Development of Self-Sorting G-Protein Coupled Receptor Microarrays

Development of Self-Sorting G-Protein Coupled Receptor Microarrays M. Bally 1, K. Bailey 2,3, B. Städler 1 and J. Vörös 1 1 Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and University Zürich, Switzerland 2 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide SA, Australia 3 CSIRO Molecular and Health Technologies, Adelaide SA, Australia

Background Microarrays are high-throughput tools for drug discovery, therapeutics and diagnostics After the success of DNA arrays, much effort is made to develop other array types Proteins are more sensitive to external perturbations, especially membrane proteins Target (sample) Fluorophore www.nature.com Capture biomolecule 1

Motivation: membrane protein arrays High throughput screening techniques for the characterization of membrane proteins are needed Membrane proteins such as e.g. GPCRs are the target of 40% of the prescription drugs There are over 700 different GPCRs but not all the endogenous ligands have been identified yet (there are still 120 orphan GPCRs) http://www.rikenresearch.riken.jp www.nature.com 2

Goal: a functional membrane protein array library.thinkquest.org Immobilize membrane proteins in their natural lipidic environment to preserve the functionality 3

Goal: a functional membrane protein array library.thinkquest.org Immobilize membrane proteins in their natural lipidic environment to preserve the functionality Arrays of synthetic phospholipid vesicles Arrays of whole cell membrane extracts 3

Goal: from DNA array to heterogeneous vesicle array Vesicle tagged with ssdna ssdna X DNA microarray vesicle microarray Single-stranded DNAs (ssdna) are the linkers for vesicle binding A universal DNA array is converted into a heterogeneous functional vesicle array by surface sorting 4

Goal: from DNA array to heterogeneous vesicle array Vesicle tagged with ssdna ssdna X DNA microarray vesicle microarray Oligonucleotides provide a strong linker with multiplexing possiblities Gentle protein immobilization, no drying steps No need to optimize the spotting conditions for each type of array 4

Goal: from DNA array to heterogeneous vesicle array Vesicle tagged with ssdna ssdna Stable linker Inert Background DNA microarray A stable linker between the vesicle and the DNA tag is required for surface sorting An inert background is required to avoid high background noise and false positive response 4

Content Surface modification strategy & DNA microarrays Heterogeneous synthetic vesicle arrays Membrane extract arrays Conclusions

Content Surface modification strategy & DNA microarrays Heterogeneous synthetic vesicle arrays Membrane extract arrays Conclusions

DNA arrays: inert background Poly(L-Lysine)-grafted-Poly(Ethylene Glycol) Biotin Surfaces coated with PLL-g-PEG are resistant against unspecific protein and vesicle adsorption PLL-g-PEG can be functionalized with a bioactive ligand, e.g. biotin Metal oxide N.-P. Huang, et al. Langmuir, 18 (2002), pp. 220-230

PLL-g-PEG-based DNA microarrays Biotinylated PLL-g-PEG Ta 2 O B. Stadler, et al. Langmuir, 20 (2004), pp. 11348-1134 B. Stadler, M. Bally et al. Biointerphases, 1(200) pp. 142-14 7

PLL-g-PEG-based DNA microarrays Biotinylated ssdna Biotinylated PLL-g-PEG Streptavidin / Neutravidin Ta 2 O B. Stadler, et al. Langmuir, 20 (2004), pp. 11348-1134 B. Stadler, M. Bally et al. Biointerphases, 1(200) pp. 142-14 7

PLL-g-PEG-based DNA microarrays DNA-tagged vesicle Biotinylated ssdna Biotinylated PLL-g-PEG Streptavidin / Neutravidin Ta 2 O B. Stadler, et al. Langmuir, 20 (2004), pp. 11348-1134 B. Stadler, M. Bally et al. Biointerphases, 1(200) pp. 142-14 7

Microarray imaging: ZeptoREADER Microarray reader using planar waveguide technology Surface bound fluorophores are excited by the evanescent field generated by total internal reflection of a laser beam G.L. Duveneck et al., Analytica Chimica Acta 49 (2002), pp 49 1 M. Bally et al., Surface and Interface Analysis, 38 (200): p. 1442-148. 8

