U.S. patent application number 12/403304 was filed with the patent office on 2009-09-03 for combinatorial artificial receptors including tether building blocks.
This patent application is currently assigned to RECEPTORS LLC. Invention is credited to Robert E. Carlson.
Application Number | 20090221439 12/403304 |
Document ID | / |
Family ID | 35613667 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090221439 |
Kind Code |
A1 |
Carlson; Robert E. |
September 3, 2009 |
COMBINATORIAL ARTIFICIAL RECEPTORS INCLUDING TETHER BUILDING
BLOCKS
Abstract
The present invention relates to artificial receptors, arrays or
microarrays of artificial receptors or candidate artificial
receptors, methods of and compositions for making them, and methods
of using them. Each artificial receptor includes a plurality of
building block compounds. In an embodiment, at least one of the
building blocks includes a tether moiety. The tether can provide
spacing or distance between the recognition element and the support
or scaffold to which the building block is immobilized. A tether
moiety can have any of a variety of characteristics or properties
including flexibility, rigidity or stiffness, ability to bond to
another tether moiety, and the like.
Inventors: |
Carlson; Robert E.;
(Minnetonka, MN) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
RECEPTORS LLC
Chaska
MN
|
Family ID: |
35613667 |
Appl. No.: |
12/403304 |
Filed: |
March 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11219515 |
Sep 1, 2005 |
7504365 |
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12403304 |
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60609160 |
Sep 11, 2004 |
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60612666 |
Sep 23, 2004 |
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60626770 |
Nov 10, 2004 |
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60645582 |
Jan 19, 2005 |
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60649729 |
Feb 3, 2005 |
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60607438 |
Sep 3, 2004 |
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60607458 |
Sep 3, 2004 |
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60608557 |
Sep 10, 2004 |
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60607457 |
Sep 3, 2004 |
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60608654 |
Sep 10, 2004 |
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Current U.S.
Class: |
506/9 ; 506/13;
506/15; 506/32 |
Current CPC
Class: |
G01N 2500/00 20130101;
G01N 33/566 20130101; G01N 33/543 20130101; G01N 2600/00 20130101;
G01N 33/6845 20130101; G01N 33/54353 20130101; G01N 33/531
20130101 |
Class at
Publication: |
506/9 ; 506/32;
506/13; 506/15 |
International
Class: |
C30B 30/04 20060101
C30B030/04; C40B 50/18 20060101 C40B050/18; C40B 40/00 20060101
C40B040/00; C40B 40/04 20060101 C40B040/04 |
Claims
1. A method of making a heterogeneous building block array, the
method comprising: forming a plurality of spots on a solid support,
the spots comprising a plurality of building blocks, at least one
building block comprising a tether; coupling a plurality of
building blocks to the solid support in the spots, at least one of
the coupled building blocks comprising a tether.
2. The method of claim 1, further comprising mixing a plurality of
activated building blocks, at least one of the building blocks
comprising a tether, and employing the mixture in forming the
plurality of spots.
3. The method of claim 1, comprising applying individual activated
building blocks on the support.
4. The method of claim 1, wherein forming comprises piezoelectric
spotting, pin spotting, or electromagnetic spotting.
5. The method of claim 1, wherein the solid support comprises a
glass plate or microscope slide.
6. A method of making a receptor surface, the method comprising:
forming a region on a solid support, the region comprising a
plurality of building blocks, at least one of the building blocks
comprising a tether; coupling the plurality of building blocks to
the solid support in the region.
7. The method of claim 6, further comprising mixing a plurality of
activated building blocks, at least one of the building blocks
comprising a tether, and employing the mixture in forming the
receptor surface.
8. The method of claim 6, comprising applying individual activated
building blocks to the support.
9. The method of claim 6, wherein the solid support comprises a
tube, plate, or well.
10. A method of making an artificial receptor, the method
comprising: forming a region on a support, the region comprising a
plurality of building blocks, at least one of the building blocks
comprising a tether; coupling the plurality of building blocks to
the support in the region.
11. The method of claim 10, wherein the region is a spot.
12. The method of claim 10, wherein the support comprises a
scaffold and the region comprises a plurality of functional groups
on the scaffold.
13. A method of using an artificial receptor comprising: contacting
a first heterogeneous molecular array with a test ligand; the array
comprising: a support; and a plurality of spots of building blocks
attached to the support, at least one of the building blocks
comprising a tether; the spots of building blocks comprising a
plurality of building blocks; and the building blocks being coupled
to the support; detecting binding of a test ligand to one or more
spots; and selecting one or more of the binding spots as the
artificial receptor; wherein the building blocks in the array
define a first set of building blocks, and the plurality of
building blocks in the one or more binding spots defines one or
more selected binding combination of building blocks.
14. The method of claim 13, wherein the artificial receptor
comprises a lead artificial receptor.
15. The method of claim 13, further comprising: determining the
combinations of building blocks in the one or more binding spots;
developing, based on the combinations determined, one or more
developed combinations of building blocks, at least one of the
building blocks in the developed combinations of building blocks
comprising a tether, the developed combination being distinct from
those in the one or more selected combinations of building blocks;
contacting a second heterogeneous molecular array with the test
ligand, the second heterogeneous molecular array comprising a
plurality of spots, the spots comprising a developed combination of
building blocks; detecting binding of a test ligand to one or more
spots of the second heterogeneous molecular array; and selecting
one or more of the spots of the second heterogeneous molecular
array as the artificial receptor; wherein the building blocks in
the second heterogeneous molecular array define a second set of
building blocks.
16. The method of claim 15, wherein the artificial receptor
comprises a lead artificial receptor.
17. The method of claim 16, further comprising varying the
structure of the lead artificial receptor to increase binding speed
or binding affinity of the test ligand.
18. The method of claim 14, further comprising varying the
structure of the lead artificial receptor to increase binding speed
or binding affinity of the test ligand.
19. The method of claim 13, wherein the first set of building
blocks comprises a subset of a larger set of building blocks.
20. The method of claim 15, wherein the first set of building
blocks comprises a subset of a larger set of building blocks, the
second subset of building blocks defines a subset of the larger set
of building blocks, and the first subset is not equivalent to the
second subset.
21. The method of claim 13, wherein the spots comprise 2, 3, or 4
building blocks.
22. The method of claim 15, wherein the spots of the second
heterogeneous molecular array comprise 3, 4, or 5 building blocks,
and the spots of the second heterogeneous molecular array comprise
more building blocks than the binding spots.
23. The method of claim 13, further comprising: identifying the
plurality of building blocks making up the artificial receptor;
coupling the identified plurality of building blocks to a scaffold
molecule; evaluating the scaffold artificial receptor for binding
of the test ligand.
24. The method of claim 23, wherein: coupling comprises making a
plurality of positional isomers of the building blocks on the
scaffold; evaluating comprises comparing the plurality of the
scaffold positional isomer artificial receptors; and selecting one
or more of the scaffold positional isomer artificial receptors as
lead or working artificial receptor.
25. The method of claim 15, further comprising: identifying the
plurality of building blocks making up the artificial receptor;
coupling the identified plurality of building blocks to a scaffold
molecule; evaluating the scaffold artificial receptor for binding
of the test ligand.
26. The method of claim 25, wherein: coupling comprises making a
plurality of positional isomers of the building blocks on the
scaffold; evaluating comprises comparing the plurality of the
scaffold positional isomer artificial receptors; and selecting one
or more of the scaffold positional isomer artificial receptors as
lead or working artificial receptor.
27. The method of claim 13, further comprising applying the test
ligand to one or more spots that function as controls for
validating or evaluating binding to an artificial receptor.
28. The method of claim 27, wherein the control spot comprises no
building block, only a single building block, only functionalized
lawn, or a combination thereof.
29. A composition comprising: a support; and a portion of the
support comprising a plurality of building blocks; the building
blocks being coupled to the support; at least one of the building
blocks comprising a tether.
30. The composition of claim 29, comprising a candidate artificial
receptor, a lead artificial receptor, a working artificial
receptor, or a combination thereof.
31. The composition of claim 30, wherein the support comprises a
scaffold molecule.
32. The composition of claim 30, wherein the artificial receptor
comprises 2, 3, 4, 5, or 6 different building blocks.
33. The composition of claim 29, comprising a plurality of spots on
the support; the spots comprising a plurality of building blocks;
and the building blocks being coupled to the support.
34. The composition of claim 33, wherein the spots are configured
in an array.
35. The composition of claim 34, wherein the array comprises more
than 1 million spots.
36. The composition of claim 33, wherein the spots comprise 2, 3,
4, 5, or 6 building blocks.
37. The composition of claim 33, wherein the support comprises a
solid support.
38. The composition of claim 37, comprising a plurality of spots on
a surface of the solid support.
39. The composition of claim 33, comprising a functionalized lawn
coupled to the support and the building blocks coupled in spots to
the lawn.
40. The composition of claim 39, comprising a functionalized glass
support.
41. The composition of claim 33, wherein the support comprises a
scaffold molecule.
42. The composition of claim 29, wherein: the support comprises a
surface; the surface comprises a region; and the region comprises a
plurality of building blocks; the building blocks being coupled to
the support.
43. The composition of claim 42, wherein the region comprises 2, 3,
4, 5, or 6 building blocks.
44. The composition of claim 42, wherein the support comprises a
tube or well.
45. The composition of claim 42, further comprising a
functionalized lawn coupled to the tube or well and the building
blocks coupled to the lawn.
