TOPICS IN (NANO) BIOTECHNOLOGY

Lecture V

29th April, 2003

PhD Course

DNA Microarrays - Mechanics -

• Orderly, high-density arrangements of nucleic-acid spots (probes) immobilized on a suitable substrate (e.g., glass).– Hundreds of identical arrays are often “printed” together.

• Probes may be of two different types: – 1) gene (cDNA) fragments;– 2) oligonucleotides.

• Probe deposition is computer aided with up to 10,000 cDNA or 250,000 oligonucleotide spots/cm2 being routinely used today.

• The “target” mRNA is reverse-transcribed into cDNA and either radiolabeled or labeled with fluorescent molecules for detection.– Fluorescence is the method of choice.– Fluorescence analysis requires the use of two target samples.

• Once labeled, the cDNA pools are hybridized to the DNA array and the signals are detected by a “reader”.

• Data is analyzed with sophisticated bioinformatics software to resolve, quantitate and group the signals in a meaningful way.

General Overview of cDNA Microarray Process

MicroarrayPreparation

cDNA ProbeHybridization

ConfocalMicroscope

ComputerAnalysis

Visualization of Fluorescence Microarrays

50 m

g/k

g25 m

g/k

g2.5

mg

/kg

0.2

5 m

g/k

g

2D Graph showing genes which were highly regulated relative to control in the

50 and 2.5 mg/kg drug treatments.

Drug

T-ceIl specific proteinPutative purine nucleotide binding proteinMtrp1 beta variant (Mtrp1)Mus musculus nimA-reIated kinase 2 (Nek2)Mouse mRNA for interleukin-6Mouse testosterone 16-alpha-hydroxylaseMouse testosterone 16-alpha-hydroxyIase (C-16-alpha)ATP Citrate LyaseCyp4a-10SODIUM/BILE ACID COTRANSPORTERLOW MOLECULAR WEIGHT GROWTH HORMONE RECEPTOR/BI“FRUCTOSE-1, 6-BISPHOSPHATASE”Mouse histidase locus histidine ammonia-lyaseGroup 1 major urinary proteinMus musculus reelinCytochrome P450 monooxygenase (CYP2J5)M.musculus spot14 gene.

Graph showing expression changes for genes that have been associated with hepatotoxicity. Genes that are colored in red were upregulated relative to control, genes that are colored green were downregulated relative to control. (B). Table showing the expression changes of genes normally regulated during an immune response.

50

mg

/kg

Drug

0.2

5

mg

/kg

2.5

mg

/kg

25

mg

/kg

CYP4B1M. musculus Cyp4 a-10 mRNACytochrome P450 (Cyp1b1)Cytochrome P450 Cyp7b1Cytochrome P450 monooxygenase (CYP2J5)Cytochrome P450 monooxygenase (CYP2J6)M.musculus CYP1A1 geneCYP4A14Glutathione S-transferaseGlutathione PeroxidaseGlutathione SynthetaseUDP GIucuronosyltransferaseAcetyl TransfereaseProliferating cell nuclear antigenCycIin B1Cyclin B2Cyclin ACycIin E

DNA Array Analysis of Drug Treated Cells

10

100

1000

10000

100000

10 100 1000 10000 100000

Drug

Dru

g

Induced by Oligonucleotide

Treatment

Inhibited by Oligonucleotide

Treatment

RNA Expression

RN

A

Exp

ressio

n

DNA Microarrays- Limitations -

• Need for standardization of the technology to enable comparisons of data between labs.

• Large amount of high quality sample RNA is generally required (50-200 g). Sensitivity limitations.

• Reproducibility of chip quality, wet methodology.

• Expensive and labor intensive.

• mRNA levels may not parallel protein levels, protein activity.

Proteomics I• Definitions

– Proteome: The total protein complement of a genome (1995).– Proteomics: A scientific discipline devoted to defining and

characterizing the proteomes of specific organisms.• Proteomics is inherently more complex than DNA-based

(e.g., arrays) technologies.– Larger alphabet (4 vs 20)– RNA splicing and editing produces different proteins from a

single gene.– Proteins are post-translationally modified in many different ways

(e.g., spliced, phosphorylation).– Methods for separating and detecting proteins is more complex.

• Proteomics is still largely centered upon two-dimensional (2-D) gel electrophoresis.– Been in existence >25 years– The only method currently available which is capable of

simultaneously separating and resolving thousands of proteins.

Sequence motifs for some of the common co- and post-translational modifications. Amino acids in italics

are those that will be modified in each case

Proteomics II• The concept of 2-D gels

– Proteins are separated first based on charge (1st

dimension):• Isoelectric Focusing (IEF).• Proteins are separated in a pH gradient until they reach a point

in which their net change = 0 (isoelectric points or pI).

– Following IEF proteins are next separated based on molecular mass (2nd dimension).

• Presence of sodium dodecyl sulphate (SDS) overrides the intrinsic change of a protein.

• Recent advances in 2-D gel electrophoresis, protein microanalysis and bioinformatics have made the large-scale, systematic analysis of proteins possible.– Immobilized pH gradients (IPGs)

• Replaces soluble ampholytes to maintain charge gradient.• Permits much larger amounts of protein to be analyzed.

Proteomics III• Commercially-available precast gels of sufficient quality permits

reproducibility.

• Major advances in Mass Spectrophotometry.– Identification of amino acid sequence and post-translational

modifications based on mass.– Highly sensitive.

• Bioinformatics permits the large-scale analysis of data over a reasonable time-frame.– Proteomics databases are widely available today on the web to

support proteomics research.

2-Dimensional Gel Electrophoresis

14 kDa

96 kDa

pH 3.5 pH 10.0

Protein Analysis Based on 2-D Gel Technology

Vector analysis

Excise protein spot from the

gel

In situ proteinase

digest (peptides passively eluted

into supernatant)

High sensitivity Edman sequencing

MS Analysism/z 366 (HexNAc-

Hex+)m/z 204 (HexNAc+)m/z 292 (NeuAc+)

m/z 79 (PO3-)fragment ions

Electrotransferonto

membrane

Endoproteinasedigest of protein

Enzymic releaseof modifications

EdmanSequence

Acid hydrolyses (monosaccharide,

phosphoamino & amino acid composition)

A B

Proteomics- Limitations -

• Expensive and labor intensive.

• Non-specific protein contamination due to ultra-sensitivity of new detection methods.

• Protein levels may not parallel protein activity.

• Standardization of technology to enable comparative studies between different investigators still in progress.

Target Validation Approaches

Gene ProteinTargetValidation

Correlative Approaches•Comparative Genomics

•Transcriptional Profiling

•Proteomics

Drug Identification

ClinicalDevelopment

Causative Approaches

•Overexpression Systems

•Gene Knockouts

•Small Molecules

•Antibody Approaches

•Antisense

•Interference RNA