Why tethered-ligand technology?

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
3
Chemical labeling can change protein-target
interactions
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

6
Miyatas Antigen-Ab hydrogels
Nature 399, 766 - 769 (1999)
7
What is fluorescence resonance transfer (FRET)
technology?
Ro

• Energy transfer between two dye molecules
• Ro distance is typically between 40 to 90
  angstroms
• Commonly used technique

Dye 1 Dye 2
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 2nd antibody
• In selected locations, bind biotinated ligand
  1st antibody complex
• Addition of unlabeled ligand will abolish Avidin
  2nd 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