46. The composition of claim 29, the plurality of building blocks
independently comprising tether, framework, linker, first
recognition element, and second recognition element.
47. The composition of claim 46, wherein the framework comprises an
amino acid.
48. The composition of claim 47, wherein the amino acid comprises
serine, threonine, or tyrosine.
49. The composition of claim 47, wherein the amino acid comprises
tyrosine.
50. The composition of claim 46, wherein the linker has the formula
(CH.sub.2).sub.nC(O)--, with n=1-16.
51. The composition of claim 46, wherein the first recognition
element and second recognition element independently are of
formulas B1, B2, B3, B4, B5, B6, B7, B8, B9, A1, A2, A3, A4, A5,
A6, A7, A8, or A9.
52. The composition of claim 47, wherein the support comprises a
support matrix and the support matrix comprises a lawn of
amines.
53. The composition of claim 29, the plurality of building blocks
independently having the formula: ##STR00007## in which: X is
absent or C.dbd.O; Y is absent, NH, or O; Z is O; R.sub.2 is H or
CH.sub.3; R.sub.3 is CH.sub.2 or CH.sub.2-phenyl; RE.sub.1 is B1,
B2, B3, B4, B5, B6, B7, B8, B9, A1, A2, A3, A4, A5, A6, A7, A8, or
A9; RE.sub.2 is A1, A2, A3, A4, A5, A6, A7, A8, A9, B1, B2, B3, B4,
B5, B6, B7, B8, or B9; and L is (CH.sub.2).sub.nCOOH, with
n=1-16.
54. The composition of claim 29, wherein the support comprises a
dendrimer.
55. The composition of claim 29, wherein the support comprises a
scaffold molecule.
56. The composition of claim 29, wherein the artificial receptor
comprises 2, 3, 4, 5, or 6 different building blocks.
57. The composition of claim 29, wherein the support comprises a
solid support.
58. The composition of claim 29, comprising a functionalized lawn
coupled to the support and the building blocks coupled in spots to
the lawn.
59. The composition of claim 58, comprising a functionalized glass
support.
60. An artificial receptor, the artificial receptor comprising a
plurality of building blocks coupled to a support; at least one of
the building blocks comprising a tether.
61. A heterogeneous building block array comprising: a support; and
a plurality of spots on the support; the spots comprising a
plurality of building blocks; the building blocks being coupled to
the support; at least one of the building blocks comprising a
tether.
62. A composition comprising: a surface; and a region on the
surface comprising a plurality of building blocks; the building
blocks being coupled to the support; at least one of the building
blocks comprising a tether.
63. A composition of matter comprising a plurality of building
blocks; the building blocks having the formula:
linker-tether-framework-(first recognition element) (second
recognition element).
64. The composition of matter of claim 63, wherein the framework
comprises an amino acid.
65. The composition of matter of claim 64, wherein the amino acid
comprises serine, threonine, or tyrosine.
66. The composition of matter of claim 64, wherein the amino acid
comprises tyrosine.
67. The composition of matter of claim 63, wherein the linker has
the formula (CH.sub.2).sub.nCO--, with n=1-16.
68. The composition of matter of claim 63, wherein the first
recognition element and second recognition element independently
are of formulas B1, B2, B3, B4, B5, B6, B7, B8, B9, A1, A2, A3, A4,
A5, A6, A7, A8, or A9.
69. The composition of matter of claim 63, the plurality of
building blocks independently having the formula: ##STR00008## in
which: X is absent or C.dbd.O; Y is absent, NH, or O; Z is O;
R.sub.2 is H or CH.sub.3; R.sub.3 is CH.sub.2 or CH.sub.2-phenyl;
RE.sub.1 is B1, B2, B3, B4, B5, B6, B7, B8, B9, A1, A2, A3, A4, A5,
A6, A7, A8, or A9; RE.sub.2 is A1, A2, A3, A4, A5, A6, A7, A8, A9,
B1, B2, B3, B4, B5, B6, B7, B8, or B9; and L is
(CH.sub.2).sub.nCOOH, with n=1-16.
70. The composition of matter of claim 63, further comprising a
support.
71. The composition of matter of claim 63, comprising about 10 to
about 200 distinct building blocks.
72. The composition of matter of claim 63, wherein the building
blocks are activated for coupling to a functional group.
73. The composition of matter of claim 63, wherein the building
blocks are coupled to a support.
74. The composition of matter of claim 63, wherein each building
block is in a container.
75. The composition of matter of claim 63, further comprising a
package containing the plurality of building blocks and
instructions for their use.
76. The composition of matter of claim 75, wherein the building
blocks are components of a heterogeneous molecular array.
77. The composition of matter of claim 63, comprising a mixture of
building blocks.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/219,515, filed Sep. 1, 2005, which claims
priority to U.S. Provisional Patent Application Nos. 60/609,160,
filed Sep. 11, 2004, entitled "ARTIFICIAL RECEPTORS, BUILDING
BLOCKS, AND METHODS", 60/612,666, filed Sep. 23, 2004, entitled
"ARTIFICIAL RECEPTORS, BUILDING BLOCKS, AND METHODS", 60/626,770,
filed Nov. 10, 2004, entitled "ARTIFICIAL RECEPTORS, BUILDING
BLOCKS, AND METHODS", 60/645,582, filed Jan. 19, 2005, entitled
"ARTIFICIAL RECEPTORS, BUILDING BLOCKS, AND METHODS", 60/649,729,
filed Feb. 3, 2005, entitled "ARTIFICIAL RECEPTORS, BUILDING
BLOCKS, AND METHODS", 60/607,438, filed Sep. 3, 2004, entitled
"COMBINATORIAL ARTIFICIAL RECEPTORS INCLUDING TETHER BUILDING
BLOCKS ON SCAFFOLDS", 60/607,458, filed Sep. 3, 2004, entitled
"COMBINATORIAL ARTIFICIAL RECEPTORS INCLUDING TETHER BUILDING
BLOCKS ON SCAFFOLDS", 60/608,557, filed Sep. 10, 2004, entitled
"COMBINATORIAL ARTIFICIAL RECEPTORS INCLUDING TETHER BUILDING
BLOCKS ON SCAFFOLDS", 60/607,457, filed Sep. 3, 2004, entitled
"SCAFFOLD-BASED ARTIFICIAL RECEPTORS AND METHODS", and 60/608,654,
filed Sep. 10, 2004, entitled "SCAFFOLD-BASED ARTIFICIAL RECEPTORS
AND METHODS".
[0002] Each of the listed applications is incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates to artificial receptors,
arrays or microarrays of artificial receptors or candidate
artificial receptors, methods of and compositions for making them,
and methods of using them. Each artificial receptor includes a
plurality of building block compounds. In an embodiment, at least
one of the building blocks includes a tether moiety. The tether can
provide spacing or distance between the recognition element and the
support or scaffold to which the building block is immobilized. A
tether moiety can have any of a variety of characteristics or
properties including flexibility, rigidity or stiffness, ability to
bond to another tether moiety, and the like.
BACKGROUND
[0004] The preparation of artificial receptors that bind ligands
like proteins, peptides, carbohydrates, microbes, pollutants,
pharmaceuticals, and the like with high sensitivity and specificity
is an active area of research. None of the conventional approaches
has been particularly successful; achieving only modest sensitivity
and specificity mainly due to low binding affinity.
[0005] Antibodies, enzymes, and natural receptors generally have
binding constants in the 10.sup.8-10.sup.12 range, which results in
both nanomolar sensitivity and targeted specificity. By contrast,
conventional artificial receptors typically have binding constants
of about 10.sup.3 to 10.sup.5, with the predictable result of
millimolar sensitivity and limited specificity.
[0006] Several conventional approaches are being pursued in
attempts to achieve highly sensitive and specific artificial
receptors. These approaches include, for example, affinity
isolation, molecular imprinting, and rational and/or combinatorial
design and synthesis of synthetic or semi-synthetic receptors.
[0007] Such rational or combinatorial approaches have been limited
by the relatively small number of receptors which are evaluated
and/or by their reliance on a design strategy which focuses on only
one building block, the homogeneous design strategy. Common
combinatorial approaches form microarrays that include 10,000 or
100,000 distinct spots on a standard microscope slide. However,
such conventional methods for combinatorial synthesis provide a
single molecule per spot. Employing a single building block in each
spot provides only a single possible receptor per spot. Synthesis
of thousands of building blocks would be required to make thousands
of possible receptors.
[0008] Further, these conventional approaches are hampered by the
currently limited understanding of the principals which lead to
efficient binding and the large number of possible structures for
receptors, which makes such an approach problematic.
[0009] There remains a need for methods for detecting test ligands
in unknown samples and for detecting compounds that disrupt one or
more binding interactions.
SUMMARY
[0010] The present invention relates to artificial receptors,
arrays or microarrays of artificial receptors or candidate
artificial receptors, methods of and compositions for making them,
and methods of using them. Each artificial receptor includes a
plurality of building block compounds. In an embodiment, at least
one of the building blocks includes a tether moiety. The tether can
provide spacing or distance between the recognition element and the
support or scaffold to which the building block is immobilized. A
tether moiety can have any of a variety of characteristics or
properties including flexibility, rigidity or stiffness, ability to
bond to another tether moiety, and the like.
[0011] The present invention relates to artificial receptors,
arrays or microarrays of artificial receptors or candidate
artificial receptors, methods of and compositions for making them,
and methods of using them. The artificial receptor includes a
plurality of building block compounds including a tether and
immobilized on a surface.
