1. Nitrogenous bases Bicyclic purines Monocyclic pyrimidine
Thymine (T) is 5-methyluracil (U)

2. 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
(糖苷键) R
Ribose or
2’-deoxyribose

3. (deoxyribose containing)
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.
Phosphate diester bonds 6 4 7 5 5 1 9 2 2 4 1
Deoxynucleotides
(deoxyribose containing)
Ribonucleotides
(ribose containing)

4. 5’end: not always has attached phosphate groups
C1 Nucleic Acid Structure
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

5. DNA double helix back Watson and Crick , 1953.
C1 Nucleic Acid Structure
DNA double helix
Watson and Crick , 1953.
Two separate strands Antiparellel (5’3’ direction)
Complementary (sequence)
Base pairing: hydrogen bonding that holds two strands together
Essential for replicating DNA and transcribing RNA
Sugar-phosphate backbones (negatively charged): outside
Planar bases (stack one above the other): inside
back

6. Base pairing via hydrogen bonds5 5 4 4 6 6 3 3 1 1 7 2 7 2 6 5 6 8 5 1 8 1 9 4 9 4 2 3 2 3
A:T
G:C
Base pairing via hydrogen bonds

7. Double helix B form: Right-handed 10 base pairs/turn 34 Å /turn
C1 Nucleic Acid Structure
Double helix
B form:
Right-handed
10 base pairs/turn
34 Å /turn
Diameter: ca. 20 Å
Other forms:
A: 11 bases/turn, base plate 20° slant
Z: 12 bases/turn, left-handed helical, one groove

8. RNA Secondary Structure
C1 Nucleic Acid Structure
RNA Secondary Structure
Single stranded, no long helical structure like double-stranded DNA
Globular conformation with local regions of helical structure formed by intramolecular hydrogen bonding and base stacking.
tRNA
(clover-like)

9. C1 Nucleic Acid 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 protein
Helical DNA
Globular RNA
Structural proteins
Enzymes,
antibodies,
receptors etc
Genetic information maintenance
Ribozome RNA
Transfer RNA (tRNA)
Signal recognition

10. 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 avoid restriction digestion of endogenous DNA sequence (Topic G3).

11. C2 Chemical and Physical Properties of Nucleic Acids
Stability of Nucleic Acids
Hydrogen bonding
Contribute to specificity, not overall stability of DNA helix
Stability lies in the stacking interactions between base pairs
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 (base stacking & hydrophobic effect).
This stacking is maximized in double-stranded DNA

12. C2 Chemical and Physical Properties of Nucleic Acids
Effect of Acid & applications
Strong acid + high temperature  completely hydrolyzed to
(perchloric acid+100°C) bases, riboses/deoxyribose, and phosphate
pH  apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ]
Maxam and Gilbert chemical DNA sequencing:
A DNA sequencing technique based on chemical removal and modification of bases specifically and then cleaving the sugar-phosphate backbone of the DNA and RNA at particular bases (J2)

13. C2 Chemical and Physical Properties of Nucleic Acids
Effect of Alkali & Application
DNA denaturation at high pH
keto form
enolate form
keto form
enolate form
Base pairing is not stable anymore because of the change of tautomeric (异构) states of the bases, resulting in DNA denaturation

14. Effect of Alkali & Application
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

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

16. Viscosity Reasons for the DNA high viscosity High axial ratio
Relatively stiff
Applications
Long DNA molecules can easily be shortened by shearing force.
When isolating very large DNA molecule, always avoid shearing problem

17. Buoyant density
1.7 g cm-3, a similar density to 8M CsCl. Rho= (GC)%
Purifications of DNA: equilibrium density gradient centrifugation
Protein floats
RNA pellets at the bottom

18. 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
Fixing of the bases in a hydrophobic environment by stacking,
which makes these bases less accessible to UV absorption.
dsDNA, ssDNA/RNA, nucleotide

19. 3. Quantitation of nucleic acids
Extinction coefficient (e): 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

20. 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.

21. Rapid cooling Only allow the formation of local base paring
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.
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)

22. DNA Supercoiling
Almost all DNA molecules in cells are on average negatively supercoiled.
Supercoiled DNA has a higher energy than relaxed DNA. Negative supercoiling may thus facilitate cellular processes which require the unwinding of the helix, such as transcription initiation or replication
Topoisomerases exist in cell the regulate the level of supercoiling of DNA molecules. (important to know in the sense of gene expression)

23. Linker number a topological property of a closed-circular DNA, which can be changed only if one or both of the DNA backbones are broken.
Topoisomer A molecule of a given linking number is known as a topoisomer. Topoisomers of the same molecule differ from each other only in their linker number.
The conformation (geometry) of the DNA can be altered while the linking number remains constant. Writhe (wrap around,缠绕) and Twist (扭转) changes are to measure the conformational change of a DNA molecule (Lk = Tw + Wr).
The topological change (Lk) in supercoiling of a DNA molecule is partitioned into a conformational change of twist (Tw )and/or a change of writhe (Wr).
For a given isomer of a circular closed DNA (Lk = 0), the increase in twist will cause a corresponding decrease in writhe.

24. Ethidium bromide (intercalator 插入物)
locally unwinding of bound DNA, resulting in a reduction in
twist and increase in writhe.
Topoisomerases
Type I break one strand of the DNA (via P-tyrosine bond) , 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. (ATP)
Bacterial gyrase (旋转酶)
introduce negative supercoiling. ATP.

25. Summary: Nucleic acid structure
bases > nucleosides (base+sugar) > nucleotides (nucleoside+phosphate) > polynucleotides /DNA/RNA (via 3’,5’-phosphodiester bond) > DNA double helix/RNA secondary structure
Chemical and physical properties
stability support, effect of acid and alkali, chemical denaturation, viscosity, buoyant density
3. Spectroscopic and thermal properties
4. DNA supercoiling: Linking number (twist and writhe))