U.S. patent application number 10/341215 was filed with the patent office on 2003-07-24 for arrays of biological membranes and methods and use thereof.
Invention is credited to Fang, Ye, Jonas, Steven J., Kalal, Peter J., Lahiri, Joydeep, Wang, Wei.
Application Number | 20030138853 10/341215 |
Document ID | / |
Family ID | 46204127 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138853 |
Kind Code |
A1 |
Lahiri, Joydeep ; et
al. |
July 24, 2003 |
Arrays of biological membranes and methods and use thereof
Abstract
The present invention overcomes the problems and disadvantages
associated with prior art arrays by providing an array comprising a
plurality of biological membrane microspots associated with a
surface of a substrate that can be produced, used and stored, not
in an aqueous environment, but in an environment exposed to air
under ambient or controlled humidities. Preferably, the biological
membrane microspots comprise a membrane bound protein. Most
preferably, the membrane bound protein is a G-protein coupled
receptor, an ion channel or a receptor tyrosine kinase.
Inventors: |
Lahiri, Joydeep; (Painted
Post, NY) ; Fang, Ye; (Painted Post, NY) ;
Jonas, Steven J.; (Ann Arbor, MI) ; Kalal, Peter
J.; (Corning, NY) ; Wang, Wei; (Pittsford,
NY) |
Correspondence
Address: |
CORNING INCORPORATED
SP-TI-3-1
CORNING
NY
14831
|
Family ID: |
46204127 |
Appl. No.: |
10/341215 |
Filed: |
January 13, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10341215 |
Jan 13, 2003 |
|
|
|
09854786 |
May 14, 2001 |
|
|
|
60224135 |
Aug 10, 2000 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
427/2.11; 435/287.2; 436/518 |
Current CPC
Class: |
B01J 2219/00725
20130101; G01N 33/566 20130101; C40B 40/10 20130101; G01N 2500/02
20130101; G01N 33/5438 20130101; B01J 2219/0061 20130101; B01J
2219/00527 20130101; G01N 2333/726 20130101; B01J 2219/00596
20130101; B01J 2219/00619 20130101; B82Y 30/00 20130101; B01J
2219/00497 20130101; B01J 2219/00612 20130101; B01J 2219/00637
20130101; G01N 33/6872 20130101; B01J 2219/00659 20130101; B01J
2219/0074 20130101; B01J 2219/00617 20130101; B01J 2219/00605
20130101; B01J 2219/00585 20130101; B01J 2219/00626 20130101; B01J
2219/0063 20130101 |
Class at
Publication: |
435/7.1 ;
435/287.2; 436/518; 427/2.11 |
International
Class: |
G01N 033/53; B05D
003/00; C12M 001/34; G01N 033/543 |
Claims
We claim:
1. An array comprising a plurality of biological membrane
microspots associated with a surface of a substrate.
2. The array of claim 1, wherein the biological membrane microspots
comprise a membrane bound protein.
3. The array of claim 2, wherein the membrane bound protein is a
G-protein coupled receptor.
4. The array of claim 2, wherein the membrane bound protein is an
ion channel.
5. The array of claim 2, wherein the membrane bound protein is a
receptor tyrosine kinase.
6. The array of claim 1, wherein the substrate comprises glass,
metal, or plastic.
7. The array of claim 1, wherein the substrate is configured as a
chip, a slide or a microplate.
8. The array of claim 1, wherein the surface is coated.
9. The array of claim 8, wherein the coating is a material that
enhances the affinity of the biological membrane microspot for the
substrate.
10. The array of claim 9, wherein the material confers a contact
angle ranging from about 15.degree. to 80.degree..
11. The array of claim 9, wherein the material is a silane, thiol,
or a polymer.
12. The array of claim 9, wherein the thiol is on a substrate
comprising a gold-coated surface.
13. The array of claim 9, wherein the thiol comprises hydrophobic
and hydrophilic moieties.
14. The array of claim 13, wherein the thiol is a thioalkyl
compound.
15. The array of claim 11, wherein the silane is on a substrate
comprising glass.
16. The array of claim 11, where in the silane presents terminal
polar moieties.
17. The array of claim 16, wherein the terminal polar moieties are
hydroxyl, carboxyl, phosphate, sulfonate, or amino groups.
18. The array of claim 16, wherein the surface is positively
charged and contains amino groups.
19. The array of claim 9, wherein the material is
.gamma.-aminopropyl-sila- ne.
20. The array of claim 9, wherein the material is a derivatized
monolayer having covalently bonded linker moieties.
21. The array of claim 20, wherein the monolayer is a self
assembled monolayer.
22. The array of claim 21, wherein the monolayer comprises a
thioalkyl compound or a silane compound.
23. The array of claim 22, wherein the thioalkyl is selected from
the group consisting of a thioalkyl acid, thioalkyl alcohol,
thioalkyl amine, and halogen containing thioalkyl compound.
24. The array of claim 23, wherein the compound is a thioalkyl
acid.
25. The array of claim 24, wherein the thioalkyl compound is
16-mercaptohexadecanoic acid.
26. The array of claim 22, wherein the silane compound is selected
from the group consisting of a silyl anhydride, silyl acid, silyl
amine, silyl alcohol, vinyl silane or silyl acrylate.
27. The array of claim 20, wherein the linker moiety comprises a
straight or branched C.sub.10-C.sub.25 alkyl, alkynyl, alkenyl,
aryl, araalkyl, heteroalkyl, heteroalkynyl, heteroalkenyl,
heteroaryl, heteroaraalkyl molecule comprising: (i) a terminal
functional group capable of reacting with the derivatized
monolayer; (ii) a hydrophilic spacer region; and (iii) a
hydrophobic membrane adhering region.
28. The array of claim 27, wherein the terminal functional group is
selected from the group consisting of a carboxylic acid, halogen,
amine, thiol, alkene, acrylate, anhydride, ester, acid halide,
isocyanate, hydrazine, maleimide and hydroxyl group.
29. The array of claim 27, wherein the hydrophilic spacer region
comprises n oxyethylene groups, wherein n=2 to 25.
30. The array of claim 27, wherein the membrane adhering region
comprises a straight or branched chain C.sub.10-C.sub.25
hydrophobic tail.
31. The array of claim 1, wherein the surface is nano-porous.
32. The array of claim 1, wherein the substrate is selected from
the group consisting of glass, polymeric materials, and metallic
substrates.
33. A method for producing an array comprising: providing a
substrate having a surface; providing a solution a biological
membrane; immersing the tip of a pin into the solution; removing
the tip from the solution to provide a solution adhered to the tip;
contacting the solution with the surface to thereby transfer the
solution from the tip to the surface; and repeating the contacting
step a plurality of times to provide biological membrane microspots
patterned in an array on the surface.
34. The method of claim 33, wherein the solution comprises a
protein.
35. The method of claim 34, wherein the protein is a G-protein
coupled receptor.
36. The method of claim 34, wherein the protein is an ion
channel.
37. The method of claim 33, further comprising the step of
contacting the microspot with a solution comprising a protein.
38. The method of claim 33, wherein the surface of the substrate is
exposed to air under ambient or controlled humidities when the tip
of the pin contacts the substrate.
39. A method for detecting a binding event between a probe and
target compound, said method comprising: contacting a solution
comprising the target compound with an array of probe biological
membrane microspots associated with a surface of a substrate, the
target compound having one or more constituents, and detecting a
binding event between at least one or more of the probes with one
or more of the constituents of the target.
40. The method of claim 39, wherein at least one of the
constituents of the target is labeled and the detection step
comprises detecting the presence of the label.
