SECTION C Properties of Nucleic Acids

C1 Nucleic Acid Structure

C2 Chemical and Physical Properties of Nucleic Acids

C3 Spectroscopic and Thermal Properties of Nucleic Acids

C4 DNA Supercoiling

Molecular Biology

Molecular Biology

Bicyclic Purines:

Thymine (T) is a 5-methyluracil (U)

C1 Nucleic Acid Structure-1Bases

Monocyclic pyrimidine:

Molecular BiologyC1 Nucleic Acid Structure-2

Nucleosides

The structures of pentose sugar

Adenosine, guanosine, cytidine, thymidine, uridine

The bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside

Molecular Biology

Molecular Biology

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.

Deoxyribonucleotides (containing deoxyribose)

Ribonucleotides (containing ribose)

Phosphate ester bonds

C1 Nucleic Acid Structure-3Nucleotides

Molecular Biology

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)

Molecular Biology

Brief Summary

Primary sequence:

5’end: may or may not have any

attached phosphate groups.

3’ end: is most likely to be a free

hydroxyl group.

Molecular Biology

C1 Nucleic Acid Structure-4Phosphodiester bonds an

d Sequence

•Deduced by James Watson

and Francis Crick in 1953.

•DNA is the genetic material

of all organisms except for some

viruses.

• Essential for replicating DNA and

transcribing RNA

•The foundation of the molecular

biology

C1 Nucleic Acid Structure-5DNA double helix

Molecular Biology

Molecular Biology

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

• The negatively charged Sugar-phosphate backbones are on the outside of helix.• Planar bases stack one above the other in the center (inside) of helix.

Molecular Biology

Base pairing

A:T

G:C

Molecular Biology

•Helical turn:

10 base pairs/turn

3.4 nm/turn

Molecular Biology

A-form B-form Z-form

Molecular BiologyC1 Nucleic Acid Structure-6

A, B and Z helices

1. Normally occurs as single stranded molecule

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.

Molecular BiologyC1 Nucleic Acid Structure-7

RNA structure

Molecular Biology

Tertiary structure

tRNA Secondary structure

Conformational variability of RNA is reflected in the more diverse roles of RNA in the cell, when compared to DNA.

Comparison of Structure and Function of protein and nucleic acids

Protein Nucleic Acids

Fibrous protein Globular protein

Double Helical DNA

Globular RNA

Structural proteins

Enzymes,antibodies,receptors etc

Genetic material

RibozymesTransfer RNA (tRNA)Signal recognition etc.

Molecular Biology

Molecular BiologyC1 Nucleic Acid Structure-8Modified Nucleic Acids

The chemical modification of bases or nucleotides in nucleic acids i

s widespread, and has a number of specific roles. In cellular DNA, t

he modifications are restricted to the methylation of the N-6 positio

n of adenine and the 4-amino group and the 5-position of cytosine.

These methylations have a role in restriction modification (see Topi

c G3), base mismatch repair (see Topic F3) and eukaryotic genome

structure (see Topic D3). A much more diverse range of modificati

ons occurs in RNA after transcription, which again reflects the diff

erent roles of RNA in the cell. These are considered in more detail i

n Topics O3 and P2 。

C2 Chemical and Physical Properties of Nucleic Acids

Molecular Biology

Stability of Nucleic Acids

1. Hydrogen bonding • Contributes to specific structures of nucleic acids or protein. For

example, -helix, -sheet, DNA double helix, RNA secondary structures

• H-bonds within a structure does not normally confer the stability, that is to say, it does not contribute the overall stability of these secondary structures

2. Stacking interaction/hydrophobic interaction between

aromatic base pairs contribute to the stability of nucleic acids.• Even in single-stranded DNA, the bases have a tendency to stack on top of each other, but this stacking is maximized in double-stranded DNA •It is energetically favorable to exclude water altogether from pairs of such

surfaces by stacking them together

Molecular Biology

Effect of Acid

In strong acid and at elevated temperatures: are hydrolyzed (水解 ) completely to bases, ribose or deoxyribose, and phosphate (e.g., perchloric acid (HClO4) at > 100°C)

In more dilute mineral acid, for example at pH 3–4, the most easily hydrolyzed bonds are selectively broken. E.g., glycosylic bonds attaching purine bases to the ribose ring are broken by formic acid.

Application: the basis of the chemical DNA sequencing method developed by Maxam and Gilbert.

Molecular Biology

Molecular Biology

Effect of Alkali-DNA

keto form enolate form keto form enolate form

3. This affects the specific hydrogen bonding between the base pairs, with the result that the double-stranded structure of the DNA breaks down; that is the DNA becomes denatured .