[0012] The present invention includes a method of making an array
of artificial receptors including tethered building blocks. This
method includes forming a plurality of spots on a solid support. At
least certain of the spots include a plurality of building blocks,
at least one of the building blocks including a tether moiety. The
method includes immobilizing building blocks on the solid support
in the spots.
[0013] The present invention includes a method of making a receptor
surface or an artificial receptor. This method includes forming a
region on a solid support. The region includes a plurality of
building blocks, at least one of the building blocks including a
tether moiety. The method includes immobilizing building blocks on
the solid support in the region.
[0014] The invention includes artificial receptors and
compositions. The compositions can include a support and a
plurality of building blocks, at least one of the building blocks
including a tether moiety. The compositions can also include a
functionalized lawn. The functionalized lawn can be coupled to the
support. Building blocks can be immobilized on the support, the
lawn, or both. In an embodiment, the present invention includes a
composition including a surface and a region on the surface. This
region includes a plurality of building blocks, at least one of the
building blocks including a tether moiety.
[0015] The present invention includes arrays of artificial
receptors and heterogeneous building block arrays. Such an array
can include a support and a plurality of building blocks, at least
one of the building blocks including a tether moiety. The array can
also include a functionalized lawn. The functionalized lawn can be
coupled to the support. The array can also include a plurality of
regions on the support. The regions can include a plurality of
building blocks, at least one of the building blocks including a
tether moiety.
[0016] The present invention includes kits and articles of
manufacture. Such an article of manufacture can include a support
and a plurality of building blocks. The article of manufacture can
also include a functionalized lawn reagent. The functionalized lawn
reagent can be configured to be coupled to the support. At least
one of the building blocks includes a tether moiety.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 schematically illustrates two dimensional
representations of an embodiment of a receptor according to the
present invention that employs 4 different building blocks to make
a ligand binding site.
[0018] FIG. 2 schematically illustrates two and three dimensional
representations of an embodiment of a molecular configuration of 4
building blocks, each building block including a recognition
element, a framework, and a linker coupled to a support
(immobilization/anchor).
[0019] FIG. 3 schematically illustrates an embodiment of the
present methods and artificial receptors employing shuffling and
exchanging building blocks.
[0020] FIG. 4 schematically illustrates an embodiment of a building
block including a flexible tether (left) and a building block
including a rigid tether (right).
[0021] FIG. 5 schematically illustrates an embodiment with two
tether building blocks coupled, for example, by a reversible
covalent bond between moieties X and Y and by noncovalent
interaction between moieties A and B.
[0022] FIG. 6 schematically illustrates an embodiment of a test
ligand bound to an embodiment of an artificial receptor including a
plurality of building blocks including a rigid tether and a
plurality of building blocks including a flexible tether.
[0023] FIG. 7 schematically illustrates the receptor and test
ligand of FIG. 15, the test ligand displaying an altered form of
feature VE, VE.sub.2.
[0024] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand, such as a molecule or cell.
[0025] FIG. 9 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors.
[0026] FIG. 10 schematically illustrates certain binding patterns
on an array of working artificial receptors.
[0027] FIG. 11 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand.
[0028] FIG. 12 schematically illustrates an embodiment of a method
for detecting an agent that disrupts a binding interaction of a
target molecule.
[0029] FIG. 13 schematically illustrates an embodiment of a method
for detecting an agent that disrupts a binding interaction of a
complex including a target molecule.
[0030] FIG. 14 schematically illustrates a candidate disruptor
disrupting a protein:protein complex.
[0031] FIG. 15 schematically illustrates an embodiment of a method
of employing the present artificial receptors to produce or as an
affinity support.
[0032] FIG. 16 schematically illustrates evaluating an array of
candidate artificial receptors for binding of a test ligand and
selecting one or more working artificial receptors for binding or
operating on a test ligand.
[0033] FIG. 17 schematically illustrates identification of a lead
artificial receptor from among candidate artificial receptors.
[0034] FIG. 18 schematically illustrates a false color fluorescence
image of a labeled microarray according to an embodiment of the
present invention.
[0035] FIG. 19 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding phycoerythrin.
[0036] FIG. 20 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding phycoerythrin.
[0037] FIG. 21 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of ovalbumin.
[0038] FIG. 22 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of ovalbumin.
[0039] FIG. 23 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of bovine serum
albumin.
[0040] FIG. 24 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of bovine serum
albumin.
[0041] FIG. 25 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding an acetylated horseradish peroxidase.
[0042] FIG. 26 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding an acetylated horseradish peroxidase.
[0043] FIG. 27 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding a TCDD derivative of horseradish peroxidase.
[0044] FIG. 28 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding a TCDD derivative of horseradish peroxidase.
[0045] FIG. 29 schematically illustrates a subset of the data
illustrated in FIG. 5.
[0046] FIG. 30 schematically illustrates a subset of the data
illustrated in FIG. 5.
[0047] FIG. 31 schematically illustrates a subset of the data
illustrated in FIG. 5.
[0048] FIG. 32 schematically illustrates a correlation of binding
data for phycoerythrin against logP for the building blocks making
up the artificial receptor.
[0049] FIG. 33 schematically illustrates a correlation of binding
data for phycoerythrin against logP for the building blocks making
up the artificial receptor.
[0050] FIG. 34 schematically illustrates a two dimensional plot
comparing data obtained for candidate artificial receptors
contacted with and/or binding phycoerythrin to data obtained for
candidate artificial receptors contacted with and/or binding a
fluorescent derivative of bovine serum albumin.
[0051] FIGS. 35, 36, and 37 schematically illustrate subsets of
data from FIGS. 5, 9, and 7, respectively, and demonstrate that the
array of artificial receptors according to the present invention
yields receptors distinguished between three analytes,
phycoerythrin, bovine serum albumin, and ovalbumin.
[0052] FIG. 38 schematically illustrates a gray scale image of the
fluorescence signal from a scan of a control plate which was
prepared by washing off the building blocks with organic solvent
before incubation with the test ligand.
[0053] FIG. 39 schematically illustrates a gray scale image of the
fluorescence signal from a scan of an experimental plate which was
incubated with 1.0 .mu.g/ml Cholera Toxin B at 23.degree. C.
[0054] FIG. 40 schematically illustrates a gray scale image of the
fluorescence signal from a scan of an experimental plate which was
incubated with 1.0 .mu.g/ml Cholera Toxin B at 3.degree. C.
[0055] FIG. 41 schematically illustrates a gray scale image of the
fluorescence signal from a scan of an experimental plate which was
incubated with 1.0 .mu.g/ml Cholera Toxin B at 43.degree. C.
[0056] FIGS. 42-44 schematically illustrate plots of the
fluorescence signals obtained from the candidate artificial
receptors illustrated in FIG. 36-41.
[0057] FIG. 45 schematically illustrate plots of the fluorescence
signals obtained from the combinations of building blocks employed
in the present studies, when those building blocks are covalently
linked to the support. Binding was conducted at 23.degree. C.
[0058] FIG. 46 schematically illustrates the changes in
fluorescence signal from individual combinations of covalently
immobilized building blocks at 4.degree. C., 23.degree. C., or
44.degree. C.
[0059] FIG. 47 schematically illustrates a graph of the changes in
fluorescence signal from individual combinations of building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C.
[0060] FIG. 48 schematically illustrates the data presented in FIG.
46 (lines marked A) and the data presented in FIG. 47 (lines marked
B).
[0061] FIG. 49 schematically illustrates a graph of the
fluorescence signal at 44.degree. C. divided by the signal at
23.degree. C. against the fluorescence signal obtained from binding
at 23.degree. C. for the artificial receptors with reversibly
immobilized receptors.
[0062] FIG. 50 illustrates fluorescence signals produced by binding
of cholera toxin to a microarray of the present candidate
artificial receptors followed by washing with buffer in an
experiment reported in Example 4.
[0063] FIG. 51 illustrates the fluorescence signals due to cholera
toxin binding that were detected upon competition with GM1 OS (0.34
.mu.M) in an experiment reported in Example 4.
[0064] FIG. 52 illustrates the ratio of the amount bound in the
absence of GM1 OS to the amount bound in competition with GM1 OS
(0.34 .mu.M) in an experiment reported in Example 4.
[0065] FIG. 53 illustrates fluorescence signals produced by binding
of cholera toxin to a microarray of the present candidate
artificial receptors followed by washing with buffer in an
experiment reported in Example 4 and for comparison with
competition experiments using 5.1 .mu.M GM1 OS.
[0066] FIG. 54 illustrates the fluorescence signals due to cholera
toxin binding that were detected upon competition with GM1 OS (5.1
.mu.M) in an experiment reported in Example 4.
[0067] FIG. 55 illustrates the ratio of the amount bound in the
absence of GM1 OS to the amount bound in competition with GM1 OS
(5.1 .mu.M) in an experiment reported in Example 4.
[0068] FIG. 56 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors alone and in competition with each of the three
concentrations of GM1 in the experiment reported in Example 5.
[0069] FIG. 57 illustrates the ratio of the amount bound in the
absence of GM1 OS to the amount bound upon competition with GM1 for
the low concentration of GM1 employed in Example 5.
[0070] FIG. 58 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors without pretreatment with GM1 in the experiment reported
in Example 6.
[0071] FIGS. 59-61 illustrate the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors with pretreatment with GM1 (100 g/ml, 10 .mu.g/ml, and 1
.mu.g/ml GM1, respectively) in the experiment reported in Example
6.