41. The method of claim 40, wherein the detection of the label is
carried out by imaging based on the radioactivity, fluorescence,
phosphorescence, chemiluminescence, or resonance light scattering
emanating from the bound target.
42. The method of claim 40, further comprising washing the
substrate of unbound target prior to the detection step.
43. The method of claim 39, wherein the array of microspots is
incubated with labeled cognate target, and an unlabeled target
compound and the binding event between the unlabeled target
compound and the probe is determined by measuring a decrease in the
signal of the label due to competition between the cognate labeled
target and the unlabeled target compound for the probe.
44. The method of claim 43, wherein the labeled cognate target is
incubated with the array before incubation with the unlabeled
target.
45. The method of claim 39, wherein the target is unlabeled and
binding event is determined by a change in physical properties at
the interface.
46. The method of claim 45, wherein the change in physical
properties at the interface is a change in refractive index or
electrical impedence.
47. The method of claim 39, wherein the target is unlabeled and the
binding of the target is detected by mass spectroscopy.
48. The method of claim 39, wherein the probe biological membrane
microspots comprises a G-protein coupled receptor.
49. The method of claim 39, wherein the probe biological membrane
microspots comprises a ion channel.
50. The method of claim 39, wherein the probe biological membrane
microspots comprises a receptor tyrosine kinase.
51. An array comprising a plurality of biological membrane
microspots associated with a surface of a glass substrate, wherein
the surface is coated with .gamma.-aminopropyl-silane and the
biological membrane microspots comprise a G-protein coupled
receptor.
Description
BACKGROUND OF THE INVENTION
[0001] DNA microarrays have become an extremely important
bioanalytical tool (e.g. for analyzing gene-expression); protein
microarray technology has, however, lagged behind. The fabrication
of protein arrays is challenging because of difficulties associated
with preserving the folded conformation of proteins in the
immobilized state, and high amounts of non-specific binding to
immobilized proteins. As a large fraction of drug targets are
membrane bound proteins (e.g., G-protein coupled receptors,
ion-channels, etc.), there is an impetusto develop tools for
high-througput screening against membrane bound proteins. Membrane
proteins maintain their folded conformation when associated with
lipids; therefore, to create arrays of such proteins it is
important to first develop surfaces that support the binding of
membranes. Bilayer-lipid membranes adsorbed onto solid supports,
referred to as supported bilayer-lipid membranes, can mimic the
structural and functional role of biological membranes. See
Sackmann, E. Science 1996, 271, 43-48; Bieri, C. et al., Nature
Biotech, 1999, 17, 1105-1108; Groves, J. T. et al., Science 1997,
275, 651-653; Lang, H. et al., Langmuir 1994, 10, 197-210; Plant,
A. L. et al., Langmuir 1999, 15, 5128-5135; and Raguse, B. et al.,
Langmuir 1998, 14, 648-659. These hybrid surfaces were developed to
overcome the fragility of black lipid membranes while preserving
aspects of lateral fluidity observed in native biological
membranes.
[0002] Surfaces binding lipid membranes can be broadly classified
into three categories:
[0003] (i) hydrophobic surfaces (e.g., self-assembled monolayers
presenting terminal methyl groups) which support the adsorption of
lipid monolayers are of limited utility as they cannot be used to
incorporate membrane-spanning proteins (Plant, A. L., Langmuir
1999, 15, 5128-5135);
[0004] (ii) hydrophilic surfaces (e.g., glass surfaces) which bind
bilayer-lipid membranes are also of limited utility as they can
only be used to incorporate membrane-spanning proteins with
extra-membrane domains that are less thicker than the layer of
adsorbed water (.about.10.degree. A) (Groves, J. T. et al., Science
1997, 275, 651-653; and Groves, J. T. et al., Langmuir 1998, 14,
3347-3350); and (iii) amphiphilic surfaces that contain hydrophobic
and hydrophilic portions that bind bilayer-lipid membranes offer
the potential for incorporating a wide variety of membrane-spanning
proteins (Lang, H. et al., Langmuir 1994, 10, 197-210; Raguse, B.
et al., Langmuir 1998, 14, 648-659; and Vanderah, D. J. et al.,
Materials Research Society Fall Meeting Abstracts, Boston,
1999).
[0005] Methods to create arrays of membranes would enable
high-throughput screening of multiple targets against multiple
drug-candidates. Arrays of membranes may be obtained by fabricating
grids of titanium oxide on a glass substrate as titanium oxide
resists the adsorption of lipids (Boxer, S. G. et al. Science 1997,
275, 651-653; and Boxer, S. G. et al. Langmuir 1998, 14,
3347-3350). Micropipeting techniques have been used to spatially
address each corralled lipid-binding region (Cremer, P. S. et al.,
J. Am. Chem. Soc. 1999, 121, 8130-8131). However, these methods are
cumbersome and require the fabrication of patterned surfaces. To
make membrane arrays by printing membranes on unpatterned surfaces,
it would be necessary to confine the membrane to the printed areas
without lateral diffusion of the membrane molecules to the
unprinted areas. Boxer et al. demonstrated that it was possible to
pattern lipids on glass surfaces by microcontact printing using
poly-dimethylsiloxane (PDMS) stamps "inked" with
phosphatidylcholine ("PC"). They attributed the lateral confinement
of the lipids to the stamped regions, to the self-limiting
expansion of PC membranes to .about.106% of the original printed
areas (Hovis, J. et al., Langmuir 2000, 16, 894-897). The methods
used by Boxer et al., however, have certain limitations. First,
Boxer and co-workers carried out the stamping of lipids on surfaces
immersed under water (Hovis 2000). Second, lipids adsorbed on the
bare-glass substrates used by Boxer and co-workers spontaneously
desorbed when drawn through an air-water interface (Cremer 1999).
Cremer et al., propose in WO01/20330 the use of spatially addressed
lipid bilayer arrays that remain submerged underwater to preserve
the planar support structure. Such systems may not be practical for
robust, high throughput, microarray based assays.
SUMMARY OF THE INVENTION
[0006] The present invention overcomes the problems and
disadvantages associated with prior art arrays by providing an
array comprising a plurality of biological membrane microspots
associated with a surface of a substrate that can be produced, used
and stored, not in an aqueous environment, but in an environment
exposed to air under ambient or controlled humidities. Preferably,
the biological membrane microspots comprise a membrane bound
protein. Most preferably, the membrane bound protein is a G-protein
coupled receptor, an ion channel or a receptor tyrosine kinase.
[0007] The substrate for use in the array of the present invention
can comprise glass, silicon, metal or polymeric materials. The
substrate can be configured as a chip, a slide or a microplate.
[0008] In certain embodiments, the surface of the substrate is
coated. Preferably, the coating is a material that enhances the
affinity of the biological membrane microspot for the substrate.
Most preferred coating material confers a contact angle ranging
from about 15.degree. to 80.degree..
[0009] The coating material can be a silane, thiol, or a polymer.
Preferably, when the material is a thiol, the substrate comprises a
gold-coated surface. Preferably, the thiol comprises hydrophobic
and hydrophilic moieties. Most preferably, the thiol is a thioalkyl
compound.
[0010] Preferably, when the coating material is a silane, the
substrate comprises glass. Preferably, the silane presents terminal
polar moieties including, for example, hydroxyl, carboxyl,
phosphate, sulfonate, or amino groups. A most preferred silane
coating material .gamma.-aminopropyl-silane.
[0011] In an alternative embodiment, the coating material is a
derivatized monolayer (or several monolayers) having covalently
bonded linker moieties. Most preferably, the monolayer comprises a
thioalkyl compound or a silane compound.
