Section C - Properties of Nucleic Acids

1
Section C - Properties of Nucleic Acids
2
Contents

• C1 Nucleic Acid Structure
• Bases, Nucleosides, Nucleotides,
  Phosphodiester bonds, DNA/RNA sequence, DNA
  double helix, A, B and Z helices, RNA secondary
  structure, Modified nucleic acids
• C2 Chemical and Physical Properties of Nucleic
  Acids
• Stability of Nucleic Acids, Effect of acid,
  Effect of alkali, Chemical denaturation,
  Viscosity, Buoyant density
• C3 Spectroscopic Properties of Nucleic Acids
• UV absorption, Hypochromicity, Quantization
  of nucleic acids, Purity of DNA, Thermal
  denaturation, Renaturation
• C4 DNA supercoiling
• Closed-circular, Supercoiling, Topoisomer,
  Twist and writhe, Intercalators, Energy of
  supercoiling, Topoisomerases

3
C1 Nucleic Acid Structure Bases
Bicyclic Purine
Monocyclic Pyrimidine
4
C1 Nucleic Acid Structure Nucleosides
Glycosidic (glycoside, glycosylic) bond (???)
5
C1 Nucleic Acid Structure 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.

6
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)
7
C1 Nucleic Acid Structure
Phosphodiester bonds
8
C1 Nucleic Acid Structure
DNA/RNA sequence
9
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

Watson
Crick
10
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
11

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

12
Base pairing
13
C1 Nucleic Acid Structure
A, B and Z helices
14
C1 Nucleic Acid Structure RNA
secondary 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.
15
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.
16
COMMON SECONDARY STRUCTURE MOTIFS
17
C1 Nucleic Acid Structure
Modified nucleic acids

• 1. Methylation
• (N-6position of adenine ,4-amino group and
  5-position of cytosine)
• Restriction modification
• 2. Base mismatch

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.
18
Types of nucleic acids Types of nucleic acids
Constituents Nucleobases Nucleosides Nucleotides Deoxynucleotides
Ribonucleic acids RNA mRNA (pre-mRNA/hnRNA) tRNA rRNA aRNA gRNA miRNA ncRNA piRNA shRNA siRNA snRNA snoRNA stRNA ta-siRNA tmRNA
Deoxyribonucleic acids DNA cDNA cpDNA gDNA msDNA mtDNA
Nucleic acid analogues GNA LNA PNA TNA morpholino
Cloning vectors phagemid plasmid lambda phage cosmid P1 phage fosmid BAC YAC HAC
19
C2 Chemical and Physical 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

20
C2 Chemical and Physical 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

21
C2 Chemical and Physical Properties of Nucleic
Acids
Effect of alkali

• RNA hydrolyzes at higher pH because of 2-OH
  groups in RNA

22

1. High pH (gt 7-8) has subtle (small) effects on
   DNA structure
2. High pH changes the tautomeric (????)state of the
   bases

Base pairing is not stable anymore because of the
change of tautomeric states of the bases,
resulting in DNA denaturation
23
C2 Chemical and Physical Properties of Nucleic
Acids Chemical
denaturation
Strong acid high temperature completely
hydrolyzed to bases, riboses/deoxyrobose, and
phosphate
Disrupting the hydrogen bonding of the bulk water
solution
Hydrophobic effect (aromatic bases) is reduced
Denaturation of strands in double helical
structure
24
C2 Chemical and Physical Properties of Nucleic
Acids
Viscosity
Viscosity Buoyant density
Physical properties

• 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.
25
C2 Chemical and Physical Properties of Nucleic
Acids
Buoyant density

• 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
26
C3 Spectroscopic Properties of Nucleic Acids
UV absorption

• 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
• Applications detection, quantitation, assessment
  of purity (A260/A280)
• Quantitation of nucleic acids
• Extinction coefficients 1 mg/ml dsDNA has an
  A260 of 20ssDNA and RNA, 25

27
C3 Spectroscopic Properties of Nucleic Acids
Hypochromicity

• 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

28
C3 Spectroscopic Properties of Nucleic Acids
Quantitation of nucleic acids

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

29
C3 Spectroscopic Properties of Nucleic Acids
Purity of DNA

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

30
C3 Spectroscopic Properties of Nucleic Acids
Thermal denaturation

• Thermal denaturation/melting
• heating leads to the destruction of
  double-stranded hydrogen-bonded regions of DNA
  and RNA.

31
C3 Spectroscopic Properties of Nucleic Acids
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.
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)
32
C4 DNA supercoiling Closed-circular

• cccDNA Covalently closed circular

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

33
C4 DNA supercoiling Supercoiling

• Most natural DNA is negatively supercoiled, that
  is the DNA is deformed in the direction of
  unwinding of double helix.

34
C4 DNA supercoiling Topoisomer

• Topoisomer
• A circular dsDNA molecule with a
  specific linking number which may not be changed
  without first breaking one or both strands.

35
C4 DNA supercoiling Twist and writhe

• Supercoiling is partitioned geometrically into a
  change in twist, the local winding up or
  unwinding of the double helix, and a change in
  writhe, the coiling of the helix axis up itself.
• Twist and writhe are interconvertible according
  to the equation

?LK?T??Wr
36
C4 DNA supercoiling Intercalators

• Ethidium bromide (intercalator)
• locally unwinding of bound DNA, resulting
  in a reduction in twist and increase in writhe.

37
C4 DNA supercoiling Energy of supercoiling

• Negatively supercoiled DNA has a high torsional
  energy, which facilitates the untwisting of DNA
  helix and can drive processes which require the
  DNA to be unwound.
• Such as transcription initiation or replication.

38
C4 DNA supercoiling Topoisomerases

• 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.

39
(No Transcript)
40
Multiple choice questions

• 1.The sequence 5'-AGTCTGACT-3' in DNA is
  equivalent to which sequence in RNA?
• A 5'-AGUCUGUGACU -3'
• B 5' -UGTCTGUTC -3'
• C 5' -UCAGUCUGA-3'
• D 5'- AGUCAGACU-3'

41

• 2. Which of the following correctly describes
  A-DNA?
• A a right-handed antiparallel double helix with
  10 bp/turn and bases lying perpendicular to
  the helix axis.
• B a left-handed antiparallel double-helix with
  12 bp/turn formed from alternating
  pyrimidine-purine sequences.
• C a right-handed antiparallel double helix with
  11 bp/turn and bases tilted with respect to the
  helix axis.
• D a globular structure formed by short
  intramolecular helices formed in a single-strand
  nucleic acid.
• 3. Denaturation of double stranded DNA involves
  .
• A preakage into short double-stranded fragments.
• B separation into single strands.
• C hydrolysis of the DNA backbone.
• D cleavage of the bases from the sugar-phosphate
  backbone.

42

• 4. Which has the highest absorption per unit
  mass at a wavelength of 260 nm?
• A double-stranded DNA.
• B mononucleotides.
• C RNA.
• D protein.
• 5. Type I DNA topoisomeraes .
• A change linking number by?2
• B require ATP.
• C break one strand of a DNA double helix.
• D are the target of antibacterial drugs.

43
THANK YOU !