Development of Self-Sorting G-Protein Coupled Receptor Microarrays

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1 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

Vörös 1 1 Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and

2 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 Capture biomolecule 1

other array types Proteins are more sensitive to external perturbations,

3 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) 2

GPCRs are the target of 40% of the prescription drugs There are over 700 different GPCRs but not

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

5 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

environment to preserve the functionality Arrays of

6 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

(ssdna) are the linkers for vesicle binding A universal DNA array is

7 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

strong linker with multiplexing possiblities Gentle protein immobilization,

8 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

between the vesicle and the DNA tag is required for surface sorting An inert

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

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

11 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

protein and vesicle adsorption PLL-g-PEG can be functionalized with a

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

13 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 B. Stadler, M. Bally et al. Biointerphases, 1(200) pp

B. Stadler, et al. Langmuir, 20 (2004), pp. 11348-1134 B.

14 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 B. Stadler, M. Bally et al. Biointerphases, 1(200) pp

Neutravidin Ta 2 O B. Stadler, et al. Langmuir, 20 (2004), pp.

15 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

internal reflection of a laser beam G.L. Duveneck et al.

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

17 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

18 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

reference 1 mm1 mm Maleimide-thiol linkage Control

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

20 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 Cryo-TEM 12

Liposome are observed Muscarinic receptor (M 2 R) Sf 9 cell

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

22 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

23 GPCR liposomes: Site specific immobilization 1

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

25 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

26 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 Ligand binding (fmoles/mg protein) [ 3 H-Scopolamine]pM 17

27 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) [ 3 H-Scopolamine]pM W. Leifert et al., Mol. Membr. 22 (200), pp fmoles 3 S-GTPγS bound Basal Agonist Antagonist 17

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

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

30 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

31 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 NMPP-NM & ETH Zürich - Project NANOCUES, FP-NMP CSIRO, Emerging Science Area for Nanotechnology funding scheme - OzNano2Life/DEST 20

32 Thank you! Contact Name: Marta Bally

UNIVERSITY OF WROCŁAW

BIO-IMAGing in research INnovation and Education UNIVERSITY OF WROCŁAW Faculty of Biotechnology Tamka 2, Przybyszewskiego 63/77, WROCŁAW, POLAND Legend I. Confocal system (LSM510 META - Zeiss) - Laboratory