[0012] Preferably, the thioalkyl compound is selected from the
group consisting of a thioalkyl acid, thioalkyl alcohol, thioalkyl
amine, and halogen containing thioalkyl compound. Most preferably,
the thioalkyl compound is a thioalkyl acid, for example,
16-mercaptohexadecanoic acid.
[0013] Preferably, the silane compound is selected from the group
consisting of a silyl anhydride, silyl acid, silyl amine, silyl
alcohol, vinyl silane or silyl acrylate.
[0014] The bonded linker moiety can comprises a straight or
branched C.sub.10-C.sub.25 alkyl, alkynyl, alkenyl, aryl, araalkyl,
heteroalkyl, heteroalkynyl, heteroalkenyl, heteroaryl,
heteroaraalkyl molecule that in turn includes:
[0015] (i) a terminal functional group capable of reacting with the
derivatized monolayer;
[0016] (ii) a hydrophilic spacer region; and
[0017] (iii) a hydrophobic membrane adhering region.
[0018] The preferably, the terminal functional group is selected
from the group consisting of a carboxylic acid, halogen, amine,
thiol, alkene, acrylate, anhydride, ester, acid halide, isocyanate,
hydrazine, maleimide and hydroxyl group. The hydrophilic spacer
region preferably comprises n oxyethylene groups, wherein n=2 to
25. The membrane adhering region preferably comprises a straight or
branched chain C.sub.10-C.sub.25 hydrophobic tail.
[0019] In further alternative embodiments the surface is
nano-porous.
[0020] The present invention also provides a method for producing
an array of biological membranes. The method comprises the steps of
providing a substrate having a surface; providing a solution of a
biological membrane (as used herein a "solution of a biological
membrane" also includes a suspension of a biological
membrane);immersing the tip of a pin into the solution; removing
the tip from the solution to provide a solution adhered to the tip;
contacting the solution with the surface to thereby transfer the
solution from the tip to the surface; and repeating the contacting
step a plurality of times (at least twice) to provide biological
membrane microspots patterned in an array on the surface.
Typically, the surface of the substrate is exposed to air under
ambient or controlled humidities when the tip of the pin contacts
the substrate.
[0021] In a preferred embodiment, the solution comprises a protein.
Preferably, the solution comprises a membrane bound protein. Most
preferably, the membrane bound protein is a G-protein coupled
receptor (GPCR), an ion channel or a receptor tyrosine kinase. In
certain embodiments, the protein contains a mutation, e.g. a point
mutation. In other embodiments, the solution comprises multiple
proteins.
[0022] In an alternative embodiment, the method includes the
additional step of contacting the microspot with a solution
comprising a protein.
[0023] The present invention further provides for detecting a
binding event between a probe array and target compounds. The
method comprises contacting a solution comprising the target
compound with an array of probe biological membrane microspots
associated with a surface of a substrate, and detecting a binding
event between at least one or more of the probe microspots with one
or more of the constituents of the target. Preferably, at least one
of the constituents of the target is labeled and the detection step
comprises detecting the presence of the label. The detection of the
label is preferably carried out by imaging based on the
radioactivity, fluorescence, phosphorescence, chemiluminescence, or
resonance light scattering emanating from the bound target. The
substrate can be washed to remove unbound target prior to the
detection step.
[0024] In an alternative embodiment, the array of microspots is
incubated with labeled cognate target, and an unlabeled target
compound and the binding event between the unlabeled target
compound and the probe is determined by measuring a decrease in the
signal of the label due to competition between the cognate labeled
target and the unlabeled target compound for the probe. Preferably,
the labeled cognate target is incubated with the array before
incubation with the unlabeled target. In other embodiments, the
target is unlabeled and binding event is determined by a change in
physical properties at the interface or by mass spectroscopy.
Preferably, the change in physical properties at the interface is a
change in refractive index or electrical impedance.
[0025] Biosensors and diagnostic devices that comprise the arrays
of the invention are also contemplated by the present
invention.
DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows a top view of an array of the present
invention.
[0027] FIGS. 2(A) and 2(B) show fluorescence images of 1.times.5
arrays of microspots of the .beta.-adrenergic receptor (subtype 1)
incubated with solutions of BT-TMR CGP12177 (5 nM). In FIG. 2(A)
the image corresponds to an array that was stored for 7 days at
4.degree. C. in a container saturated with water vapor. In FIG.
2(B) the images correspond to arrays that were stored for 1,6, and
14 days in a dessicator at 4.degree. C.
[0028] FIGS. 3(A) and 3(B) are fluorescent images of microarrays of
the present invention. FIG. 3(A) fluorescence images of microarrays
of DMP/DPPC (1:4) lipids doped with FITC-DHPE (2%) on GAPS slides
that were subject to repeated immersions in buffer and withdrawn
through air-water interfaces. (I) Fluorescence image of the lipid
array immersed in buffer. (II) Fluorescence image of the array
immersed in buffer after being withdrawn five times through an
air-water interface. (III) Fluorescence image of the same array
immersed in buffer after being withdrawn five more times through an
air-water interface. (IV) Fluorescence image of the array in air
after drying. (V) Fluorescence image of the same array under buffer
after reimmersion. FIG. 3(B) fluorescence images of microarrays of
egg PC (1:4) lipids doped with FITC-DHPE (2%) on GAPS slides that
were subject to repeated immersions in buffer and withdrawn through
air-water interfaces, as described above for (I)-(V). The data were
collected using a ScanArray 5000 scanner. The buffer was used was
50 mM sodium phosphate, pH 7.5.
[0029] FIGS. 4(A)-4(E) show fluorescence images of GPCR arrays, in
which each array contains three columns and each column consists of
five replicate microspots. Each column of microspots corresponds to
a different GPCR. From left to right, these receptors are the
.beta.-adrenergic receptor subtype I (.beta.1), the neurotensin
receptor subtype I (NTR1), and the dopamine receptor subtype I
(D1). FIG. 4(A) fluorescence image of an array incubated with
binding buffer only; this image serves as a negative control. FIG.
4(B) fluorescence image of a second array incubated with a solution
of BT-NT (1 nM). FIG. 4(C) fluorescence image of an array incubated
with a solution of BT-NT (1 nM) and CGP12177 (1 .mu.M). FIG. 4(D)
fluorescence image of an array incubated with a solution of BT-NT
(1 .mu.M) and SCH23390 (1 .mu.M). FIG. 4(E) fluorescence image of
an array incubated with a solution of BT-NT (1 nM) and neurotensin
(1 .mu.M). CGP12177 and SCH23390 are ligands that are known not to
bind to NTR1 receptors; neurotensin is the cognate ligand for
NTR1.
[0030] FIGS. 5(A) and 5(B) show fluorescent images of arrays of the
present invention. FIG. 5(A) fluorescence images of 1.times.5
arrays of microspots of NTR1 incubated in solutions containing
different concentrations of BT-neurotensin, as indicated in the
figure. FIG. 5(B) fluorescence images of 1.times.5 arrays of
microspots of the galanin receptor incubated in solutions
containing different concentrations of cy5-labeled antagonist D, as
indicated in the figure. The binding buffer was 50 mM Tris-HCl, 10
mM MgCl.sub.2, 2 mM EDTA, 0.1% BSA, at pH 7.4.
[0031] FIG. 6 shows fluorescence images of NTR1 receptor arrays
incubated in solutions containing BT-neurotensin at fixed
concentration (2 nM) and unlabeled neurotensin at different
concentrations in the binding buffer (FIG. 6(A)).