[0072] FIG. 62 illustrates the ratio of the amount bound in the
presence of 1 .mu.g/ml GM1 to the amount bound in the absence of
GM1 in the experiment reported in Example 6.
DETAILED DESCRIPTION
Definitions
[0073] As used herein, the term "peptide" refers to a compound
including two or more amino acid residues joined by amide
bond(s).
[0074] As used herein, the terms "polypeptide" and "protein" refer
to a peptide including more than about 20 amino acid residues
connected by peptide linkages.
[0075] As used herein, the term "proteome" refers to the expression
profile of the proteins of an organism, tissue, organ, or cell. The
proteome can be specific to a particular status (e.g., development,
health, etc.) of the organism, tissue, organ, or cell.
[0076] As used herein, the term "support" refers to a solid support
that is, typically, macroscopic.
[0077] As used herein, the term scaffold refers to a molecular
scale structure to which a plurality of building blocks can
covalently bind.
[0078] Reversibly immobilizing building blocks on a support couples
the building blocks to the support through a mechanism that allows
the building blocks to be uncoupled from the support without
destroying or unacceptably degrading the building block or the
support. That is, immobilization can be reversed without destroying
or unacceptably degrading the building block or the support. In an
embodiment, immobilization can be reversed with only negligible or
ineffective levels of degradation of the building block or the
support. Reversible immobilization can employ readily reversible
covalent bonding or noncovalent interactions. Suitable noncovalent
interactions include interactions between ions, hydrogen bonding,
van der Waals interactions, and the like. Readily reversible
covalent bonding refers to covalent bonds that can be formed and
broken under conditions that do not destroy or unacceptably degrade
the building block or the support.
[0079] A combination of building blocks immobilized on, for
example, a support can be a candidate artificial receptor, a lead
artificial receptor, or a working artificial receptor. That is, a
heterogeneous building block spot on a slide or a plurality of
building blocks coated on a tube or well can be a candidate
artificial receptor, a lead artificial receptor, or a working
artificial receptor. A candidate artificial receptor can become a
lead artificial receptor, which can become a working artificial
receptor.
[0080] As used herein the phrase "candidate artificial receptor"
refers to an immobilized combination of building blocks that can be
tested to determine whether or not a particular test ligand binds
to that combination. In an embodiment, the combination includes one
or more reversibly immobilized building blocks. In an embodiment,
the candidate artificial receptor can be a heterogeneous building
block spot on a slide or a plurality of building blocks coated on a
tube or well.
[0081] As used herein the phrase "lead artificial receptor" refers
to an immobilized combination of building blocks that binds a test
ligand at a predetermined concentration of test ligand, for example
at 10, 1, 0.1, or 0.01 .mu.g/ml, or at 1, 0.1, or 0.01 ng/ml. In an
embodiment, the combination includes one or more reversibly
immobilized building blocks. In an embodiment, the lead artificial
receptor can be a heterogeneous building block spot on a slide or a
plurality of building blocks coated on a tube or well.
[0082] As used herein the phrase "working artificial receptor"
refers to a combination of building blocks that binds a test ligand
with a selectivity and/or sensitivity effective for categorizing or
identifying the test ligand. That is, binding to that combination
of building blocks describes the test ligand as belonging to a
category of test ligands or as being a particular test ligand. A
working artificial receptor can, for example, bind the ligand at a
concentration of, for example, 100, 10, 1, 0.1, 0.01, or 0.001
ng/ml. In an embodiment, the combination includes one or more
reversibly immobilized building blocks. In an embodiment, the
working artificial receptor can be a heterogeneous building block
spot on a slide or a plurality of building blocks coated on a tube,
well, slide, or other support or on a scaffold.
[0083] As used herein the phrase "working artificial receptor
complex" refers to a plurality of artificial receptors, each a
combination of building blocks, that binds a test ligand with a
pattern of selectivity and/or sensitivity effective for
categorizing or identifying the test ligand. That is, binding to
the several receptors of the complex describes the test ligand as
belonging to a category of test ligands or as being a particular
test ligand. The individual receptors in the complex can each bind
the ligand at different concentrations or with different
affinities. For example, the individual receptors in the complex
each bind the ligand at concentrations of 100, 10, 1, 0.1, 0.01 or
0.001 ng/ml. In an embodiment, the combination includes one or more
reversibly immobilized building blocks. In an embodiment, the
working artificial receptor complex can be a plurality of
heterogeneous building block spots or regions on a slide; a
plurality of wells, each coated with a different combination of
building blocks; or a plurality of tubes, each coated with a
different combination of building blocks.
[0084] As used herein, the phrase "significant number of candidate
artificial receptors" refers to sufficient candidate artificial
receptors to provide an opportunity to find a working artificial
receptor, working artificial receptor complex, or lead artificial
receptor. As few as about 100 to about 200 candidate artificial
receptors can be a significant number for finding working
artificial receptor complexes suitable for distinguishing two
proteins (e.g., cholera toxin and phycoerythrin). In other
embodiments, a significant number of candidate artificial receptors
can include about 1,000 candidate artificial receptors, about
10,000 candidate artificial receptors, about 100,000 candidate
artificial receptors, or more.
[0085] Although not limiting to the present invention, it is
believed that the significant number of candidate artificial
receptors required to provide an opportunity to find a working
artificial receptor may be larger than the significant number
required to find a working artificial receptor complex. Although
not limiting to the present invention, it is believed that the
significant number of candidate artificial receptors required to
provide an opportunity to find a lead artificial receptor may be
larger than the significant number required to find a working
artificial receptor. Although not limiting to the present
invention, it is believed that the significant number of candidate
artificial receptors required to provide an opportunity to find a
working artificial receptor for a test ligand with few features may
be more than for a test ligand with many features.
[0086] As used herein, the term "building block" refers to a
molecular component of an artificial receptor including portions
that can be envisioned as or that include one or more linkers, one
or more frameworks, and one or more recognition elements. In an
embodiment, the building block includes a linker, a framework, and
one or more recognition elements. In an embodiment, the linker
includes a moiety suitable for reversibly immobilizing the building
block, for example, on a support, surface or lawn. The building
block interacts with the ligand.
[0087] As used herein, the term "linker" refers to a portion of or
functional group on a building block that can be employed to or
that does (e.g., reversibly) couple the building block to a
support, for example, through covalent link, ionic interaction,
electrostatic interaction, or hydrophobic interaction.
[0088] As used herein, the term "framework" refers to a portion of
a building block including the linker or to which the linker is
coupled and to which one or more recognition elements are
coupled.
[0089] As used herein, the term "recognition element" refers to a
portion of a building block coupled to the framework but not
covalently coupled to the support. Although not limiting to the
present invention, the recognition element can provide or form one
or more groups, surfaces, or spaces for interacting with the
ligand.
[0090] As used herein, the phrase "plurality of building blocks"
refers to two or more building blocks of different structure in a
mixture, in a kit, or on a support or scaffold. Each building block
has a particular structure, and use of building blocks in the
plural, or of a plurality of building blocks, refers to more than
one of these particular structures. Building blocks or plurality of
building blocks does not refer to a plurality of molecules each
having the same structure.
[0091] As used herein, the phrase "combination of building blocks"
refers to a plurality of building blocks that together are in a
spot, region, or a candidate, lead, or working artificial receptor.
A combination of building blocks can be a subset of a set of
building blocks. For example, a combination of building blocks can
be one of the possible combinations of 2, 3, 4, 5, or 6 building
blocks from a set of N (e.g., N=10-200) building blocks.
[0092] As used herein, the phrases "homogenous immobilized building
block" and "homogenous immobilized building blocks" refer to a
support or spot having immobilized on or within it only a single
building block.
[0093] As used herein, the phrase "activated building block" refers
to a building block activated to make it ready to form a covalent
bond to a functional group, for example, on a support. A building
block including a carboxyl group can be converted to a building
block including an activated ester group, which is an activated
building block. An activated building block including an activated
ester group can react, for example, with an amine to form a
covalent bond.
[0094] As used herein, the term "naive" used with respect to one or
more building blocks refers to a building block that has not
previously been determined or known to bind to a test ligand of
interest. For example, the recognition element(s) on a naive
building block has not previously been determined or known to bind
to a test ligand of interest. A building block that is or includes
a known ligand (e.g., GM1) for a particular protein (test ligand)
of interest (e.g., cholera toxin) is not naive with respect to that
protein (test ligand).
[0095] As used herein, the term "immobilized" used with respect to
building blocks coupled to a support refers to building blocks
being stably oriented on the support so that they do not migrate on
the support or release from the support. Building blocks can be
immobilized by covalent coupling, by ionic interactions, by
electrostatic interactions, such as ion pairing, or by hydrophobic
interactions, such as van der Waals interactions.
[0096] As used herein a "region" of a support, tube, well, or
surface refers to a contiguous portion of the support, tube, well,
or surface. Building blocks coupled to a region can refer to
building blocks in proximity to one another in that region.
[0097] As used herein, a "bulky" group on a molecule is larger than
a moiety including 7 or 8 carbon atoms.
[0098] As used herein, a "small" group on a molecule is hydrogen,
methyl, or another group smaller than a moiety including 4 carbon
atoms.
[0099] As used herein, the term "lawn" refers to a layer, spot, or
region of functional groups on a support, for example, at a density
sufficient to place coupled building blocks in proximity to one
another. The functional groups can include groups capable of
forming covalent, ionic, electrostatic, or hydrophobic interactions
with building blocks.
