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 ( 糖苷键 )

R Ribose or 2’-deoxyribose

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.

Deoxynucleotides (deoxyribose containing)

Ribonucleotides (ribose containing)

Phosphate diester bonds

4

5 12

67

9 21

54

C1 Nucleic Acid Structure

Phosphodiester bonds

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

3’ end: free hydroxyl (-OH) group

5’end: not always has attached phosphate groups

C1 Nucleic Acid Structure

DNA double helix

•Watson and Crick , 1953.

•Two separate strands Antipa

rellel (5’3’ direction)

Complementary (sequence)

Base pairing: hydrogen bondin

g 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

Base pairing via hydrogen bonds

A:TG:C

1

234

567

8

9

12

3

4 5

6

2

1

34

5 67

89

2 13

4 5

6

•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

C1 Nucleic Acid 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)

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 RNATransfer RNA (tRNA)Signal recognition

C1 Nucleic Acid Structure

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).

C2 Chemical and Physical Properties of Nucleic Acids

Stability of Nucleic Acids

1. 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

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 3-4 apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broke

n ]

Maxam and Gilbert chemical DNA sequencing:A DNA sequencing technique based on chemical removal and modificati

on of bases specifically and then cleaving the sugar-phosphate backbone of the DNA and RNA at particular bases (J2)

C2 Chemical and Physical Properties of Nucleic Acids

Effect of Alkali & Application

DNA denaturation at high pH

keto form enolate formketo 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

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

Effect of Alkali & Application

RNA is unstable at higher pH

OH free 5’-OH

2’, 3’-cyclic phosphodiester

alkali

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

Viscosity

Reasons for the DNA high viscosity 1. High axial ratio2. Relatively stiff

Applications1. Long DNA molecules can easily be shortened by

shearing force.2. When isolating very large DNA molecule, always

avoid shearing problem

Buoyant density

1.7 g cm-3, a similar density to 8M CsCl. Rho=1.66+0.098 (GC)%

Purifications of DNA: equilibrium density gradient centrifugation

RNA pellets at the bottom

Protein floats

Spectroscopic and Thermal Properties of Nucleic Acids

1. 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 (max = 260 nm) - Applications: detection, quantitation, assessment of purity (A260/280)

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

3. Quantitation of nucleic acids Extinction coefficient (): 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

( : purines > pyrimidines)

- The absorbance values also depend on the amount of secondary structures

due to hypochromicity.

4. Purity of DNA

A260/280:

dsDNA--1.8

pure RNA--2.0

protein--0.5

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 irregularlyDNA the absorbance increases cooperativelyMelting temperature (Tm) the temperature at which 40% increase in

absorbance is achieved.

6. Renaturation:

Rapid cooling Only allow the formation of local base paring Absorbance is slightly decreasedSlow 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)

DNA Supercoiling

1. Almost all DNA molecules in cells are on average negatively

supercoiled.

2. Supercoiled DNA has a higher energy than relaxed DNA. Ne

gative supercoiling may thus facilitate cellular processes whi

ch require the unwinding of the helix, such as transcription i

nitiation or replication

3. Topoisomerases exist in cell the regulate the level of supercoi

ling of DNA molecules. (important to know in the sense of gen

e expression)

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).

1. 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).

2. For a given isomer of a circular closed DNA (Lk = 0), the increase in twist will cause a corresponding decrease in writhe.

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.

Summary:

1. Nucleic acid structure

bases > nucleosides (base+sugar) > nucleotides (nucleoside+phosphate) > polynucleotides /DNA/RNA (via 3’,5’-phosphodiester bond) > DNA double helix/RNA secondary structure

2. 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))