[0032] FIG. 7 shows the concentrations of neurotensin used in the
example illustrated in FIG. 6.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Biological membrane arrays, as well as methods for their
preparation and use, are provided. In the arrays of the present
invention, a plurality of biological membrane probe spots are
stably associated with the surface of a solid support. The arrays
of the present invention find particular use in identification of
ligands for membrane bound proteins, such as G-protein coupled
receptors. Additionally, the arrays of the present invention offer
tremendous possibilities for high-throughput screening of multiple
membrane bound targets against multiple drug-candidates, thereby
greatly accelerating the process of drug discovery. In further
describing the subject invention, the arrays themselves are first
discussed, followed by a description of methods for their
preparation. Next, a review of representative applications in which
the subject arrays may be employed is provided.
[0034] It is to be understood that the invention is not limited to
the particular embodiments of the invention described below, as
variations of the particular embodiments may be made and still fall
within the scope of the appended claims. It is also to be
understood that the terminology employed is for the purpose of
describing particular embodiments, and is not intended to be
limiting. Instead, the scope of the present invention will be
established by the appended claims.
[0035] In this specification and the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0036] Arrays of the Present Invention
[0037] As illustrated in FIG. 1, the array (10) of the present
invention includes a substrate (12) having a surface (14) having a
plurality of biological membrane probe microspots (16) covering the
surface (14). Each probe microspot on the array comprises a
biological membrane of known composition and, in preferred
embodiments, comprise a membrane bound protein. The microspot may
comprise multiple different proteins. For example, two different
proteins involved in a heterodimer pair can be included in one
microspot. The probe microspots on the array may be any convenient
shape, but will typically be circular, elliptoid, oval, annular, or
some other analogously curved shape, where the shape may, in
certain embodiments, be a result of the particular method employed
to produce the array. The density of the all of the microspots on
the surface of the substrate, i.e. both probe spots and non-probe
spots, e.g. calibration spots, control spots, etc., is at least
about 5/cm.sup.2 and usually at least about 10/cm.sup.2 but does
not exceed about 1000/cm.sup.2, and in many embodiments does not
exceed about 500/cm.sup.2, where in certain preferred embodiments,
the density does not exceed about 400/cm.sup.2, usually does not
exceed about 300/cm.sup.2, and more usually does not exceed about
60/cm.sup.2. The microspots may be arranged in any convenient
pattern across or over the surface of the array, such as in rows
and columns so as to form a grid, in a circular pattern, and the
like, where generally the pattern of spots will be present in the
form of a grid across the surface of the solid support.
[0038] In the arrays of the present invention, the microspots are
stably associated with the surface of a substrate. By "stably
associated" is meant that the biological membranes of the spots
maintain their position relative to the substrate under binding and
washing conditions, e.g., the membrane remains adsorbed when drawn
through an air-water interface. As such, the biological membranes
which make up the spots can be non-covalently or covalently stably
associated with the substrate surface. Examples of non-covalent
association include non-specific adsorption, binding based on
electrostatic (e.g. ion, ion pair interactions), hydrophobic
interactions, hydrogen bonding interactions, surface hydration
forces and the like. Examples of covalent binding include covalent
bonds formed between the spot biological membranes and a functional
group-present on the surface of the substrate, e.g. --NH.sub.2,
where the functional group may be naturally occurring or present as
a member of an introduced coating material. In another example,
histidine-tagged mutations of GPCRs or membrane proteins can bind
to Ni-presenting surfaces through chelating bonds.
[0039] Typically, when the biological membrane microspot comprises
a membrane bound protein, only one type of protein is included in
each microspot of the array. However, in certain situations more
than one type of protein is included in each microspot. For
example, some GPCRs heterodimerize for their biological functions.
(Angers, S. et al., Proc. Natl. Acad. Sci. USA, 2000, 97,
3684-3689.) In a preferred embodiment of the array, the protein
included in the microspot differs from the protein included on a
second microspot of the same array. In such an embodiment, a
plurality of different proteins are present on separate microspots
of the array. Typically the array comprises at least about ten
different proteins. Preferably, the array comprises at least about
50 different proteins. More preferably, the array comprises at
least about 100 different proteins. Alternative preferred arrays
comprise more than about 10.sup.3 different proteins or more than
about 10.sup.4 different proteins. The array may even optionally
comprise more than about 10.sup.5 different proteins.
[0040] In one embodiment of the array, each of the microspots of
the array comprises a different protein. For instance, an array
comprising about 100 microspots could comprise about 100 different
proteins. Likewise, an array of about 10,000 microspots could
comprise about 10,000 different proteins. In an alternative
embodiment, however, each different protein is included on more
than one separate microspot on the array. For instance, each
different protein may optionally be present on two to six different
microspots. An array of the invention, therefore, may comprise
about three-thousand microspots, but only comprise about one
thousand different proteins since each different protein is present
on three different microspots.
[0041] In a further alternative embodiment, the array comprises
identical microspots or a series of identical microspots that in
use are treated with a different analyte (target). For example, an
array of the invention can include a "mini array" of 20 microspots,
each microspot containing a different membrane bound protein,
wherein the mini array is repeated 20 times as part of the larger
array.
[0042] In another embodiment of the present invention, although the
protein of one microspot is different from that of another, the
proteins are related. In a preferred embodiment, the two different
proteins are members of the same protein family. The different
proteins on the invention array may be either functionally related
or just suspected of being functionally related. In another
embodiment of the invention array, however, the function of the
immobilized proteins may be unknown. In this case, the different
proteins on the different microspots of the array share a
similarity in structure or sequence or are simply suspected of
sharing a similarity in structure or sequence. Alternatively, the
proteins may be fragments of different members of a protein
family.
[0043] Substrate
[0044] The substrates of the subject arrays comprise at least one
surface on which the pattern of probe spots is present, where the
surface may be smooth or substantially planar, or have
irregularities, such as depressions or elevations. The surface on
which the pattern of spots is present may be modified with one or
more different layers of compounds that serve to modify the
properties of the surface in a desirable manner and will be
discussed in more detail below. The surface may also be
nano-porous.
[0045] The substrate may consist of a ceramic substance, a glass, a
metal, a crystalline material, a plastic, a polymer or co-polymer,
any combinations thereof, or a coating of one material on another.
For example, but not limited to, (semi) noble metals such as gold
or silver; glass materials such as soda glass, pyrex glass, vycor
glass, quartz glass; metallic or non-metallic oxides; silicon,
monoammonium phosphate, and other such crystalline materials;
transition metals; plastics or polymers, including dendritic
polymers, such as poly(vinyl chloride), poly(vinyl alcohol),
poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride),
poly(dimethylsiloxane) monomethacrylate, polystyrenes,
polypropylene, polyethyleneimine; copolymers such as poly(vinyl
acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride),
poly(ethylene-co-acrylic acid) or the like.
[0046] The substrate may take a variety of configurations ranging
from simple to complex, depending on the intended use of the array.
Thus, the substrate could have an overall slide or plate
configuration, such as a rectangular or disc configuration. In many
embodiments, the substrate will have a rectangular cross-sectional
shape, having a length of from about 10 mm to 200 mm, usually from
about 40 to 150 mm and more usually from about 75 to 125 mm and a
width of from about 10 mm to 200 mm, usually from about 20 mm to
120 mm and more usually from about 25 to 80 mm, and a thickness of
from about 0.01 mm to 5.0 mm, usually from about 0.1 mm to 2 mm and
more usually from about 0.2 to 1 mm.