[0100] As used herein, the term "alkyl" refers to saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In certain embodiments, a straight chain or branched chain
alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.12 for straight chain, C.sub.1-C.sub.6 for branched
chain). Likewise, cycloalkyls can have from 3-10 carbon atoms in
their ring structure, for example, 5, 6 or 7 carbons in the ring
structure.
[0101] The term "alkyl" as used herein refers to both
"unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, a halogen, a hydroxyl, a
carbonyl (such as a carboxyl, an ester, a formyl, or a ketone), a
thiocarbonyl (such as a thioester, a thioacetate, or a
thioformate), an alkoxyl, a phosphoryl, a phosphonate, a
phosphinate, an amino, an amido, an amidine, an imine, a cyano, a
nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a
sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl,
an aryl alkyl, or an aromatic or heteroaromatic moiety. The
moieties substituted on the hydrocarbon chain can themselves be
substituted, if appropriate. For example, the substituents of a
substituted alkyl can include substituted and unsubstituted forms
of the groups listed above.
[0102] The phrase "aryl alkyl", as used herein, refers to an alkyl
group substituted with an aryl group (e.g., an aromatic or
heteroaromatic group).
[0103] As used herein, the terms "alkenyl" and "alkynyl" refer to
unsaturated aliphatic groups analogous in length and optional
substitution to the alkyls groups described above, but that contain
at least one double or triple bond respectively.
[0104] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics". The
aromatic ring can be substituted at one or more ring positions with
such substituents such as those described above for alkyl groups.
The term "aryl" also includes polycyclic ring systems having two or
more cyclic rings in which two or more carbons are common to two
adjoining rings (the rings are "fused rings") wherein at least one
of the rings is aromatic, e.g., the other cyclic ring(s) can be
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
heterocyclyls.
[0105] As used herein, the terms "heterocycle" or "heterocyclic
group" refer to 3- to 12-membered ring structures, e.g., 3- to
7-membered rings, whose ring structures include one to four
heteroatoms. Heterocyclyl groups include, for example, thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,
phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,
pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,
indole, indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine,
pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,
furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,
piperidine, piperazine, morpholine, lactones, lactams such as
azetidinones and pyrrolidinones, sultams, sultones, and the like.
The heterocyclic ring can be substituted at one or more positions
with such substituents such as those described for alkyl
groups.
[0106] As used herein, the term "heteroatom" as used herein means
an atom of any element other than carbon or hydrogen, such as
nitrogen, oxygen, sulfur and phosphorous.
Overview of the Artificial Receptor
[0107] FIG. 1 schematically illustrates an embodiment employing 4
distinct building blocks in a spot on a microarray to make a ligand
binding site. This Figure illustrates a group of 4 building blocks
at the corners of a square forming a unit cell. A group of four
building blocks can be envisioned as the vertices on any
quadrilateral. FIG. 1 illustrates that spots or regions of building
blocks can be envisioned as multiple unit cells, in this
illustration square unit cells. Groups of unit cells of four
building blocks in the shape of other quadrilaterals can also be
formed on a support.
[0108] Each immobilized building block molecule can provide one or
more "arms" extending from a "framework" and each can include
groups that interact with a ligand or with portions of another
immobilized building block. FIG. 2 illustrates that combinations of
four building blocks, each including a framework with two arms
(called "recognition elements"), provides a molecular configuration
of building blocks that form a site for binding a ligand. Such a
site formed by building blocks such as those exemplified below can
bind a small molecule, such as a drug, metabolite, pollutant, or
the like, and/or can bind a larger ligand such as a macromolecule
or microbe.
[0109] The present artificial receptors can include building blocks
reversibly immobilized on a support or surface. Reversing
immobilization of the building blocks can allow movement of
building blocks to a different location on the support or surface,
or exchange of building blocks onto and off of the surface. For
example, the combinations of building blocks can bind a ligand when
reversibly coupled to or immobilized on the support. Reversing the
coupling or immobilization of the building blocks provides
opportunity for rearranging the building blocks, which can improve
binding of the ligand. Further, the present invention can allow for
adding additional or different building blocks, which can further
improve binding of a ligand.
[0110] FIG. 3 schematically illustrates an embodiment employing an
initial artificial receptor surface (A) with four different
building blocks on the surface, which are represented by shaded
shapes. This initial artificial receptor surface (A) undergoes (1)
binding of a ligand to an artificial receptor and (2) shuffling the
building blocks on the receptor surface to yield a lead artificial
receptor (B). Shuffling refers to reversing the coupling or
immobilization of the building blocks and allowing their
rearrangement on the receptor surface. After forming a lead
artificial receptor, additional building blocks can be (3)
exchanged onto and/or off of the receptor surface (C). Exchanging
refers to building blocks leaving the surface and entering a
solution contacting the surface and/or building blocks leaving a
solution contacting the surface and becoming part of the artificial
receptor. The additional building blocks can be selected for
structural diversity (e.g., randomly) or selected based on the
structure of the building blocks in the lead artificial receptor to
provide additional avenues for improving binding. The original and
additional building blocks can then be (4) shuffled and exchanged
to provide higher affinity artificial receptors on the surface
(D).
Adaptive Artificial Receptor
[0111] In an embodiment, the present invention relates to an
artificial receptor including a tethered building block. A tethered
building block can include one or more recognition elements at an
end of the building block. Such a building block can be envisioned
as including a framework moiety located at or forming that end of
the building block. The recognition elements can be coupled to the
framework moiety. Such a building block can also include a tether
moiety.
[0112] The tether moiety can provide spacing or distance between
the recognition element and the support or scaffold to which the
building block is immobilized. A tether moiety can have any of a
variety of characteristics or properties including flexibility,
rigidity or stiffness, ability to bond to another tether moiety,
and the like. The tether moiety can include the linker. The
framework moiety be envisioned as forming all or part of the tether
moiety. FIG. 4 schematically illustrates an embodiment of a
building block including a flexible tether (left) and a building
block including a rigid tether (right).
[0113] The tether can include groups suitable for coupling one
tether building block to another or one tether to another. Such
coupling can provide, for example, rigidity or positioning to a
building block with a flexible tether. Such coupling can maintain,
for example, two building blocks in proximity to one another. The
coupling can be reversible, which can allow the coupled building
blocks to "change partners" and couple to no or a different
building block. FIG. 5 schematically illustrates an embodiment with
two tether building blocks coupled, for example, by a reversible
covalent bond between moieties X and Y and by noncovalent
interaction between moieties A and B. The specification below
describes suitable groups for forming reversible covalent bonds and
noncovalent interactions.
[0114] An artificial receptor can include a plurality of building
blocks with one or more of the building blocks having a tether
moiety. For example, an artificial receptor can include at least
one building block without a tether moiety, at least one building
block with a linker suitable for reversible immobilization on a
support, or at least one tether building block. For example, an
artificial receptor can include a plurality of tether building
blocks, which can include at least one building block including a
rigid tether or at least one building block including a flexible
tether. In an embodiment, the artificial receptor can include at
least one building block including a rigid tether and at least one
building block including a flexible tether. In an embodiment, the
artificial receptor can include a plurality of building blocks
including a rigid tether and a plurality of building blocks
including a flexible tether.
[0115] FIG. 6 schematically illustrates an embodiment of a test
ligand bound to an embodiment of an artificial receptor including a
plurality of building blocks including a rigid tether and a
plurality of building blocks including a flexible tether. The
illustrated test ligand includes three features of interest, FE1,
FE2, and VE1. These features can be thought of as epitopes that can
be recognized by the artificial receptor, which can be considered
analogous to an antibody. Two of the features can be considered to
have fixed structures. One of the features can be considered to
have a structure that can change or be altered. For example,
features FE1 and FE2 can be visualized as fixed epitopes on the
surface of a microbe. For example, feature VE1 can be visualized as
a variable epitope on the surface of a microbe.
[0116] The artificial receptor schematically illustrated in FIG. 6
includes 12 different building blocks, 6 with rigid tethers and 6
with flexible tethers, each coupled to a support. In this
illustration, the 6 building blocks with rigid tethers bind to
features FE1 and FE2, which can be considered fixed epitopes.
Heterogeneous building block combinations that bind to each of
features FE1 and FE2 can be selected by methods described herein
and in greater detail in Applicant's co-pending applications. In
this illustration, 3 of the building blocks with flexible tethers
bind to feature VE.sub.1, which can be considered form 1 of a
variable epitope. Combinations of building blocks including
flexible tethers and that bind to feature VE.sub.1 can be selected
by methods including those described herein and those employed for
selecting combinations of other building blocks.
[0117] As schematically illustrated in FIG. 6, the artificial
receptor includes 3 building blocks with flexible tethers not bound
to any feature of the test ligand. In practice, an artificial
receptor could include any number of different unbound building
blocks with flexible tethers. The unbound flexible tether building
blocks can be selected for their ability to bind to different forms
of the variable feature, to other features of the test ligand, to
another test ligand, or the like. The unbound flexible tether
building blocks can be selected to be naive to the variable feature
or the test ligand. That is, the unbound flexible tether building
blocks can be selected so that one or more of them has the
possibility of binding to, for example, a different form of the
variable feature.
