1. Why tethered-ligand technology? It is easy to bind targets to microarrays, but in order to detect interactions, the fluorescence of a spot must either increase or decrease Detection is easy for genomic microarrays – standard DNA/RNA enzymatic methods amplify and fluorescently label nucleic acid samples Detection is very difficult for proteomic microarrays –no equivalent enzymatic amplification and duplication methods Direct chemical labeling methods are unsatisfactory

2. Labeling: genomic vs proteomic Genomic microarray Sample: Amplification and labeling Binding: Proteomic microarray

3. Chemical labeling can change protein-target interactions Sample: Binding: Labeling: Without labelingWith labeling

4. How to detect binding of unlabeled proteins and ligands? Want to simply take a cell extract or diagnostic sample, dump it on a microarray, and go The microarray itself must contain the detection means in an inactive state

5. Tethered-ligand detection techniques Relies on fluorescence resonance transfer (FRET) technology An FRET labeled receptor- ligand reagent is prebound to the microarray Analytes are detected as they perturb the bound receptor-ligand pair Tether Receptor Ligand

6. Miyata’s Antigen-Ab hydrogels Nature 399, 766 - 769 (1999)

7. What is fluorescence resonance transfer (FRET) technology? Dye 1 Dye 2 RoRo Energy transfer between two dye molecules R o distance is typically between 40 to 90 angstroms Commonly used technique

8. Tethered-ligand chemistry Here, a labeled ligand is displaced from a receptor by an unlabeled “test sample” ligand

9. Enzymatic detection -P Here, a kinase phosphorylates a group, abolishing target binding, and creating a fluorescent signal.

10. General purpose reagent platform? Cover microarray surface with FRET coupled Avidin and 2 nd antibody In selected locations, bind biotinated ligand – 1 st antibody complex Addition of unlabeled ligand will abolish Avidin – 2 nd antibody FRET

11. Tetered-ligand patent application 20030108972 A1 Concept of tethered-ligand methods for all microarray applications All detection methodologies and enzymatic types claimed Specific applications for proteases, kinases, sepsis, apoptosis, angiogenesis