Nucleic acid Basics

Hybridization

Electrophoresis

PCRDiagnostic

tools

DNA-Proteininteractions

Chromatin

Gene expression

Six Nucleosides

Guanosine(base: guanine)

Cytidine(base: cytosine)

Thymidine (base: thymine) thymidine is deoxynucleotide

Uridine(bsae: uracil)

Adenosine(base: adenine)

5-methyl Cytidine(base: 5-methy cytosine)

Features of Nucleosides

NO

N

OOH

OHNH2

OH

1’ carbon forms aglycosidic linkageto a base (adenineis shown here

2’ carbon is connected to: - H in DNA - OH in RNAIn RNA the OH may functionas a catalyst in some reactions.

3’ oxygen forms a phosphoester bond.

5’ oxygen forms a phosphoester bond.

Cytidine

3’

1’

2’

4’5’

A Dinucleotide

5’ end

3’ end

O

O

ON

NH

O

O

CH3

P OO

NO

N

N

ON

NH2

Ophosphodiester

Single Stranded Nucleic Acids

• In cells, RNAs are the most abundant single stranded nucleic acids– secondary structure is largely in the form of

“hairpin loops”.– tertiary structures are important for catalysis.

The 2’OH as a catalyst

NO

N

OOH

OHNH2

OH

1’ carbon forms aglycosidic linkageto a base (adenineis shown here

2’ carbon is connected to: - H in DNA - OH in RNAIn RNA the OH may functionas a catalyst in some reactions.

3’ oxygen forms a phosphoester bond.

5’ oxygen forms a phosphoester bond.

Cytidine

3’

1’

2’

4’5’

Single Stranded Nucleic Acids

• Tertiary structures are important for interactions with proteins and can be manipulated to produce designer drugs:– Interference RNAs– Aptamers.

Rusconi et al, 2002 Nature 419:90-94

RNA inhibitors of clotting factor IXa

RNA inhibitor of clotting factor IXa

and its antidote

Rusconi et al, 2002 Nature 419:90-94

Single Stranded Nucleic Acids

• Single stranded DNAs are important in clinical and scientific investigations. Probes and primers are synthetic single stranded DNAs

Double Stranded Polynucleotides

G:CThree H-bonds

A:TTwo H-bonds

N

N

N

O

N

O

N

O

O

H

H

H

NO

N

O

O

O

N

H

H

NO

N

N

O

N

N

O

HH

O

O

O

NN

O

OCH3

H

Important Forces

Negative charges on phosphates destabilize

H-bonds stabilize

Base-base stacking interactions stabilize

(bases at the ends lack this stabilizing force)

Nucleic acid Basics

Hybridization

Electrophoresis

PCRDiagnostic

tools

DNA-Proteininteractions

Chromatin

Gene expression

DNA “Melting”The DNA strands separate when heated

Strand separation occurs over a narrow temperature range.The midpoint is Tm, the “melting temperature”.

Factors That Influence TmProperties of the helix

• Base composition:– C:G rich is more stable than A:T rich

• Mismatches:– Sequences with perfect complementarity are

more stable than those with mismatches.

• Length of the helix– Very short helicies are less stable that

moderately long ones.

Factors That Influence TmProperties of the solution

• Ionic conditions– Solutons with high ionic strength will stabilize.

• Extremes of pH

• Chemicals that disrupt H-bonds– Urea, formamide, formaldehyde

Factors That Influence TmProperties of cells

• Helix-destabilizing proteins– These proteins play physiologically important

roles in a number of cellular processes.

Separated Strands Can Rehybridize

- Duplex formation is a bimolecular reaction:thermodynamically favored

- Hair-pin helix formation is a monomolecular reaction:kinetically favored

Hybridization:Conditions are important

• Concentration is important– Hydridization is a bimolecular reaction. A high

concentration of DNA will favor duplex formation.

Hybridization:Conditions are important

• Temperature is important– Slow cooling will favor the formation of DNA

duplexes.– Fast cooling will favor the formation of hair-pin

loops, which may prevent duplex formation.– The temperature must be near the Tm if high

stringency is desired (formation of duplexes with perfect complementarity).

Fluorescence in situ hybridization

FISH

Biopsy from a patient with breast cancer showing HER-2 amplification

Control probe

HER-2 probe

HER-2 probe

Control probe