[0047] Coating Material
[0048] An array of the present invention may optionally further
comprise a coating material on a portion of the substrate
comprising the probe microspots. Preferably the coating material
enhances the affinity of the biological membrane microspot for the
substrate. Most preferably, the coating material confers a contact
angle ranging from about 15.degree. to 80.degree..
[0049] In one embodiment, the coating material is a silane, thiol,
or a polymer. Preferably, when the material is a thiol, the
substrate comprises a gold-coated surface. Preferably, the thiol
comprises hydrophobic and hydrophilic moieties. Most preferably,
the thiol is a thioalkyl compound.
[0050] Preferably, when the coating material is a silane, the
substrate comprises glass. Preferably, the silane presents terminal
polar moieties including, for example, hydroxyl, carboxyl,
phosphate, sulfonate, or amino groups. A most preferred silane
coating material .gamma.-aminopropyl-silane.
.gamma.-aminopropyl-silane coated slides (CMT-GAPS.TM. glass
slides) are available commercially from Corning Inc.
[0051] In an alternative embodiment, the coating material is a
derivatized monolayer or multilayer having covalently bonded linker
moieties. The monolayer coating, for example, comprising of long
chain hydrocarbon moieties, may have for example, but not limited
to, thiol (e.g., thioalkyl), disulfide or silane groups that
produce a chemical or physicochemical bonding to the substrate. The
attachment of the monolayer to the substrate may also be achieved
by non-covalent interactions or by covalent reactions.
[0052] Preferably, the thiol is a thioalkyl compound and is
selected from the group consisting of a thioalkyl acid, thioalkyl
alcohol, thioalkyl amine, and halogen containing thioalkyl
compound. Most preferably, the thioalkyl compound is a thioalkyl
acid, for example, 16-mercaptohexadecanoic acid. Such compounds can
be readily synthesized and/or purchased from commercial
sources.
[0053] After attachment to the substrate the monolayer has at least
one reactive functional group. Examples of reactive functional
groups on the monolayer coating are, but not limited to, carboxyl,
isocyanate, halogen, amine or hydroxyl groups. In one embodiment,
these reactive functional groups on the monolayer coating may be
activated by standard chemical techniques to corresponding
activated functional groups on the monolayer coating (for example,
conversion of carboxyl groups to anhydrides or acid halides, etc.).
The activated functional groups of the monolayer coating on the
substrate may be, but not limited to, anhydrides,
N-hydroxysuccinimide esters or other common activated esters or
acid halides, for covalent coupling to terminal amino groups of the
linker compound. In another embodiment, the activated functional
groups on the monolayer coating may be, but not limited to,
anhydride derivatives for coupling with a terminal hydroxyl group
of the linker compound; hydrazine derivatives for coupling onto
oxidized sugar residues of the linker compound; or maleimide
derivatives for covalent attachment to thiol groups of the linker
compound. To produce a derivatized monolayer coating at least one
terminal carboxyl group on the monolayer coating is first activated
to an anhydride group and then reacted with a linker compound.
[0054] Alternatively, the reactive functional groups on the
monolayer coating may be reacted with a linker compound having
activated functional groups, for example, but not limited to,
N-hydroxysuccinimide esters, acid halides, anhydrides, and
isocyonates for covalent coupling to reactive amino groups on the
monolayer coating.
[0055] The linker compound has one terminal functional group, a
spacer region and a membrane adhering region. The terminal
functional groups for reacting with the activated functional groups
on the activated monolayer coating are for example, but not limited
to, halogen, amino, hydroxyl, or thiol groups. Preferably, the
terminal functional group is selected from the group consisting of
a carboxylic acid, halogen, amine, thiol, alkene, acrylate,
anhydride, ester, acid halide, isocyanate, hydrazine, maleimide and
hydroxyl group.
[0056] The spacer region may consist of, but not limited to,
oligo/poly ethers, oligo/poly peptides, oligo/poly amides,
oligo/poly amines, oligo/poly esters, oligo/poly saccharides,
polyols, multiple charged species or any other combinations
thereof. For example, but not limited to, oligomers of ethylene
glycols, peptides, glycerol, ethanolamine, serine, inositol, etc.,
and is such that membranes freely adhere to the membrane adhering
region of the linker moiety. The spacer region may be hydrophilic
in nature. In one preferred embodiment, the spacer has n
oxyethylene groups, where n is between 2 and 25. In the most
preferred embodiment, the spacer has ten oxyethylene groups. In a
preferred embodiment the membrane adhering region or "hydrophobic
tail" of the linker compound is hydrophobic or amphiphilic with
straight or branched chain alkyl, alkynyl, alkenyl, aryl, araalkyl,
heteroalkyl, heteroalkynyl, heteroalkenyl, heteroaryl, or
heteroaraalkyl. In a preferred embodiment, the membrane adhering
region comprises of a C.sub.10 to C.sub.25 straight or branched
chain alkyl or heteroalkyl hydrophobic tail. In the most preferred
embodiment, the hydrophobic tail comprises a C.sub.10 to C.sub.20
straight or branched chain alkyl fragment.
[0057] In another embodiment, the linker compound has a terminal
functional group on one end, a spacer, a linker/membrane adhering
region and a hydrophilic group on another end. The hydrophilic
group at one end of the linker compound may be a single group or a
straight or branched chain of multiple hydrophilic groups. For
example, but not limited to, a single hydroxyl group or a chain of
multiple ethylene glycol units.
[0058] Biological Membranes
[0059] In accordance with the present invention, a "biological
membrane" as referred to in the present invention comprises a
membrane which may be synthetic or naturally occuiring, for
example, but not limited to, vesicles, liposomes, monolayer lipid
membranes, bilayer-lipid membranes, membranes incorporated with
receptors, whole or part of cell membranes, or liposomes containing
re-folded proteins, or detergent micelles containing re-folded
proteins, or the like. Membranes suitable for use with the present
invention are amphiphilic molecules, for example, but not limited
to, phospholipids, sphingomyelins, cholesterol or their
derivatives. In a preferred embodiment, the membrane includes a
membrane-protein. Such membrane proteins include, for example,
integral membrane proteins, peripheral membrane proteins and
receptors (e.g., G protein-coupled receptors, ion-channel
receptors, tyrosine kinase-linked receptors, cytokine receptors,
and receptors with intrinsic enzymatic activity). In another
embodiment, the membrane may be bilayer-lipid membranes
incorporated with, but not limited to, ionophores (for example, but
not limited to, valinomycin, nonactin, methyl monesin, coronands,
cryptands or their derivatives), ion-channels (for example, but not
limited to, protein ionophores, etc.) or synthetic or naturally
occurring analytes, for example, but not limited to, antibody,
enzyme, lectin, dye, chelating agent and the like.
[0060] Proteins
[0061] The proteins incorporated on the array may be produced by
any of the variety of means known to those of ordinary skill in the
art. In preparation for incorporation on the arrays of the present
invention, the protein may be obtained from natural sources or
optionally be overexpressed using recombinant DNA methods. Proteins
include, for example, GPCRs (e.g. the aderenergic receptor,
angiotensin receptor, cholecystokinin receptor, muscarinic
acetylcholine receptor, neurotensin receptor, galanin receptor,
dopamine receptor, opioid receptor, erotonin receptor, somatostatin
receptor, etc), ion-channels (nicotinic acetylcholine receptor,
sodium and potassium channels, etc), receptor tyrosine kinases,
receptors for growth factors and hormones (epidermal growth factor
(EGF) receptor), and other membrane-bound proteins. Mutants or
modifications of such proteins may also be used. For example, some
single or multiple point mutations of GPCRs retain function and may
be involved in disease. (See, Stadel, et al., Trends in
Pharmocological Review, 1997, 18, 430-437.)