[0118] FIG. 7 schematically illustrates the receptor and test
ligand of FIG. 6, the test ligand displaying an altered form of
feature VE, VE.sub.2. The alternate forms of feature VE can be
considered, for example, as different forms of a variable protein
expressed on the surface of a microbe. The alternate forms of
feature VE can be considered, for example, as different structural
features on isoforms or variants of a protein. The alternate forms
of feature VE can be considered, for example, as different
structural features on isomeric or homomorphic compounds.
[0119] As schematically illustrated in FIG. 7, the rigid tether
building blocks each still bind features FE1 and FE2. Binding of
the flexible tether building blocks to form VE.sub.2 of feature VE
has changed. The flexible tether building blocks that had bound to
VE1 are now unbound. The flexible tether building blocks that had
been unbound are now bound to VE.sub.2. The artificial receptor
including flexible tether building blocks can bind a plurality of
forms of the test ligand. As illustrated, the artificial receptor
binds to different forms of the test ligand including versions
VE.sub.1 and VE.sub.2 of feature VE. An artificial receptor
including one or more flexible tether building blocks can be
referred to as an adaptive receptor.
Methods of Making an Adaptive Artificial Receptor
[0120] The present invention relates to a method of making an
artificial receptor or a candidate artificial receptor. In an
embodiment, this method includes preparing a spot or region on a
support, the spot or region including a plurality of building
blocks immobilized on the support. In an embodiment, at least one
of the building blocks includes a tether moiety. The method can
include forming a plurality of spots on a solid support, each spot
including a plurality of building blocks, and immobilizing (e.g.,
reversibly) a plurality of building blocks on the solid support in
each spot. In an embodiment, at least one of the building blocks in
at least one of the spots includes a tether. In an embodiment, an
array of such spots is referred to as a heterogeneous building
block array.
[0121] The method can include mixing a plurality of building blocks
and employing the mixture in forming the spot(s). In an embodiment,
at least one of the building blocks includes a tether moiety.
Alternatively, the method can include spotting individual building
blocks on the support. Coupling building blocks to the support can
employ covalent bonding or noncovalent interactions. Suitable
noncovalent interactions include interactions between ions,
hydrogen bonding, van der Waals interactions, and the like. In an
embodiment, the support can be functionalized with moieties that
can engage in covalent bonding or noncovalent interactions. Forming
spots can yield a microarray of spots of heterogeneous combinations
of building blocks (at least one spot including at least one tether
building block), each of which can be a candidate artificial
receptor. The method can apply or spot building blocks onto a
support in combinations of 2, 3, 4, or more building blocks. In an
embodiment, at least one of the building blocks includes a tether
moiety.
[0122] In an embodiment, the present method can be employed to
produce a solid support having on its surface a plurality of
regions or spots, each region or spot including a plurality of
building blocks. In an embodiment, at least one of the building
blocks includes a tether moiety. For example, the method can
include spotting a glass slide with a plurality of spots, each spot
including a plurality of building blocks. Such a spot can be
referred to as including heterogeneous building blocks. A plurality
of spots of building blocks can be referred to as an array of
spots.
[0123] In an embodiment, the present method includes making a
receptor surface. Making a receptor surface can include forming a
region on a solid support, the region including a plurality of
building blocks, and immobilizing (e.g., reversibly) the plurality
of building blocks to the solid support in the region. In an
embodiment, at least one of the building blocks includes a tether
moiety. The method can include mixing a plurality of building
blocks and employing the mixture in forming the region or regions.
Alternatively, the method can include applying individual building
blocks in a region on the support. Forming a region on a support
can be accomplished, for example, by soaking a portion of the
support with the building block solution. The resulting coating
including building blocks can be referred to as including
heterogeneous building blocks. In an embodiment, at least one of
the building blocks includes a tether moiety.
[0124] A region including a plurality of building blocks can be
independent and distinct from other regions including a plurality
of building blocks. In an embodiment, one or more regions including
a plurality of building blocks can overlap to produce a region
including the combined pluralities of building blocks. In an
embodiment, two or more regions including a single building block
can overlap to form one or more regions each including a plurality
of building blocks. In an embodiment, at least one of the building
blocks includes a tether moiety. The overlapping regions can be
envisioned, for example, as portions of overlap in a Venn diagram,
or as portions of overlap in a pattern like a plaid or tweed.
[0125] In an embodiment, the method produces a spot or surface with
a density of building blocks sufficient to provide interactions of
more than one building block with a ligand. That is, the building
blocks can be in proximity to one another. Proximity of different
building blocks can be detected by determining different (e.g.,
greater) binding of a test ligand to a spot or surface including a
plurality of building blocks compared to a spot or surface
including only one of the building blocks.
[0126] In an embodiment, the method includes forming an array of
heterogeneous spots made from combinations of a subset of the total
building blocks and/or smaller groups of the building blocks in
each spot. That is, the method forms spots including only, for
example, 2 or 3 building blocks, rather than 4 or 5. For example,
the method can form spots from combinations of a full set of
building blocks (e.g. 81 of a set of 81) in groups of 2 and/or 3.
For example, the method can form spots from combinations of a
subset of the building blocks (e.g., 25 of the set of 81) in groups
of 4 or 5. For example, the method can form spots from combinations
of a subset of the building blocks (e.g., 25 of a set of 81) in
groups of 2 or 3. The method can include forming additional arrays
incorporating building blocks, lead artificial receptors, or
structurally similar building blocks.
[0127] In an embodiment, the method includes forming an array
including one or more spots that function as controls for
validating or evaluating binding to artificial receptors of the
present invention. In an embodiment, the method includes forming
one or more regions, tubes, or wells that function as controls for
validating or evaluating binding to artificial receptors of the
present invention. Such a control spot, region, tube, or well can
include no building block, only a single building block, only
functionalized lawn, or combinations thereof.
[0128] The method can immobilize (e.g., reversibly) building blocks
on supports using known methods for immobilizing compounds of the
types employed as building blocks. Coupling building blocks to the
support can employ covalent bonding or noncovalent interactions.
Suitable noncovalent interactions include interactions between
ions, hydrogen bonding, van der Waals interactions, and the like.
In an embodiment, the support can be functionalized with moieties
that can engage in reversible covalent bonding, moieties that can
engage in noncovalent interactions, a mixture of these moieties, or
the like.
[0129] In an embodiment, the support can be functionalized with
moieties that can engage in covalent bonding, e.g., reversible
covalent bonding. The present invention can employ any of a variety
of the numerous known functional groups, reagents, and reactions
for forming reversible covalent bonds. Suitable reagents for
forming reversible covalent bonds include those described in Green,
T W; Wuts, P G M (1999), Protective Groups in Organic Synthesis
Third Edition, Wiley-Interscience, New York, 779 pp. For example,
the support can include functional groups such as a carbonyl group,
a carboxyl group, a silane group, boric acid or ester, an amine
group (e.g., a primary, secondary, or tertiary amine, a
hydroxylamine, a hydrazine, or the like), a thiol group, an alcohol
group (e.g., primary, secondary, or tertiary alcohol), a diol group
(e.g., a 1,2 diol or a 1,3 diol), a phenol group, a catechol group,
or the like. These functional groups can form groups with
reversible covalent bonds, such as ether (e.g., alkyl ether, silyl
ether, thioether, or the like), ester (e.g., alkyl ester, phenol
ester, cyclic ester, thioester, or the like), acetal (e.g., cyclic
acetal), ketal (e.g., cyclic ketal), silyl derivative (e.g., silyl
ether), boronate (e.g., cyclic boronate), amide, hydrazide, imine,
carbamate, or the like. Such a functional group can be referred to
as a covalent bonding moiety, e.g., a first covalent bonding
moiety.
[0130] A carbonyl group on the support and an amine group on a
building block can form an imine or Schiff's base. The same is true
of an amine group on the support and a carbonyl group on a building
block. A carbonyl group on the support and an alcohol group on a
building block can form an acetal or ketal. The same is true of an
alcohol group on the support and a carbonyl group on a building
block. A thiol (e.g., a first thiol) on the support and a thiol
(e.g., a second thiol) on the building block can form a
disulfide.
[0131] A carboxyl group on the support and an alcohol group on a
building block can form an ester. The same is true of an alcohol
group on the support and a carboxyl group on a building block. Any
of a variety of alcohols and carboxylic acids can form esters that
provide covalent bonding that can be reversed in the context of the
present invention. For example, reversible ester linkages can be
formed from alcohols such as phenols with electron withdrawing
groups on the aryl ring, other alcohols with electron withdrawing
groups acting on the hydroxyl-bearing carbon, other alcohols, or
the like; and/or carboxyl groups such as those with electron
withdrawing groups acting on the acyl carbon (e.g., nitrobenzylic
acid, R--CF.sub.2--COOH, R--CCl.sub.2--COOH, and the like), other
carboxylic acids, or the like.
[0132] In an embodiment, the support, matrix, or lawn can be
functionalized with moieties that can engage in noncovalent
interactions. For example, the support can include functional
groups such as an ionic group, a group that can hydrogen bond, or a
group that can engage in van der Waals or other hydrophobic
interactions. Such functional groups can include cationic groups,
anionic groups, lipophilic groups, amphiphilic groups, and the
like.
[0133] In an embodiment, the support, matrix, or lawn includes a
charged moiety (e.g., a first charged moiety). Suitable charged
moieties include positively charged moieties and negatively charged
moieties. Suitable positively charged moieties (e.g., at neutral pH
in aqueous compositions) include amines, quaternary ammonium
moieties, ferrocene, or the like. Suitable negatively charged
moieties (e.g., at neutral pH in aqueous compositions) include
carboxylates, phenols substituted with strongly electron
withdrawing groups (e.g., tetrachlorophenols), phosphates,
phosphonates, phosphinates, sulphates, sulphonates,
thiocarboxylates, hydroxamic acids, or the like.