1. Increasing pH (> 7-8) has more subtle effects on DNA structure2. The effect of alkali is to change the tautomeric (互变异构 )state of

the bases

RNA are hydrolyzed at higher pH because of the present of 2’-OH group in RNA

RNA is unstable at higher pH

HO

2’, 3’-cyclic phosphodiester

Alkali

Molecular Biology

Effect of Alkali-RNA

Chemical DenaturationA number of chemical agents can cause the denaturation of DNA or RNA at neutral pH, e.g. Urea (H2NCONH2) is used in denaturing PAGE; Formamide (HCONH2) is used Southern and Northern blotting.

Disrupting the hydrogen bonding of the bulk water solution

Hydrophobic effect (stacking interaction) is reduced

Denaturation of the strands

Molecular Biology

Mechanism

Viscosity（粘性）

Consequence of the DNA high viscosity 1. A high axial ratio (2 nm in diameter, and a length

of micrometers, millimeters or even several centimeters in the Eukaryotic chromosomes)

2. Relatively stiff

Applications:Long DNA molecules can easily be damaged by shearing force, or by sonication.

Pay attention to avoid shearing problem when need to isolate intact very large DNA molecule.

Molecular Biology

Molecular Biology

Buoyant density

DNA and 8M CsCl has a similar density, around 1.7 g cm-3

Purifications of DNA: equilibrium density gradient centrifugation

RNA pellets at the bottom

Protein floats

ΡDNA=1.66+0.098%(G+C)

Molecular Biology

C3 Spectroscopic and Thermal Properties of

Nucleic Acids

Molecular Biology

UV absorption

• Nucleic acids absorb UV light due to the conjugated a

romatic nature of the bases

• The wavelength of maximum absorption of light by bo

th DNA and RNA is 260 nm (max = 260 nm)

• Applications: can be used for detection, quantitation a

nd assessment of purity (A260/280)

Molecular Biology

Hypochromicity

Caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. A260 value: dsDNA<ssDNA or RNA<nucleotide

Quantitation of nucleic acidsExtinction coefficients: 1 mg/ml dsDNA has an A260 of 20;

The corresponding value for ssDNA and RNA is approximately 25The values for ssDNA and RNA are approximate for two reason:(1) The values are the sum of absorbance contributed by the differen

t bases ( : purines > pyrimidines)(2) The absorbance values also depend on the amount of secondary

structures in a given molecule.

A260/280:

dsDNA = 1.8

pure RNA = 2.0

Protein <1, 0.5 or so

Molecular Biology

Purity of DNA

The approximate purity of dsDNA preparations (see Topic G2) may be estimated by determination of the ratio of absorbance at 260 and 280 nm (A260/A280).

Thermal denaturation/melting

Heating also leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA.

RNA: the absorbance increases gradually and irregularlyDNA: the absorbance increases cooperatively.

melting temperature (Tm): the temperature at the mid-point of the smooth transition, which has a 20% increase in absorbance. 80-100 °C for long DNA molecules

Molecular Biology

Molecular Biology

Renaturation

Rapid cooling: allows only the formation of local region of base paring. The decreased in A260 is rather small.Slow cooling: allows time for whole complementation of dsDNA. Absorbance in 260nm decreases greatly and cooperatively.

Note: Relative concepts

Annealing: base paring of short regions of complementarity wit

hin or between DNA strands. (example: annealing step in PCR rea

ction)

Hybridization: renaturation of complementary sequences betwe

en different nucleic acid molecules.

(examples: Northern or Southern hybridization)

C4 DNA SupercoilingMolecular Biology

1. Closed-circular DNA: Almost all DNA molecules in cells can be considered as circular

2. Supercoiling: Most natural DNA is negatively supercoiled, that is the DNA is deformed in the direction of unwinding of the double helix. (Why?)

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

Learn about several concepts:

1, Linking Number (Lk): the number of double-helical turns in the DNA molecule.

2. The level of supercoiling may be quantified in terms of the change in linking number (ΔLk) from that of the unconstrained (relaxed) closed-circular molecule (Lk°).

2, Twist（缠绕） :the local winding up or unwinding of the double helix

3, writhe（扭曲 ) : the coiling of the helix axis upon itself.

Twist and writhe are interconvertible according to the equation ΔLk = ΔTw + ΔWr.

Molecular Biology

Molecular Biology

A rubber tubing model for DNA supercoiling, which illustrates the change in DNA conformation. The changes in linking number are indicated (see text for details).

Conformational flexibility in supercoiled DNA as modeled by rubber tubing. The changes in twist and writhe at constant linking number are shown (see text for detail

s).

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.

Molecular Biology

Molecular Biology

The mechanisms of (a) type I and (b) type II topoisomerases (see text for details).

Molecular Biology

Intercalator Ethidium bromide locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.

(a) Ethidium bromide; (b) the process of intercalation, illustrating the lengthening and untwisting of the DNA helix.