Content Surface modification strategy & DNA microarrays Heterogeneous synthetic vesicle arrays Membrane extract arrays Conclusions 9

Heterogeneous functional synthetic vesicles arrays Annexin V Cy3 DOPS Bovine Serum Albumin 47 Maleimide-thiol linkage Ta 2 O O Maleimide O HS N DNA O S DNA N O 10

Heterogeneous functional synthetic vesicles arrays Annexin V Cy3 DOPS Bovine Serum Albumin 47 DNA1 DNA2 reference 1 mm1 mm Maleimide-thiol linkage Control (streptavidin) Ta 2 O Maleimide O HS N O DNA O S DNA N O 10

Content Surface modification strategy & DNA microarrays Heterogeneous synthetic vesicle arrays Membrane extract arrays Conclusions 11

GPCR liposomes Membrane extracts were isolated from sf9 (spodoptera frugiperda) overexpressed the GPCR of interest. Liposome are observed Muscarinic receptor (M 2 R) Sf 9 cell expressing a GPCR Membrane extract 200 nm W. Leifert et al., Mol. Membr. 22 (200), pp 07 47. Cryo-TEM 12

GPCR liposomes: surface immobilization 13 GPCR Liposome Cholesterol-ssDNA (cdna) Biotinylated-ssDNA Neutravidin Biotinylated PLL-g-PEG Cholesterol-ssDNA

Quartz crystal microbalance with dissipation (QCM-D) In situ monitoring of mass changes in thin films Measures crystal resonance frequency and crystal oscilliation damping Frequency shift ~ Mass changes Energy dissipation ~ Viscoelasticity f 1 > f 2 Bare crystal Adlayer Rigid adlayer Soft adlayer 14

GPCR liposomes: Site specific immobilization 1

GPCR liposomes: Site specific immobilization - cdna + cdna + cdna - cdna 1

GPCR microarrays: Fluorescent ligand binding BODIPY-pirenzepine DNA1 DNA2 ref 0 μm ZeptoREADER 0 μm Confocal microscopy Unspecific signal on the spots presenting non-complementary DNA <20% 1

GPCR liposomes: Functionality Ligand binding properties are preserved 1 Ligand outside 2 G-proteins activation GDP 2 GTP α β γ inside 3 Rearrangement / separation 7000 000 Ligand binding (fmoles/mg protein) 000 4000 3000 2000 1000 0 0 1000 2000 3000 4000 000 000 7000 [ 3 H-Scopolamine]pM 17

GPCR liposomes: Functionality Ligand binding properties are preserved 1 Agonist outside Receptor signaling activity is preserved 2 G-proteins activation GDP 2 GTP α 3 β γ inside Rearrangement / separation Ligand binding (fmoles/mg protein) 7000 000 000 4000 3000 2000 1000 0 0 1000 2000 3000 4000 000 000 7000 [ 3 H-Scopolamine]pM W. Leifert et al., Mol. Membr. 22 (200), pp 07 47. fmoles 3 S-GTPγS bound 2.0 1. 1.0 0. 0.0 Basal Agonist Antagonist 17

GPCR microarrays: Fluorescent ligand binding BODIPY-pirenzepine No GPCR GPCR 18

Content Surface modification strategy & DNA microarrays Heterogeneous synthetic vesicle arrays Membrane extract arrays Conclusions

Conclusions A DNA array for vesicle immobilization is presented Parallel sorting of synthetic vesicles from solution is possible DNA directed immobilization of liposomes containing GPCRs is achieved GPCR extracts preserve their functionality both in solution and on the surface 19

Acknowledgement Connie Darmanin Prof. Alfred Gilman (University of Texas, USA) Prof. Ted Mc Murchie, Dr. Wayne Leifert (CSIRO Molecular and Health Technologies, Australia) Dr. Ekkehard Kauffmann (Zeptosens - A Division of Bayer (Schweiz) AG, Switzerland) Funding: - CTI, no. 7241.1 NMPP-NM & ETH Zürich - Project NANOCUES, FP-NMP-2002-3.4.1.2-1 - CSIRO, Emerging Science Area for Nanotechnology funding scheme - OzNano2Life/DEST 20

Thank you! Contact Name: Marta Bally Email: bally@biomed.ee.ethz.ch www.lbb.ethz.ch