[0134] In an embodiment, the support, matrix, or lawn includes
groups that can hydrogen bond (e.g., a first hydrogen bonding
group), either as donors or acceptors. The support, matrix, or lawn
can include a surface or region with groups that can hydrogen bond.
For example, the support, matrix, or lawn can include a surface or
region including one or more carboxyl groups, amine groups,
hydroxyl groups, carbonyl groups, or the like. Ionic groups can
also participate in hydrogen bonding.
[0135] In an embodiment, the support, matrix, or lawn includes a
lipophilic moiety (e.g., a first lipophilic moiety). Suitable
lipophilic moieties include branched or straight chain C.sub.6-36
alkyl, C.sub.8-24 alkyl, C.sub.12-24 alkyl, C.sub.12-18 alkyl, or
the like; C.sub.6-36 alkenyl, C.sub.8-24 alkenyl, C.sub.12-24
alkenyl, C.sub.12-18 alkenyl, or the like, with, for example, 1 to
4 double bonds; C.sub.6-36 alkynyl, C.sub.8-24 alkynyl, C.sub.12-24
alkynyl, C.sub.12-18 alkynyl, or the like, with, for example, 1 to
4 triple bonds; chains with 1-4 double or triple bonds; chains
including aryl or substituted aryl moieties (e.g., phenyl or
naphthyl moieties at the end or middle of a chain); polyaromatic
hydrocarbon moieties; cycloalkane or substituted alkane moieties
with numbers of carbons as described for chains; combinations or
mixtures thereof; or the like. The alkyl, alkenyl, or alkynyl group
can include branching; within chain functionality like an ether
group; terminal functionality like alcohol, amide, carboxylate or
the like; or the like. A lipophilic moiety like a quaternary
ammonium lipophilic moiety can also include a positive charge.
Building Blocks for Adaptive Artificial Receptors
[0136] The present invention relates to building blocks for making
or forming candidate artificial receptors. Building blocks can be
designed, made, and selected to provide a variety of structural
characteristics among a small number of compounds. A building block
can provide one or more structural characteristics such as positive
charge, negative charge, acid, base, electron acceptor, electron
donor, hydrogen bond donor, hydrogen bond acceptor, free electron
pair, .pi. electrons, charge polarization, hydrophilicity,
hydrophobicity, and the like. A building block can be bulky or it
can be small.
[0137] A building block can be visualized as including several
components, such as one or more frameworks, one or more linkers,
one or more recognition elements, and/or one or more tethers. The
framework can be covalently coupled to each of the other building
block components. The linker can be covalently coupled to the
framework. The linker can be coupled to a support through one or
more of covalent, electrostatic, hydrogen bonding, van der Waals,
or like interactions. The recognition element can be covalently
coupled to the framework. The tether can be covalently coupled to
the linker and to the framework. In an embodiment, a building block
includes a framework, a linker, a recognition element, and a
tether. In an embodiment, a building block includes a framework, a
linker, a tether, and two recognition elements.
[0138] A description of general and specific features and functions
of a variety of building blocks and their synthesis can be found in
copending U.S. patent application Ser. Nos. 10/244,727, filed Sep.
16, 2002, 10/813,568, filed Mar. 29, 2004, and Application No.
PCT/US03/05328, filed Feb. 19, 2003, each entitled "ARTIFICIAL
RECEPTORS, BUILDING BLOCKS, AND METHODS"; U.S. patent application
Ser. Nos. 10/812,850 and 10/813,612, and application No.
PCT/US2004/009649, each filed Mar. 29, 2004 and each entitled
"ARTIFICIAL RECEPTORS INCLUDING REVERSIBLY IMMOBILIZED BUILDING
BLOCKS, THE BUILDING BLOCKS, AND METHODS"; and U.S. Provisional
Patent Application Nos. 60/499,965, filed Sep. 3, 2003, and
60/526,699, filed Dec. 2, 2003, each entitled BUILDING BLOCKS FOR
ARTIFICIAL RECEPTORS; the disclosures of which are incorporated
herein by reference. These patent documents include, in particular,
a detailed written description of: function, structure, and
configuration of building blocks, framework moieties, recognition
elements, synthesis of building blocks, specific embodiments of
building blocks, specific embodiments of recognition elements, and
sets of building blocks.
Framework
[0139] The framework can be selected for functional groups that
provide for coupling to the recognition moiety and for coupling to
or being the tether and/or linking moieties. The framework can
interact with the ligand as part of the artificial receptor. In an
embodiment, the framework includes multiple reaction sites with
orthogonal and reliable functional groups and with controlled
stereochemistry. Suitable functional groups with orthogonal and
reliable chemistries include, for example, carboxyl, amine,
hydroxyl, phenol, carbonyl, and thiol groups, which can be
individually protected, deprotected, and derivatized. In an
embodiment, the framework has two, three, or four functional groups
with orthogonal and reliable chemistries. In an embodiment, the
framework has three functional groups. In such an embodiment, the
three functional groups can be independently selected, for example,
from carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol group.
The framework can include alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, and like moieties.
[0140] A general structure for a framework with three functional
groups can be represented by Formula 1a:
##STR00001##
A general structure for a framework with four functional groups can
be represented by Formula 1b:
##STR00002##
In these general structures: R.sub.1 can be a 1-12, a 1-6, or a 1-4
carbon alkyl, substituted alkyl, cycloalkyl, heterocyclic,
substituted heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl
alkyl, or like group; and F.sub.1, F.sub.2, F.sub.3, or F.sub.4 can
independently be a carboxyl, amine, hydroxyl, phenol, carbonyl, or
thiol group. F.sub.1, F.sub.2, F.sub.3, or F.sub.4 can
independently be a 1-12, a 1-6, a 1-4 carbon alkyl, substituted
alkyl, cycloalkyl, heterocyclic, substituted heterocyclic, aryl
alkyl, aryl, heteroaryl, heteroaryl alkyl, or inorganic group
substituted with carboxyl, amine, hydroxyl, phenol, carbonyl, or
thiol group. F.sub.3 and/or F.sub.4 can be absent.
[0141] A variety of compounds fit the formulas and text describing
the framework including amino acids, and naturally occurring or
synthetic compounds including, for example, oxygen and sulfur
functional groups. The compounds can be racemic, optically active,
or achiral. For example, the compounds can be natural or synthetic
amino acids, .alpha.-hydroxy acids, thioic acids, and the like.
[0142] Suitable molecules for use as a framework include a natural
or synthetic amino acid, particularly an amino acid with a
functional group (e.g., third functional group) on its side chain.
Amino acids include carboxyl and amine functional groups. The side
chain functional group can include, for natural amino acids, an
amine (e.g., alkyl amine, heteroaryl amine), hydroxyl, phenol,
carboxyl, thiol, thioether, or amidino group. Natural amino acids
suitable for use as frameworks include, for example, serine,
threonine, tyrosine, aspartic acid, glutamic acid, asparagine,
glutamine, cysteine, lysine, arginine, histidine. Synthetic amino
acids can include the naturally occurring side chain functional
groups or synthetic side chain functional groups which modify or
extend the natural amino acids with alkyl, substituted alkyl,
cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,
aryl, heteroaryl, heteroaryl alkyl, and like moieties as framework
and with carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol
functional groups. Suitable synthetic amino acids include
.beta.-amino acids and homo or .beta. analogs of natural amino
acids. In an embodiment, the framework amino acid can be serine,
threonine, or tyrosine, e.g., serine or tyrosine, e.g.,
tyrosine.
[0143] Although not limiting to the present invention, a framework
amino acid, such as serine, threonine, or tyrosine, with a linker
and two recognition elements can be visualized with one of the
recognition elements in a pendant orientation and the other in an
equatorial orientation, relative to the extended carbon chain of
the framework.
[0144] All of the naturally occurring and many synthetic amino
acids are commercially available. Further, forms of these amino
acids derivatized or protected to be suitable for reactions for
coupling to recognition element(s) and/or linkers can be purchased
or made by known methods (see, e.g., Green, T W; Wuts, P G M
(1999), Protective Groups in Organic Synthesis Third Edition,
Wiley-Interscience, New York, 779 pp.; Bodanszky, M.; Bodanszky, A.
(1994), The Practice of Peptide Synthesis Second Edition,
Springer-Verlag, New York, 217 pp.).
Tether
[0145] In an embodiment, the present invention relates to a
building block including a tether moiety. The tether can include
the framework. The tether moiety can provide spacing or distance
between the recognition element and the support or scaffold to
which the building block is immobilized. A tether moiety can have
any of a variety of characteristics or properties including
flexibility, rigidity or stiffness, ability to bond to another
tether moiety, and the like. The tether moiety can include the
linker. The framework moiety be envisioned as forming all or part
of the tether moiety.
[0146] Suitable tether moieties can include a polyethylene glycol,
a polyamide, a linear polymer, a peptide, a polypeptide, an
oligosaccharide, a polysaccharide, a semifunctionalized oligo- or
polyglycine. In an embodiment, the tether is or includes a polymer
of up to 2000 carbon atoms (e.g., up to 48 carbon atoms). Such a
polymer can be naturally occurring or synthetic. Suitable polymers
include a polyether or like polymer, such as a PEG, a
polyethyleneimine, polyacrylate (e.g., substituted polyacrylates),
salt thereof, a mixture or combination thereof, or the like.
