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•Biochemistry Course

Chapter 2
Nucleic Acid Structure
and Analyais
Section 1 Chemical Composition of
Nucleic Acids
Section 2 DNA Structure
Section 3 RNA Structure
Section 4 Chemical and Physical
Properties of Nucleic Acids
Section 1 Chemical Composition of Nucleic Acid

Comparisons of names of bases, nucleosides and nucleotides


BASES NUCLEOSIDES NUCLEOTIDES
Adenine (A) Adenosine Adenosine 5’-triphosphate (ATP)
Deoxyadenosine Deoxyadenosine 5’-triphosphate (dATP)
Guanine (G) Guanosine Guanosine 5’-triphosphate (GTP)
Deoxy-guanosine Deoxy-guanosine 5’-triphosphate (dGTP)
Cytosine (C) Cytidine Cytidine 5’-triphosphate (CTP)
Deoxy-cytidine Deoxy-cytidine 5’-triphosphate (dCTP)
Uracil (U) Uridine Uridine 5’-triphosphate (UTP)
Thymine (T) Thymidine/ Thymidine/deoxythymidine
deoxythymidine 5’-triphosphate (dTTP)

Purine: A & G; Pyrimidine: C & T/U; (deoxy)-ribose,


Nitrogenous bases

Bicyclic purines:

Monocyclic pyrimidine:

Thymine (T) is 5-methyluracil (U)


Nucleosides
In nucleic acids, the bases are covalently attached to the 1’ position
of a pentose sugar ring, to form a nucleoside

Glycosidic (glycoside, glycosylic) bond

R Ribose or 2’-deoxyribose

Adenosine, guanosine, cytidine, thymidine, uridine


Nucleotides
A nucleotide is a nucleoside with one or more phosphate groups bound covalently to
the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up
to three phosphates may be attached.

Phosphate diester bonds


7 6 4
5 1 5
9 4 2 1 2

Deoxynucleotides Ribonucleotides
(deoxyribose containing) (ribose containing)
5’end: not always has attached phosphate groups

DNA/RNA sequence:
From 5’ end to 3’ end
Example:
5’-UCAGGCUA-3’
= UCAGGCUA

Phosphodiester bonds

3’ end: free hydroxyl (-OH) group


Section 2 DNA Structure
1. Primary structure
of DNA:
5’end: free phosphate
group
3’ end: free hydroxyl
(-OH) group

Phosphodiester bond
2. Secondary structure of DNA

•Watson and Crick , 1953


•The genetic material of all
organisms except for some
viruses
•The foundation of the
molecular biology
Essential for replicating DNA and
•Two separate strands transcribing RNA
Antiparellel (5’→3’
direction) 3’
5’
Complementary
(sequence)
Base pairing: hydrogen
bonding that holds two
strands together

• Sugar-phosphate backbones
(negatively charged): outside
• Planner bases (stack one
above the other): inside 3’
back 5’
A:T G:C

Base pairing
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•Helical turn:
10 base
pairs/turn

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Section 3 : RNA structure
1. Single stranded nucleic acid
2. Secondary structure are formed
some time
3. Globular tertiary structure are
important for many functional
RNAs, such as tRNA, rRNA and
ribozyme RNA

Forces for secondary and tertiary structure:


intramolecular hydrogen bonding and base stacking.
Ribozyme RNA
tRNA Secondary
structure

Tertiary
structure
Conformational variability of RNA is important for the
much more diverse roles of RNA in the cell, when
compared to DNA.
Structure and Function correspondence of protein and nucleic acids

Protein Nucleic Acids


Fibrous protein Globular Helical DNA Globular RNA
protein
Structural •Enzymes, Genetic •Ribozymes
proteins •antibodies, information •Transfer RNA
•receptors etc maintenance (tRNA)
•Signal recognition
etc.
Section 4 Chemical and Physical Properties
of Nucleic Acids
1. Effect of Acid & applications

2.Effect of alkali & applications

3.Chemical denaturation

4.Viscosity & applications

5.Buorant density & application

6.Spectroscopic property

7.Thermal denaturation

8.Renaturation

9.Hybridization
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1.Effect of Acid & Application

(1)Strong acid + high temperature: completely


hydrolyzed to bases, riboses/deoxyrobose, and
phosphate

(2)pH 3-4 : apurinic nucleic acids [glycosylic bonds


attaching purine (A and G) bases to the ribose ring
are broken ], can be generated by formic acid

(3)Appication:Maxam and Gilbert DNA sequencing


2.Effect of Alkali & Application
(1)pH (7-8) has subtle (small) effects on DNA structure
(2)High pH changes the tautomeric state of the bases
enolate form
keto form enolate form keto form

Base pairing is not stable anymore because of the change of


tautomeric states of the bases, resulting in DNA denaturation
RNA hydrolyzes at higher pH because of 2’-OH groups
in RNA

2’, 3’-cyclic
phosphodiester
alkali OH free 5’-OH

RNA is unstable at higher pH


3.Chemical Denaturation &
Application
Urea (H2NCONH2): denaturing PAGE
Formamide (HCONH2): Northern blot

Disrupting the hydrogen bonding of the bulk water solution

Denaturation of strands in double helical structure


4. Viscosity & Application

Reasons for the DNA high viscosity


(1) High axial ratio
(2) Relatively stiff

Applications:
Long DNA molecules can easily be shortened by
shearing force. Remember to avoid shearing
problem when isolating very large DNA molecule.
5.Buoyant density (DNA)
1.7 g cm-3 , a similar density to 8M CsCl
Purifications of DNA: equilibrium density gradient centrifugation

Protein floats

RNA pellets at the bottom

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6.Spectroscopic property & Application

UV absorption:
• nucleic acids absorb UV light due to the
aromatic bases
• The wavelength of maximum absorption by
both DNA and RNA is 260 nm (λ max = 260
nm)
• Applications: detection, quantitation,
assessment of purity (A260 /Α 280 )
(1)Quantitation of nucleic acids
Extinction coefficients: 1 mg/ml dsDNA has an A260 of 20
ssDNA and RNA, 25
The values for ssDNA and RNA are approximate
The values are the sum of absorbance contributed by the
different bases (ε : purines > pyrimidines)
The absorbance values also depend on the amount of secondary
structures due to hypochromicity.

(2)Purity of DNA and RNA


A260 /Α 280 :
dsDNA--1.8
pure RNA--2.0
protein--0.5
7.Thermal denaturation/melting: heating leads to the
destruction of double-stranded hydrogen-bonded regions of
DNA and RNA.

RNA: the absorbance increases gradually and irregularly


DNA: the absorbance increases cooperatively.
melting temperature (Tm): the temperature at which 40% increase in
absorbance is achieved.
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8.Renaturation:
Rapid cooling: only allow the formation of local base
paring. Absorbance is slightly decreased
Slow cooling: whole complementation of dsDNA.
Absorbance decreases greatly and cooperatively.
Fig. 2.

Annealing: base paring of short regions of complementarity


within or between DNA strands. (example: annealing step in PCR
reaction)
Hybridization: renaturation of complementary sequences
between different nucleic acid molecules.
(examples: Northern or Southern hybridization)