[0062] Additionally, as discussed above, modifications include
histidine tagging of a GPCR at the c-terminus. Such modified
protein can be attached to the substrate surface in a specific
orientation e.g., the intracellular domain of the GPCR facing the
substrate. Moreover, the proteins can also (or independently) be
modified to include an agonist (or peptide) attached at the
N-terminus. GPCRs modified in such a way can be constitutively
activated (Nielsen, S. M. et al., Proc. Natl. Acad. Sci. USA, 2000,
97, 10277-10281).
[0063] Preparation of the Arrays
[0064] The arrays of the present invention are prepared using
micropaterning techniques. Such techniques are well known in the
art. In a preferred method of preparation, the tip of a probe (also
referred to as a "pin") is immersed into a solution of biological
membrane. The tip is removed from the solution to provide solution
adhered to the tip. The lipid solution is contacted with the
surface of a substrate to thereby transfer the solution from the
tip to the surface.
[0065] A "pin" as used in the invention may be of any shape, size,
and dimension. For example, the pin printing process may involve
ring shaped pins, square pins, or point pins, etc. In another
embodiment, the direct contact printing may involve single
pin-printing or multiple pin printing, i.e. a single pin printing
method involving a source plate or multiple pin-printing using a
laid out array of multiple pins patterned in any format.
[0066] The printing apparatus may include a print head, plate,
substrate handling unit, XY or XYZ positioning stage, environmental
control, instrument control. software, sample tracking software,
etc. For example, a quill pin-printer sold by Cartesian
Technologies, Inc.
[0067] A typographical probe array having a matrix of probes
aligned such that each probe from the matrix fits into a
corresponding source well, e.g., a well from a microtiter plate, is
preferably used to form a high density array.
[0068] Uses of the Arrays
[0069] The present invention also provides for methods of using the
biological membrane array. The arrays of the present invention are
particularly suited for the use in drug development, medical
diagnostics, proteomics and biosensors.
[0070] The sample which is delivered to the array is typically a
fluid.
[0071] A wide range of detection methods is applicable to the
methods of the invention. As desired, detection may be either
quantitative, semiquantitative, or qualitative. The invention array
can be interfaced with optical detection methods such as absorption
in the visible or infrared range, chemoluminescence, and
fluorescence (including lifetime, polarization, fluorescence
correlation spectroscopy (FCS), and fluorescence-resonance energy
transfer (FRET)). Furthermore, other modes of detection such as
those based on optical waveguides (PCT Publication WO96/26432 and
U.S. Pat. No. 5,677,196), surface plasmon resonance, surface charge
sensors, and surface force sensors are compatible with many
embodiments of the invention.
[0072] The assays used on these arrays may be direct,
non-competitive assays or indirect, competitive assays. In the
noncompetitive method, the affinity for binding sites on the probe
is determined directly. In this method, the proteins in the
microspots are directly exposed to the analyte ("the target"). The
analyte may be labeled or unlabeled. If the analyte is labeled, the
methods of detection would include fluorescence, luminescence,
radioactivity, etc. If the analyte is unlabeled, the detection of
binding would be based on a change in some physical property at the
probe surface. This physical property could be refractive index, or
electrical impedance. The detection of binding of unlabeled targets
could also be carried out by mass spectroscopy. In the competitive
method, binding-site occupancy is determined indirectly. In this
method, the proteins of the array are exposed to a solution
containing a cognate labeled ligand for the probe array and an
unlabled target. The labeled cognate ligand and the unlabled target
compete for the binding sites on the probe protein microspots. The
affinity of the target for the probe microspot relative to the
cognate ligand is determined by the decrease in the amount of
binding of the cognate labeled ligand. The detection of binding of
the target can also be carried out using sandwich assays in which
after the initial binding, the array is incubated with a second
solution containing molecules such as labeled antibodies that have
an affinity for the bound target, and the amount of binding of the
target is determined based on the amount of binding of the labeled
antibodies to the probe-target complex.
[0073] Another aspect of the invention provides for a method for
screening a plurality of proteins for their ability to bind a
particular component of a target sample. This method comprises
delivering the sample to an array of the invention comprising the
proteins to be screened and detecting, either directly or
indirectly, for the presence or amount of the particular component
retained at each microspot. In a preferred embodiment, the method
further comprises the intermediate step of washing the array to
remove any unbound or nonspecifically bound components of the
sample from the array before the detection step. In another
embodiment, the method further comprises the additional step of
further characterizing the particular component retained on at
least one microspot.
[0074] In another embodiment of the invention, a method of assaying
for protein-protein binding interactions is provided which
comprises the following steps: first, delivering a sample
comprising at least one protein to be assayed for binding to the
array of the invention; and then detecting, either directly, or
indirectly, for the presence or amount of the protein from the
sample that is retained at each microspot.
[0075] Another embodiment of the invention provides a method of
assaying in parallel for the presence of a plurality of analytes in
a sample which can react with one or more of the proteins on the
array. This method comprises delivering the sample to the array and
detecting for the interaction of the analyte with the protein at
each microspot.
[0076] In still another embodiment of the invention, a method of
assaying in parallel for the presence of a plurality of analytes in
a sample which can bind one or more of the proteins on the array
comprises delivering the fluid sample to the array and detecting,
either directly or indirectly, for the presence or amount of
analyte retained at each microspot. In a preferred embodiment, the
method further comprises the step of washing the array to remove
any unbound or non-specifically bound components of the sample from
the array.
[0077] The array may be used in a diagnostic manner when the
plurality of analytes being assayed are indicative of a disease
condition or the presence of a pathogen in an organism. In such
embodiments, the sample which is delivered to the array will then
typically be derived from a body fluid or a cellular extract from
the organism.
[0078] The array may be used for drug screening when a potential
drug candidate is screened directly for its ability to bind or
otherwise interact with a plurality of proteins on the array.
Alternatively, a plurality of potential drug candidates may be
screened in parallel for their ability to bind or otherwise
interact with one or more proteins on the array. The drug screening
process may optionally involve assaying for the interaction, such
as binding, of at least one analyte or component of a sample with
one or more proteins on an array, both in the presence and absence
of the potential drug candidate. This allows for the potential drug
candidate to be tested for its ability to act as an inhibitor of
the interaction or interactions originally being assayed.
[0079] In general, delivery of solutions containing proteins to be
bound by the proteins of the array may optionally be preceded,
followed, or accompanied by delivery of a blocking solution. A
blocking solution contains protein or another moiety which will
adhere to sites of non-specific binding on the array. For instance,
solutions of bovine serum albumin or milk may be used as blocking
solutions.
[0080] In the following, the invention is illustrated by
non-limiting examples which describe the invention.
EXAMPLES
[0081] Materials
[0082] Membrane preparations of human .beta.-adrenergic receptor
subtype I (.beta.1) and dopamine receptor subtype I (D1) were
purchased from Biosignal Packard (Montral, Canada). These
receptor-associated membranes came suspended in a buffer solution
containing 10 mM Tris-HCl, pH 7.4 and 10% glycerol. Human cloned
neurotensin receptor subtype 1 (NT1R) and BODIPY-TMR-neurotensin
(BT-NT) were purchased from Perkin Elmer Life Science (Boston,
Mass.) and were received as membrane associated suspensions in a
buffer solution containing 10 mM Tris-HCl (pH 7.4) and 10% sucrose.
