SECTION C

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• Molecular Biology Course

SECTION C Properties of Nucleic Acids
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The diffraction pattern from the DNA optical
transform slide shows the central cross pattern
indicative of the helical arrangement of the
strands of DNA. It also shows a missing 4th layer
line which is the result of the two strands of
the double helix being offset by 3/8 of a period.
The strongly diffracting phosphorus atoms create
the diamonds. The satelites above and below the
cross are attributed to the base pairs.
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• Molecular Biology Course

C1 Nucleic Acid Structure (DNA RNA)
bases?nucleosides ?nucleotide ? phosphodiester
bonds ? primary sequence ? structure, modified
nucleic acids
C2 Chemical and Physical Properties of Nucleic
Acids (DNA RNA) stability (force), chemical
properties (acid, alkali, chemical denaturation),
physical properties (viscosity, buoyant density)
C3 Spectroscopic and Thermal Properties of
Nucleic Acids (DNA RNA) UV absorption,
hyperchromocity, quantitation and purity
C4 DNA Supercoiling (DNA) closed circular
molecule, supercoiling energy, topoisomer
topoisomerase
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C. Properties of nucleic acids
C1 Nucleic Acid Structure (DNA RNA) Bases
Bicyclic Purines
Monocyclic pyrimidine
Thymine (T) is a 5-methyluracil (U)
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C. Properties of nucleic acids
C1 Nucleic Acid Structure (DNA RNA) Nucleosides
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
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C. Properties of nucleic acids
C1 Nucleic Acid Structure (DNA RNA) 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 ester bonds
Ribonucleotides (containing ribose)
Deoxynucleotides (containing deoxyribose)
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C. Properties of nucleic acids
BASES NUCLEOSIDES NUCLEOTIDES
Adenine (A) Adenosine Adenosine 5-triphosphate (ATP)
Adenine (A) Deoxyadenosine Deoxyadenosine 5-triphosphate (dATP)
Guanine (G) Guanosine Guanosine 5-triphosphate (GTP)
Guanine (G) Deoxyguanosine Deoxy-guanosine 5-triphosphate (dGTP)
Cytosine (C) Cytidine Cytidine 5-triphosphate (CTP)
Cytosine (C) Deoxycytidine Deoxy-cytidine 5-triphosphate (dCTP)
Uracil (U) Uridine Uridine 5-triphosphate (UTP)
Thymine (T) Thymidine/ Deoxythymidie Thymidine/deoxythymidie 5-triphosphate (dTTP)
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C. Properties of nucleic acids
C1 Nucleic Acid Structure (DNA RNA)
Phosphodiester bonds primary sequence
Primary sequence
5end not always has attached phosphate
groups 3 end free hydroxyl (-OH) group
Phosphodiester bond
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C. Properties of nucleic acids
C1 Nucleic Acid Structure DNA double helix

• Watson and Crick , 1953
• The genetic material of all organisms except for
  some viruses
• The foundation of the molecular biology

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C. Properties of nucleic acids
Essential for replicating DNA and transcribing RNA

• Two separate strands Antiparellel (5?3
  direction)
• Complementary (sequence)
• Base pairing hydrogen bonding that holds two
  strands together

3
5

• Sugar-phosphate backbones (negatively charged)
  outside
• Planner bases (stack one above the other) inside

3
5
back
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C. Properties of nucleic acids
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1
3
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5
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3
AT
GC
Base pairing
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C. Properties of nucleic acids

• Helical turn
• 10 base pairs/turn
• 34 Ao/turn

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C. Properties of nucleic acids
C1 Nucleic Acid Structure 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.
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Ribozyme RNA
tRNA
Secondary structure
Tertiary structure
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C. Properties of nucleic acids
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 Protein Nucleic Acids Nucleic Acids
Fibrous protein Globular protein Helical DNA Globular RNA
Structural proteins Enzymes, antibodies, receptors etc Genetic information maintenance Ribozymes Transfer RNA (tRNA) Signal recognition etc.
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C. Properties of nucleic acids
C1 Nucleic Acid Structure Modified Nucleic
Acids
Modifications correspond to numbers of specific
roles. We will discuss them in some related
topics. For example, methylation of A and C to
can avoid restriction digestion of endogenous DNA
sequence (Topic G3).
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• Molecular Biology Course

