or 3’
Phosphorylated nucleotide.
The RNA sugar phosphate
backbone is vulnerable to base
attack
-O
-O
P
O
H 2O
H
O
O-
H
H
O
OH
H
H
Base
O
P
O
H
H
OH
OH
H
H
The RNA sugar phosphate
backbone is vulnerable to base
attack
Base
-O
P
O
H 2O
O
H
H
H
H+
H
O
Base
δ+
P
O
O
H+
H
OH-
O
O-
H
O
O
O
O
H
O-
P
O-
Base
O
Base
O
OBase
δ+
O
O
P
O
O
O
H
H
H
O
O
H
OH
OH
H
H
OH
OH
H
The RNA sugar phosphate
backbone is vulnerable to base
attack
H
The DNA sugar phosphate
backbone is stable to base attack
Base
O
Base
O
O
P
O
O
H
O-
The O- now
attacks the
phosphorus
atom which is
δ+ve
H
H
H
O
O
The backbone
is cleaved and
this complex
breaks down to
a 2’ or 3’
phosphate
O
P
O
O
O-
H
H
O
H
H
H
Deoxyribose has
no OH at position
2’ so the base
cannot attack. A
special enzyme
removes the OH
from the ribose.
P
O
O-
2
Properties which make DNA
ideal as a store of genetic
information:
• The two strands, which gives a double
copy and a template for repair.
• The information (in the form of charged
and polar groups on the bases) is found,
“buried” at the very center of the double
stranded helix, protected by a hydrophilic
sugar phosphate backbone and then the
hydrophobic bases.
Cross section of DNA
Phoshpate
-ve
Sugar
polar
Bases
hydrophobic
Sugar
polar
Phoshpate
-ve
Polar
groups
-ve polar hydrophobic polar hydrophobic polar -ve
Corruptions of the Code
Corruptions of the Code
• In the silicon IT world a corruption of
the code is when a “1” turns into a “0”
or vice versa.
• In the carbon IT world it occurs when
one base spontaneously turns into
another.
• The bases are chemically stable
except for…..
• Cytosine is deaminated to uracil
spontaneously by a process called
oxidative deamination.
• In a given day about 100 cytosines
are deaminated to uracils in each cell.
These uracils are a corruption of the
code and need to be removed and
replaced.
The deamination of cytosine
Why Thymine and not Uracil?
NH2
O
N
NH
+ NH3
+ H2O
N
H
Cytosine
O
N
H
O
Uracil
• But if uracil was a normal constituent of
DNA there would be no way of knowing
whether the uracils were supposed to be
there or not.
• By converting uracils to thymines (by a
methylation) before incorporation into
DNA, any uracils found in the DNA must
have come from cytosine and can thus be
excised and replaced.
3
UV Absorption
• This is the most commonly used technique
to measure the concentration of DNA and
RNA
• It is not sequence or source dependent
• An absorbance of 1 at 260 nm gives 50
µg/mL DNA.
• An absorbance of 1 at 260 nm gives 40
µg/mL RNA.
Absorption Spectrum: Guanine
Absorption Spectrum: Adenine
0.5
1.0
0.4
0.8
Abs orbance
Absorbance
UV absorbance of bases
0.3
0.2
0.1
0
220
240
260
280
300
0.4
0.2
Wave length (nm )
0.4
0.5
Absorbance
Abs orbance
0.6
0.3
0.2
0.1
240
260
280
Wave le ngth (nm )
260
280
300
320
Absorption Spectrum: Thymine
0.5
0.0
220
240
Wave le ngth (nm )
Absorption Spectrum: Cytosine
300
320
• The conjugated ring structure of the
bases.
• Each base has similar absorption
properties, hence the final absorbance is
not sequence dependent.
The Negative Charge
0.6
0.0
220
320
What structural feature of
the nucleic acid gives it its
absorbance properties?
0.4
0.3
Both strands
of the
backbone
have a
negative
charge for
each
nucleotide.