BODIPY-TMR-CGP12177 (BT-CGP) and BODIPY-FL-SCH23390 (BF-SCH) were
purchased from Molecular Probes (Eugene, Oreg.). CGP12177 and
SCH23390 were purchased from Tocris Cookson, Inc (Ballwin, Mo.).
Neurotensin was purchased from Sigma Chemical Co. (St. Louis, Mo.).
Corning CMT-GAPS slides were used as received. Brij 76 derivatized
gold-coated substrates were prepared as described previously. The
fluorescently labeled ("hot") ligands and neurotensin were
dissolved in DMSO and stored at -20.degree. C. Before use, the
ligand solution was diluted using a binding buffer consisting of 50
mM Tris-HCl, 2 mM EDTA, 1 mM MgCl.sub.2, pH 7.4 and 0.1% bovine
serum albumin (BSA).
[0083] 1,2-dilauroyl-sn-glycero-2-phosphocholine (DLPC),
L-.alpha.-dimyristoylphosphatidylcholine (DMPC),
L-.alpha.-dipalmitoylpho- sphatidycholine (DPPC), and egg
phosphatidylcholine (egg PC), were purchased from Avanti Polar
Lipids (Alabaster, Ala.).
FITC-1,2-dihexadecanoul-sn-glycero-3-phosphoethanolamine
(FITC-DHPE) and Texas
Red-1,2-dihexadecanoul-sn-glycero-3-phosphoethanolamine (TR-DHPE)
were purchased from Molecular Probes Inc.
[0084] GPCR and Lipid Printing
[0085] Multiple arrays of GPCRs or lipids were printed on each
slide (Corning CMT-GAPS slides) using a robotic pin printer (Model
PS 5000, Cartesian Technologies Inc.) equipped with quill pins
(Telechem). Each 3.times.3 or 5.times.5 element array was separated
from its neighboring array by at least 6 mm. Membrane preparations
containing GPCRs were used for printing as received from the
manufacturer without further purification or dilution. After
printing the arrays were incubated in a humid chamber at room
temperature for one hour, and then used for ligand binding
experiments. For longer term storage, the arrays were stored in a
dessicator at 4.degree. C.
[0086] Ligand Binding
[0087] Each array on a given slide was incubated for one hour with
10 .mu.L of a buffered solution (50mM Tris-HCl, 2 mM EDTA, 1 mM
MgCl.sub.2, pH 7.4, 0.1% BSA) containing ligand. After incubation,
the solutions were carefully removed using a pipette tip attached
to a vacuum pump. The slides were rinsed briefly with water and
dried under a stream of nitrogen. The slides were imaged in a
GenPix 4000 scanner (Axon Instruments, Foster City, Calif.).
[0088] Fluorescence Recovery after Photobleaching (FRAP)
[0089] Small unilamellar vesicles (SUVs) of
1,2-dilauroyl-sn-glycero-2-pho- sphocholine (DLPC) mixed with 2%
(mol) Texas Red DHPE were generated by sonicating a suspension of
the lipids (1 mg/ml) in buffer; these vesicles were then incubated
with the substrate. After extensive and careful washing, supported
lipid membranes were formed on these surfaces. FRAP experiments
were carried out on these supported lipid membranes on bare glass
and GAPS slides using an Olympus AX70 epifluorescence microscope
equipped with a CCD detector (Princeton Instruments).
[0090] Results and Discussion
[0091] Fabrication and Storage of GPCR Arrays
[0092] Arrays of GPCRs were fabricated by conventional robotic pin
printing, using a quill-pin printer as described in the
Experimental Section. Boxer and co-workers have described the
importance of transferring membranes onto the solid-support under
water; we were, however, concerned that the lipid solution wetted
onto the pin would partially dissociate from the pin under water
and cause cross-contamination during printing. Moreover, slide
racks in commercially available printers are not set up for
printing under water. The ability to use of-the-shelf printing
equipment for fabricating membrane-protein arrays is an important
step towards the widespread fabrication and development of these
arrays for bioanalytical applications.
[0093] In order to investigate the stability of printed GPCR
proteins, arrays of the adrenergic .beta.1 receptor were printed as
targets. We first investigated the storage of these arrays under
high-humidity at various temperatures (room temp to -80.degree.
C.). These high-humidity conditions were chosen because there was a
significant body of literature that suggested the importance of an
aqueous environment for maintaining the structure of the
membrane-protein complex. (Macbeath G., Schreiber, S. L. Science
2000, 289, 1760-1761; Cremer, P. S. Boxer, S. G. J. Phys. Chem. B
1999, 103, 2554-2559). The functional stability of the arrays was
evaluated in binding assays using fluorescently labeled cognate
ligands and inhibitors using protocols described in Experimental.
No ligand binding to the arrays was observed after storage for a
week (FIG. 2 A). Therefore, we decided to test the stability of
these arrays under desiccation. We felt that desiccation would
reduce possible protease-induced degradation; we also found out
that flash-plates with immobilized GPCRs when stored desiccated at
4.degree. C. were stable for up to 3 months. Under the new
conditions, the slides with printed GPCR arrays were air dried at
room temp for a couple of hours, put into slide holders under
nitrogen, and stored in desiccators at 4.degree. C. in the dark.
Our observations indicate that, over a 2-week period, the
adrenergic .beta.1 receptors retained their ligand-binding affinity
(FIG. 2B). These stability experiments are a significant
feasibility milestone for the manufacture of GPCR arrays.
[0094] Mechanical Stability of Membrane Arrays on GAPS
Substrates
[0095] We were interested in the development of robust binding
assays for membrane-protein arrays. Boxer and co-workers have
reported that lipids adsorbed onto bare-glass substrates
spontaneously desorbed when drawn through an air-water interface
(Cremer and Boxer, 1999). We felt that this behavior was a
limitation to the use of membrane-protein arrays for bioassays,
which often requires protocols in which the slides are withdrawn
from solution (e.g. during washes by successive immersions). We
therefore investigated surfaces that supported the adsorption of
mechanically stable supported membranes; our criterion for
stability was that the supported membrane would remain adsorbed
when withdrawn through an air-water interface. Among the several
surfaces tested, the CMT-GAPS surfaces offered the most stable
supported lipids. FIG. 3A shows fluorescence images of arrays of
supported membranes consisting of DPPC/DMPC doped with
fluorescein-DHPE immersed in buffer that were withdrawn through an
air-water interface, immersed in water, dried, and again immersed
under water. We do not see any decrease in the fluorescence
intensities of these lipid microspots through these successive
immersions and withdrawals; these observations indicate that the
bound lipids are stable. FIG. 3B shows data on lipids consisting of
egg PC; arrays of these lipids are also stable when subject to
successive immersions and withdrawals. At room temperature,
DMPC/DPPC lipids are in the gel-phase, whereas egg-PC is in the
fluid phase. These experiments demonstrate that supported lipid
arrays are mechanically robust on GAPS-coated substrates,
independent of whether they are in the gel or fluid phase.
[0096] We were also interested in determining whether the lipids
adsorbed on GAPS substrates had long-range lateral fluidity. This
fluidity is an important characteristic of native biological
membranes, and is a property that is considered to be
physiologically significant (e.g. for processes such ligand induced
receptor dimerization at surfaces). Although it is not clear
whether this fluidity is required for ligand screening experiments
on supported biological membranes, we nevertheless wanted to
investigate whether the high mechanical stability of the supported
lipids described above necessarily implied that the lipids were not
laterally mobile. We made vesicles from fluorescently labeled DLPC
lipids and formed supported lipids on the GAPS substrates by
vesicle fusion. Using a fluorescence microscope, we were able to
observe fluorescence recovery of a photobleached spot on the
supported lipid in a FRAP experiment. A comparative experiment with
DLPC vesicles on bare-glass suggested that the recovery was much
slower on the GAPS substrate; there was also a certain fraction of
lipids on the GAPS substrate that did not "recover", suggesting
that a certain fraction of the supported membrane was immobile.
