High-Throughput Screening

1. High-Throughput Screening Assays on Solid Supports or Chips -> Array Technology

2. Types of Arrays – Applications: • DNA Microarrays: Expression profiles, disease research (cancer), DNA sequencing, mutation analysis, gene discovery, diagnosis, drug discovery,… • RNA Microarrays: RNA-protein interactions, biological function of proteins, drug discovery,… • Protein Microarray (chips): Enzyme profiling, Protein-protein Interaction, Protein-ligand interaction ,...

3. What is Microarray ? Microarrays are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel screening studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously - a dramatic increase in throughput.

4. Importance for High Troughput Screening

6. Microarray Landscape Slide spot pingroup subarray Gene/Protein

12. DNA Microarrays – Applications: • Monitor gene expression • Study regulatory networks • Drug discovery - mechanism of action • Diagnostics - tumor diagnosis • etc. • Genomic DNA hybridizations • Explore microbial diversity • Whole genome comparisons - genome evolution • Identify DNA binding sites • Diagnostics - tumor diagnosis • ?

13. DNA Microarray Technologies: • Microarray allows to simultaneously analyze 1000s of sequences • Developed in early 1990s • Two ways to construct microarrays: 1. DNA chips (developed at Affymetrix, Inc. ): an array of oligonucleotide (20~80-mer oligos) probes is synthesized either in situ (on-chip by photolithography) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined. 2. DNA Microarrays (developed at Stanford University): Obtain cDNA from cDNA sequencing projects, libraries,… -> ssDNA of cDNA spotted onto glass slides (around 20,000 spotes/cm) -> not so high density possible -> hybridization more specific

14. AffymetrixGeneChip • Oligonucleotide: • 11-25 mer (short oligo) • 50-70 mer (long oligo)

15. Steps in the design and implementation of the microarray experiment

16. Drought stress (Kathiresan, 2005) DNA microarray experiment: Water is a major limiting factor in agricultural production systems in several parts of the world. Due to their sessile nature, plants have had to develop efficient strategies to cope with limited water. Tolerant plants adapt to drought by invoking a battery of changes in their physiological and metabolic activities -> Microarrays used to find metabolic changes (gene expression changes)

17. Not watered 1 2 3 5 6 7 8 9 12 18 20 days Fl Field experimental procedure control Azucena Apo IR64 Assess drought-induced expression among three varieties of rice (IR64, Apo, Azucena) Stress was applied by withdrawing water from the plots for 9 days. Apo, IR64 Azucena (Katherisan, 2005).

18. Cy3 (green) -> cyber green Control/ well-watered Cy5 (red) -> cyber red Experimental/ stressed cDNA synthesis and fluorescent dye labeling 1. Total RNA isolation 2. cDNA Synthesis 3. Dyes are incorporated

21. IR64 well-watered panicles IR64 stress extract Total RNA cDNA library PCR 15 cycles genes Construction of cDNA library (Katherisan, 2005).

22. Spotting of cDNA library on slides Microtiter plates Glass slides PCR products from >9000 genes 3000 spots per slide Images by Dr. John Bennett, IRRI

23. Hybridize process GeneTAC Hyb station

24. Image processing by Laser scanning The microscope slide containing the microarray is placed inside a microarray scanner, where the slide is scanned with two lasers to detect the bound green and red cDNAs.

25. Slides Scanning 59 K oligo array from BGI, Beijing 10K rice panicle cDNA library printed at IRRI 22K chips from Agilent Images by Dr. John Bennett, IRRI

27. Data analysis What accounts for the varying colors? These actually correspond to the amount of cDNA that binds to the complementary strands on the spot.

28. Data analysis Expressed in both conditions Repressed Induced Merged images R G

29. Expression ratio - Normalization R Test/ Experimental T= ∆ Gene expression G Reference/ Control 600 1200 5600 11600 13000 15500 18000 R G 17500 16500 13500 10900 6500 2500 800 0/0 0.07 0.4 1.0 6.2 22.5 0.03 2.0

30. Microarray Gene Reporting your results The expression ratios for every gene can be organized into a table where each column is a microarray and each row is a gene. This representation however is overwhelming in experiments involving thousands of genes and data.