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
0.2
0.1
0.0
220
240
260
280
300
320
Wave le ngth (nm )
How can we use the charge?
• Gel Electrophoresis. DNA has a constant
charge to mass ratio so when subjected to
an electric field it will move to the positive
electrode (anode).
• The migration (in mm) will depend on the
number of bases in the piece of DNA.
• We can use gel electrophoresis to
separate fragments of DNA by size.
How can we use the charge?
High mol. Wt.
Low mol. Wt.
+
4
Detecting DNA by Fluorescence
• DNA can be detected by ethidium
bromide.
• This dye intercalates with the double
stranded DNA.
• It fluoresces under UV light.
• It is very sensitive, detecting as little as 10
ng of DNA in one band.
Techniques that disrupt the
double helix:
• Increased temperature will
disrupt the H bonds and base
stacking
• Increased pH will remove the
ring N proton from Thymine and
Guanine, thus preventing the Hbonding.
Melting DNA
• DNA melting is the separation of the 2
strands..also called denaturing DNA.
• This process can be monitored by
absorbance at 260 nm.
• This is known as the hyperchromic effect.
The ordered base stacking in double
stranded DNA hinders photon access.
• Single stranded DNA has 1.4 * higher
absorbance at 260 nm.
Detecting DNA by Fluorescence
The Ethidium
bromide is
green and the
DNA is purple.
It has the right
dimensions to
fit between
the base pairs
Techniques that disrupt the
double helix:
• Ethanol Precipitation. Nucleic acid, but
not nucleotides, is insoluble in ethanol
concentrations above 66% (v/v).
• This enables you to concentrate the DNA
• The ethanol is able to disrupt the base
stacking and the bases slide off each other
(into a mess!!).
The melting temperature:
Tm
• The Tm or melting temperature is the
midpoint of the transition from double
stranded to single stranded. This transition
is very sharp.
• this is dependent on the GC content of the
DNA sample. The higher the GC content
the higher is the Tm.
• The reverse process is called reannealing. This temperature, the Ta is
~15o < Tm
5
Melting DNA
Melting DNA
Single stranded DNA
Increasing GC content
A260
A260
Double stranded
DNA
Tm
Tm
Temperature oC
Promoting Base Pairing.
• Increasing ionic strength promotes base
pairing as the cations (positive ions e.g.
Na+, Mg2+) shield the phosphates and thus
reduce the repulsive effect of the strong
negative charges.
• Mg2+ ions give the best shielding but are
necessary for reactions which hydrolyse
the phosphodiester bond.
Figure 28.1
Watson and Crick’s
famous paper, in its
entirety. (Reprinted
with permission from
Watson,J.D., and
Crick,F.H.C., Molecular
structure of nucleic acid,
1953. Nature 171:737738. Copyright 1953
Macmillan Publishers
Ltd.)
Tm
Tm
Temperature oC
The Flow of Genetic
Information
replication
DNA
DNA
Transcription
RNA
Translation
Folding, modification,
translocation
Protein
Functional protein
Replication
• Is the process :
–Conservative,
–Semi-conservative OR
–Dispersive?
6
The Messelson Stahl Experiment
The Messelson Stahl Experiment
• Cells were grown up on the normal “light”
isotope of N, 14N, abbreviated as L
• then the medium was changed to one
containing the 15N (heavy or H) as sole
nitrogen source.
• DNA was isolated at various time points
after the media change and applied to a
CsCl density gradient.
• This technique separates by buoyant
density
• DNA containing 2 light strands (L:L) will
sediment at a different density to a hybrid
Heavy:Light (H:L) nucleic acid or the
Heavy:Heavy (H:H) form of DNA.
• Old parent DNA will be light (L)
• Newly synthesised DNA will be heavy (H).
The Messelson Stahl Experiment
LL
HL
HH
Concentrated CsCl
solutions, when
centrifuged really
fast form a gradient.
Compounds
separate by their
buoyant density in
such a gradient.
7