1. Properties of Nucleic Acids
Molecular Biology Course
SECTION C
Properties of Nucleic Acids

2. The Race for the Double Helix (1994)

3. 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.

4. 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

5. Bicyclic Purines: Monocyclic pyrimidine:
C. Properties of nucleic acids
C1 Nucleic Acid Structure (DNA & RNA): Bases
Bicyclic Purines:
Monocyclic
pyrimidine:
Thymine (T) is a 5-methyluracil (U)

6. Adenosine, guanosine, cytidine, thymidine, uridine
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
(糖苷键) R
Ribose or
2’-deoxyribose
Adenosine, guanosine, cytidine, thymidine, uridine

7. (containing deoxyribose)
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
Deoxynucleotides
(containing deoxyribose)
Ribonucleotides
(containing ribose)

8. C. Properties of nucleic acids BASES NUCLEOSIDES NUCLEOTIDES
Adenine (A)
Adenosine
Adenosine 5’-triphosphate (ATP)
Deoxyadenosine
Deoxyadenosine 5’-triphosphate (dATP)
Guanine (G)
Guanosine
Guanosine 5’-triphosphate (GTP)
Deoxyguanosine
Deoxy-guanosine 5’-triphosphate (dGTP)
Cytosine (C)
Cytidine
Cytidine 5’-triphosphate (CTP)
Deoxycytidine
Deoxy-cytidine 5’-triphosphate (dCTP)
Uracil (U)
Uridine
Uridine 5’-triphosphate (UTP)
Thymine (T)
Thymidine/
Deoxythymidie
Thymidine/deoxythymidie
5’-triphosphate (dTTP)

9. 5’end: not always has attached phosphate groups 3’ end: free hydroxyl
C. Properties of nucleic acids
C1 Nucleic Acid Structure (DNA & RNA): Phosphodiester bonds & primary sequence
Primary sequence:
5’end: not always has attached phosphate groups
3’ end: free hydroxyl
(-OH) group
Phosphodiester bond

10. The genetic material of all organisms except for some viruses
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

11. 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’
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12. A:T G:C Base pairing C. Properties of nucleic acids 4 1 3 2 6 7 5 8 19 4 2 3
A:T
G:C
Base pairing
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13. Helical turn: 10 base pairs/turn 34 Ao/turn
C. Properties of nucleic acids
Helical turn:
10 base pairs/turn
34 Ao/turn
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14. C1 Nucleic Acid Structure : RNA structure
C. Properties of nucleic acids
C1 Nucleic Acid Structure : RNA structure
Single stranded nucleic acid
Secondary structure are formed some time
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. Ribozyme RNA
tRNA
Secondary structure
Tertiary structure

16. 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
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.

17. 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).

18. C2 Chemical and Physical Properties of Nucleic Acids
Molecular Biology Course
C2 Chemical and Physical Properties of Nucleic Acids
Stability of Nucleic Acids
Effect of Acid & applications
Effect of alkali & applications
Chemical denaturation
Viscosity & applications
Buorant density & application
Chemical properties
Physical properties
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19. Stability of Nucleic Acids
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

20. 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

21. Effect of Alkali & Application
C. Properties of nucleic acids
Effect of Alkali & Application
High pH (> 7-8) has subtle (small) effects on DNA structure
High pH changes the tautomeric (互变异构)state of the bases
enolate form
enolate form
keto form
keto form
Base pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation

22. RNA hydrolyzes at higher pH because of 2’-OH groups in RNA
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

23. Disrupting the hydrogen bonding of the bulk water solution
C. Properties of nucleic acids
Chemical Denaturation
Urea (H2NCONH2) (尿素）: denaturing PAGE
Formamide (HCONH2) （甲酰胺）: Northern blot
Disrupting the hydrogen bonding of the bulk water solution
Hydrophobic effect (aromatic bases) is reduced
Denaturation of strands in double helical structure

24. Viscosity（粘性） Reasons for the DNA high viscosity Applications:
C. Properties of nucleic acids
Viscosity（粘性）
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. Buoyant density (DNA) 1.7 g cm-3, a similar density to 8M CsCl
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
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26. C3 Spectroscopic and Thermal Properties of Nucleic Acids
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

27. 3. Quantitation of nucleic acids
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 > 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

28. 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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29. 6. Renaturation: C. Properties of nucleic acids
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)

30. DNA Supercoiling Closed circular molecule Supercoiling & energy
C. Properties of nucleic acids
DNA Supercoiling
Closed circular molecule
Supercoiling & energy
Topoisomer & topoisomerase

31. C. Properties of nucleic acids
Almost all DNA molecules in cells can be considered as circular, and are on average negatively supercoiled.
Counter helical turn

32. C. Properties of nucleic acids
2. Negative supercoiled DNA has a higher torsional (扭转的) 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.

33. 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.

34. 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.