31. DNA Chips (Microarray) made by Photolithography

32. DNA Chips (Microarray) made by Photolithograpgy

34. DNA Chips (Microarray) Recent improvments: -> use ink-jet printer technology (Agilent) to deliver nucleotides for synthesis -> ”maskless synthesis” by photolitography (NimbleGen Systems) -> ultra-high density -> new technologies: produce longer oligos (50-100 bp)

35. DNA Microarray Direct comparision of expression patterns possible -> expression level of which genes changes during wound healing -> which genes are involved in which biochemical and cellular pathway

36. Gene expression and cancer • Hierarchical clustering • Method for analyzing microarray data • Gene level analysis • Experiment level analysis

37. DNA diagnostics • Uses of microarrays in cancer research and diagnosis. • 2733 papers published on microarrays and cancer • 1038 papers published on microarrays, gene expression, cancer diagnosis • Gene expression profiling • Identify genes involved in cancer diagnosis. • Identify gene expression patterns that are associated with disease outcome. • Gene content analysis • Identify genomic regions that are lost or amplified in tumors.

38. Identifying replication origins in yeast • Only 5% of the genome previously screened for replication origins. • Used known replication initiation factors to perform ChIP/chip analysis • Identified hundreds of additional replication origins in a single experiment.

39. Chromatin immunoprecipitation assay (ChIP) -> to analyze mechanism of control of gene expression -> binding of transcription factors (DNA binding proteins) ChIP used to analyse a lot of TF at the same time ->In vivo experiment -> can be used to test complexity of the cell

40. Chromatin immunoprecipitation assay (ChIP)

41. DNA – Protein Interaction - Microarray • Identification of DNA regions bound by a protein. • Compare a wild-type strain to a ∆gene (DNA-binding protein). • Do not need any prior knowledge of the sequence the protein binds. Iyer et al. 2001 Nature, 409:533-538

42. DNA Microarray • Problems: • signal based on hybridization -> cross-hybridization (specially with DNA chips) • If expression level high in cell -> maybe not enough targets for hybridization available -> signal not proportional to mRNA level • -> better method to analyse expression level: • High-Throughput Sequencing of cDNA or small fragments of cDNA • -> Sequences are analyzed -> number of times a sequence is present -> expression level

44. Why study proteins? • They are the machines that make cells function. • RNA levels do not always accurately predict protein levels. • Often processes are regulated at the transcriptional level. • Some processes are controlled post-transcriptionally. • Proteins are the targets of drugs.

45. Protein-protein array Different type arrays Protein-activity array Small molecule array

46. Protein microarrays • Analysis of thousands of proteins at one time. • Many different types • Antibody arrayed - detect many proteins • Proteins arrayed - detect interacting proteins • Proteins arrayed - detect interacting small molecules • Proteins arrayed - detect enzymatic activity (Enzyme profiling) • Peptides arrayed – substrate for enzymes, interaction • Small molecules arrayed – detect enzymatic activity (enzyme profiling) • Etc.

47. Requirements • Proper folding and orientation of immobilized proteins or small molecules • Solvent (presence of ions, hydrophilicity etc.) • Cofactors • pH • Temperature • This makes protein/peptide immobilization more difficult than DNA immobilization

48. Comparison DNA-Protein Microarray: Templin et al. 2002 Trend in Biotch. Vol 20

49. Quality Images Actual Spots Boundary, Regional Background Correction

50. Noncovalent Immobilization Physical adsorption Mainly hydrophobic interactions Nonspecific Capturing methods Biotin-Avidin  site-specific Antibody-antigen  site-specificity depends on method Antibody developed against enzyme Antibody developed against antigen bound to enzyme Polyclonal  several epitopes; monoclonal  one epitope His-tag binding to nickel-nitrilotriacetic acid Entrapment in gels Immobilized molecules in aqueous environment Long incubation time required