C2 Chemical and Physical Properties of Nucleic
Acids

1. Stability of Nucleic Acids
2. Effect of Acid applications
3. Effect of alkali applications
4. Chemical denaturation
5. Viscosity applications
6. Buorant density application

Chemical properties
Physical properties
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C. Properties of nucleic acids
Stability of Nucleic Acids

• Hydrogen bonding
• Does not normally contribute the stability of
  nucleic acids or protein
• Contributes to specific structures of these
  macromolecules. For example, a-helix, b-sheet,
  DNA double helix, RNA secondary structure

• 2. Stacking interaction/hydrophobic interaction
  between aromatic base pairs/bases contribute to
  the stability of nucleic acids.
• It is energetically favorable for the
  hydrophobic bases to exclude waters and stack on
  top of each other
• This stacking is maximized in double-stranded
  DNA

Fig
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C. Properties of nucleic acids
Effect of Acid
Strong acid high temperature completely
hydrolyzed to bases, riboses/deoxyrobose, and
phosphate
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
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C. Properties of nucleic acids
Effect of Alkali Application

1. High pH (gt 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
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C. Properties of nucleic acids
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
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C. Properties of nucleic acids
Chemical Denaturation
Urea (H2NCONH2) (??) denaturing PAGE Formamide
(HCONH2) (???) Northern blot
Disrupting the hydrogen bonding of the bulk water
solution
Hydrophobic effect (aromatic bases) is reduced
Denaturation of strands in double helical
structure
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C. Properties of nucleic acids
Viscosity(??)

• Reasons for the DNA high viscosity
• High axial ratio
• Relatively stiff

Applications Long DNA molecules can easily be
shortened by shearing force. Remember to avoid
shearing problem when isolating very large DNA
molecule.
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C. Properties of nucleic acids
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
back
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• Molecular Biology Course

C3 Spectroscopic and Thermal Properties of
Nucleic Acids

• 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 (lmax 260 nm)
• Applications detection, quantitation, assessment
  of purity (A260/A280)

2. Hypochromicity caused by the fixing of the
bases in a hydrophobic environment by stacking,
which makes these bases less accessible to UV
absorption. dsDNA, ssDNA/RNA, nucleotide
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C. Properties of nucleic acids

• 3. 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 (e purines gt
  pyrimidines)
• The absorbance values also depend on the amount
  of secondary structures due to hypochromicity.

• Purity of DNA
• A260/A280
• dsDNA--1.8
• pure RNA--2.0
• protein--0.5

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C. Properties of nucleic acids
5. 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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C. Properties of nucleic acids
6. 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)
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C. Properties of nucleic acids
DNA Supercoiling
Closed circular molecule Supercoiling energy
Topoisomer topoisomerase
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C. Properties of nucleic acids

1. Almost all DNA molecules in cells can be
   considered as circular, and are on average
   negatively supercoiled.

Counter helical turn
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C. Properties of nucleic acids

• 2. Negative supercoiled DNA has a higher
  torsional (???) energy than relaxed DNA, which
  facilitates the unwinding of the helix, such as
  during transcription initiation or replication
• Topoisomer A circular dsDNA molecule with a
  specific linking number which may not be changed
  without first breaking one or both strands.

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C. Properties of nucleic acids
Topoisomerases exist in cell to regulate the
level of supercoiling of DNA molecules. Type I
topoisomerase breaks one strand and change the
linking number in steps of 1. TypeII
topoisomerase breaks both strands and change
the linking number in steps of 2. Gyrase
introduce the negative supercoiling (resolving
the positive one and using the energy from ATP
hydrolysis.
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C. Properties of nucleic acids
Ethidium bromide (intercalator) locally
unwinding of bound DNA, resulting in a reduction
in twist and increase in writhe. Topoisomerases
Type I break one strand of the DNA , and change
the linking number in steps of 1. Type II break
both strands of the DNA , and change the linking
number in steps of 2.