Nevertheless, these experiments suggest there is some lateral
fluidity associated with the supported lipids on the GAPS
substrates. Our observations on the GAPS substrates are in
agreement with the lower and limited mobility of supported
membranes on polymer cushions reported by Shen et al (William W
Shen, Steven G. Boxer, Wolfgang Knoll, Curtis W. Frank;
Biomacromolecules 2001, vol 2, pp 70-79).
[0097] Biospecific Binding to GPCR Arrays
[0098] Arrays of GPCRs were fabricated by using a quill-pin
printer, as described above. The arrays were then incubated with
their fluorescently labeled cognate ligands in direct or
competition assays. FIG. 4 shows fluorescence false-color images of
five separate arrays printed on a single CMT-GAPS slide; each
individual array contains three columns containing 5 replicate
spots; each column represents a different GPCR protein. These
proteins, from left to right, are the adrenergic receptor
(.beta.1), the neurotensin receptor (NTR1) and the dopamine (D1)
receptor, respectively. The first array (FIG. 4A) was incubated
with the binding buffer only. As expected, no fluorescence is
observed. The second array (FIG. 4B) was incubated with a solution
containing fluorescently labeled neurotensin (BT-NT, 1 nM). The
image shows that only the array corresponding to NTR1 shows a
strong fluorescence signal; this observation suggests that the
binding of BT-NT to NTR1 is selective. The specificity of the
interaction was further demonstrated by incubating the arrays with
solutions containing BT-NT (1 nM) and either CGP12177 (1 .mu.M)
(FIG. 4C), SCH 23390 (1 .mu.M) (FIG. 4D), or neurotensin (1 .mu.M)
(FIG. 4E). Relative to FIG. 4B, there is no significant decrease in
the intensities of spots corresponding to NTR1 in FIG. 4C and 4D.
CGP 12177 and SCH 23390 do not bind to NTR1; hence, their addition
to the binding solution should not inhibit the interaction of BT-NT
with NTR1, in agreement with our observations. Neurotensin is the
cognate ligand for NTR1, hence, it competes for binding sites on
the NTR1 array. In FIG. 4E, the array was incubated with a solution
that contained neurotensin in 1000-fold excess over BT-NT; at these
ratios, the neurotensin is expected to completely inhibit the
binding of BT-NT to NTR1. We do not observe any signal
corresponding to the NTR1 array; hence, neurotensin is able to
specifically inhibit binding to NTR1. These experiments demonstrate
that assays to test the binding of ligands and inhibitors are
feasible using GPCR arrays.
[0099] Dose Dependent Binding
[0100] We have investigated the response of the printed GPCR arrays
to cognate ligands at different concentrations. FIG. 5A shows
fluorescence images of arrays of the neurotensin receptor treated
with BT-NT; the data shows that there an increase in the
fluorescence intensity of the arrays when treated with higher
concentrations of the fluorescently labeled ligand. For the binding
of BT-neurotensin to NTR1 arrays, the limit of detection was
.about.0.1-0.2 nM BT-NT. These results suggest that the dynamic
range of GPCR arrays utilizing fluorescently labeled ligands is
.about.2 logs for this system. FIG. 5B shows data for the binding
of cy5-labeled antagonist D to arrays of the galanin receptor--the
images show that the fluorescence intensity of the microspots are
dependent on the concentration of the ligand.
[0101] The inhibition of binding of the fluorescent ligands to the
array is dependent on the relative concentrations of the inhibitor
and the labeled ligands, and their respective dissociation
(K.sub.d) and inhibition constants (K.sub.i). FIG. 6 shows
fluorescence images of NTR1 arrays incubated with solutions
containing BT-NT (2 nM) and different concentrations of neurotensin
(0-250 nM). The data shows that there is a decrease in the
fluorescence as the concentration of neurotensin is increased. A
plot of the fluorescence intensities versus concentration is shown
in FIG. 7; based on this plot, we estimate that the inhibition
constant (K.sub.i).about.2.5 nM. This value is consistent with the
reported value of K.sub.i (2 nM) for neurotensin obtained from
fluorescence polarization experiments. These experiments
demonstrate that it is possible to obtain estimates of binding
constants of ligands and inhibitors using GPCR arrays.
[0102] While the invention has been described in connection with
specific embodiments, it will be understood that it is capable of
further modifications. Therefore, this application is intended to
cover any variations, uses, or adaptations of the invention that
follow, in general, the principles of the invention, including
departures from the present disclosure that come within known or
customary practice within the art.
[0103] Other embodiments are within the claims.
[0104] The references cited throughout the specification including
those set forth below are incorporated herein by reference.
[0105] 1. Malbon, C. C. and Morris, A. J. (1999). "Physiological
regulation of G protein-linked signaling." Physiol. Rev. 79,
1373-1430.
[0106] 2. Drews, J. (2000). "Drug discovery: a historical
perspective". Science 287, 1960-1963.
[0107] 3. Howard, A. D., McAllister, G., Feighner, S. D., Liu, Q.,
Nargund, R. P., Van der Ploeg, L. H. T. and Patchett, A. A. (2001)
"Orphan G-protein coupled receptors and natural ligand discovery".
Trends in Pharmaco.Sci. 22, 132-140.
[0108] 4. Stadel, J. M., Wilson, S. and Bergsma, D. J. (1997).
"Orphan G protein-coupled receptors: a neglected opportunity for
pioneer drug discovery." TiPS 18, 430-437.
[0109] 5. Civelli, O., Reinscheid, R. K., Nothacker, H.-P. (1999).
"Orphan receptors, novel neuropeptides and reverse pharmaceutical
research". Brain Res. 848, 63-65.
[0110] 6. Gurevich, V. V., Pals-Rylaarsdam, R., Benovic, J. L.,
Hosey, M. M. and Onorato, J. J. (1997). "Agonist-receptor-arrestin,
an alternative ternary complex with high agonist affinity."
J.Biol.Chem. 272, 28849.
[0111] 7. Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G.,
Barak, L. S. (2000) "Differential affinities of visual arrestin,
.beta.-arrestin1, and .beta.-arrestin2 for G protein-coupled
receptors delineate two major classes of receptors." J.Biol.Chem.
275, 17201-17210.
[0112] 8. Barak, L. S., Ferguson, S. S. G., Zhang, J. and Caron, M.
G. (1997). "A .beta.-arrestin/green fluorescent protein biosensor
for detecting G protein-coupled receptor activation.` J. Biol.
Chem. 272, 27497-27500.
[0113] 9. Kovoor, A., Celver, J., Abdryashitov, R., Chavkin, C.,
Gurevich, V. V. (1999). "Targeted construction of
phosphorylation-independent .beta.-arrestin mutants with
constitutive activity in cells." J.Biol.Chem. 274, 6831-6834.
[0114] 10. Lahiri, J., Kalal, P., Frutos, A. G., Jonas, S. J. and
Schaeffler, R. (2000). "Method for fabricating supported bilayer
lipid membranes on gold". Langmuir 16, 7805-7812.
[0115] It will be apparent to those skilled in the art that various
modifications and variations can be made to the present invention
without departing from the spirit and scope of the invention. Thus,
it is intended that the present invention cover the modifications
and variations of this invention provided they come within the
scope of the appended claims and their equivalents.
* * * * *