Suitable PEGs include PEG 1500 up to PEG 20,000, for example, PEG
1450, PEG 3350, PEG 4500, PEG 8000, PEG 20,000, and the like.
[0147] Suitable tether moieties can include one or more branched or
straight chain C.sub.6-36 alkyl, C.sub.8-24 alkyl, C.sub.12-24
alkyl, C.sub.12-18 alkyl, or the like; C.sub.6-36 alkenyl,
C.sub.8-24 alkenyl, C.sub.12-24 alkenyl, C.sub.12-18 alkenyl, or
the like, with, for example, 1 to 4 double bonds; C.sub.6-36
alkynyl, C.sub.8-24 alkynyl, C.sub.12-24 alkynyl, C.sub.12-18
alkynyl, or the like, with, for example, 1 to 4 triple bonds;
chains with 1-4 double or triple bonds; chains including aryl or
substituted aryl moieties (e.g., phenyl or naphthyl moieties at the
end or middle of a chain); polyaromatic hydrocarbon moieties;
cycloalkane or substituted alkane moieties with numbers of carbons
as described for chains; combinations or mixtures thereof; or the
like. The alkyl, alkenyl, or alkynyl group can include branching;
within chain functionality like an ether group; terminal
functionality like alcohol, amide, carboxylate or the like; or the
like. In an embodiment, the lipophilic moiety includes or is a
12-carbon aliphatic moiety.
[0148] Rigid tether moieties can include conformationally
restricted groups such as imines, aromatics, and polyaromatics.
Rigid tether moieties can include one or more branched or straight
chain C.sub.6-36 alkenyl, C.sub.8-24 alkenyl, C.sub.12-24 alkenyl,
C.sub.12-18 alkenyl, or the like, with, for example, 2 to 8 double
bonds; C.sub.6-36 alkynyl, C.sub.8-24 alkynyl, C.sub.12-24 alkynyl,
C.sub.12-18 alkynyl, or the like, with, for example, 1 to 8 triple
bonds; chains with 3-8 double or triple bonds; chains including
aryl or substituted aryl moieties (e.g., phenyl or naphthyl
moieties at the end or middle of a chain); polyaromatic hydrocarbon
moieties; and the like. The alkenyl or alkynyl group can include
branching; within chain functionality like an ether group; terminal
functionality like alcohol, amide, carboxylate or the like; or the
like. Rigid tether moieties can include a steroid moiety, such as
cholesterol, a corrin or another porphyrin, a polynuclear aromatic
moiety, a polar polymer fixed with metal ions, or the like.
[0149] In an embodiment, a rigid tether moiety can include more
than one tether moiety. For example, a rigid tether moiety can
include a plurality of hydrophobic chains, such as those described
in the paragraph above and in the paragraph below. The hydrophobic
chains if held in sufficient proximity on the support or scaffold
will, in a hydrophobic solvent, form a grouping sufficiently rigid
to hold one or more sets of recognition elements in place. In
another embodiment, a rigid tether moiety can include a plurality
of otherwise flexible tether moieties crosslinked to one another.
The crosslinking can include, for example, covalent bonding,
electrostatic interactions, hydrogen bonding, or hydrophobic
interactions. Groups for forming such interactions are disclosed
herein.
[0150] Flexible tether moieties can include one or more branched or
straight chain C.sub.6-36 alkyl, C.sub.8-24 alkyl, C.sub.12-24
alkyl, C.sub.12-18 alkyl, or the like; C.sub.6-36 alkenyl,
C.sub.8-24 alkenyl, C.sub.12-24 alkenyl, C.sub.12-18 alkenyl, or
the like, with, for example, 1 to 2 double bonds; C.sub.6-36
alkynyl, C.sub.8-24 alkynyl, C.sub.12-24 alkynyl, C.sub.12-18
alkynyl, or the like, with, for example, 1 to 2 triple bonds;
chains with 1-2 double or triple bonds; chains including 1 to 2
aryl or substituted aryl moieties (e.g., phenyl or naphthyl
moieties at the end or middle of a chain); cycloalkane or
substituted alkane moieties with numbers of carbons as described
for chains; combinations or mixtures thereof; or the like. The
alkyl, alkenyl, or alkynyl group can include branching; within
chain functionality like an ether group; terminal functionality
like alcohol, amide, carboxylate or the like; or the like. In an
embodiment, the lipophilic moiety includes or is a 12-carbon
aliphatic moiety.
[0151] In an embodiment, the tether forms or can be visualized as
forming a covalent bond with an alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. Between the bond to the
framework and the group participating in or formed by the
interaction with the support or lawn, the linker can include an
alkyl, substituted alkyl, cycloalkyl, heterocyclic, substituted
heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl,
ethoxy or propoxy oligomer, a glycoside, or like moiety.
[0152] Suitable tethers can include, for example: the functional
group participating in or formed by the bond to the framework, the
functional group or groups participating in or formed by the
interaction with the support or lawn, and a tether backbone moiety.
The tether backbone moiety can include about 8 to about 200 carbon
or heteroatoms, about 12 to about 150 carbon or heteroatoms, about
16 to about 100 carbon or heteroatoms, about 16 to about 50 carbon
or heteroatoms, or the like. The tether backbone can include an
alkyl, substituted alkyl, cycloalkyl, heterocyclic, substituted
heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl,
ethoxy or propoxy oligomer, a glycoside, mixtures thereof, or like
moiety. Suitable tethers have structures such as
(CH.sub.2).sub.nCOOH, with n=12-24, n=17-24, or n=16-18.
[0153] The tether can interact with the ligand as part of the
artificial receptor. The tether can also provide bulk, distance
from the support, hydrophobicity, hydrophilicity, and like
structural characteristics to the building block. In an embodiment,
the tether forms a covalent bond with a functional group on the
framework. In an embodiment, the tether also includes a functional
group that can couple to the tether or to the support or lawn,
e.g., through covalent bonding or noncovalent interactions.
[0154] In an embodiment, the tether includes one or more moieties
for forming a reversible covalent bond, a hydrogen bond, or an
ionic interaction, e.g., with another tether moiety. For example,
the linker can include about 1 to about 20 reversible
bond/interaction moieties or about 2 to about 10 reversible
bond/interaction moieties.
[0155] In an embodiment, the tether includes one or more moieties
that can engage in reversible covalent bonding. Suitable groups for
reversible covalent bonding include those described hereinabove.
Such groups for reversible covalent bonds can be part of links
between tether moieties. The tether-tether links can include, for
example, imine, acetal, ketal, disulfide, ester, or like linkages.
Such functional groups can engage in reversible covalent bonding.
Such a functional group can be referred to as a covalent bonding
moiety.
[0156] In an embodiment, the tether can be functionalized with
moieties that can engage in noncovalent interactions. For example,
the tether can include functional groups such as an ionic group, a
group that can hydrogen bond, or a group that can engage in van der
Waals or other hydrophobic interactions. Such functional groups can
include cationic groups, anionic groups, lipophilic groups,
amphiphilic groups, and the like.
[0157] In an embodiment, the present methods and compositions can
employ a tether including a charged moiety. Suitable charged
moieties include positively charged moieties and negatively charged
moieties. Suitable positively charged moieties include protonated
amines, quaternary ammonium moieties, sulfonium, sulfoxonium,
phosphonium, ferrocene, and the like. Suitable negatively charged
moieties (e.g., at neutral pH in aqueous compositions) include
carboxylates, phenols substituted with strongly electron
withdrawing groups (e.g., tetrachlorophenols), phosphates,
phosphonates, phosphinates, sulphates, sulphonates,
thiocarboxylates, and hydroxamic acids.
[0158] In an embodiment, the present methods and compositions can
employ a tether including a group that can hydrogen bond, either as
donor or acceptor (e.g., a second hydrogen bonding group). For
example, the tether can include one or more carboxyl groups, amine
groups, hydroxyl groups, carbonyl groups, or the like. Ionic groups
can also participate in hydrogen bonding.
Recognition Element
[0159] The recognition element can be selected to provide one or
more structural characteristics to the building block. The
recognition element can interact with the ligand as part of the
artificial receptor. For example, the recognition element can
provide one or more structural characteristics such as positive
charge, negative charge, acid, base, electron acceptor, electron
donor, hydrogen bond donor, hydrogen bond acceptor, free electron
pair, .pi. electrons, charge polarization, hydrophilicity,
hydrophobicity, and the like. A recognition element can be a small
group or it can be bulky.
[0160] In an embodiment the recognition element can be a 1-12, a
1-6, or a 1-4 carbon alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, or like group. The recognition
element can be substituted with a group that includes or imparts
positive charge, negative charge, acid, base, electron acceptor,
electron donor, hydrogen bond donor, hydrogen bond acceptor, free
electron pair, .pi. electrons, charge polarization, hydrophilicity,
hydrophobicity, and the like.
[0161] Recognition elements with a positive charge (e.g., at
neutral pH in aqueous compositions) include protonated amines,
quaternary ammonium moieties, sulfonium, sulfoxonium, phosphonium,
ferrocene, and the like. Suitable amines include alkyl amines,
alkyl diamines, heteroalkyl amines, aryl amines, heteroaryl amines,
aryl alkyl amines, pyridines, heterocyclic amines (saturated or
unsaturated, the nitrogen in the ring or not), amidines,
hydrazines, and the like. Alkyl amines generally have 1 to 12
carbons, e.g., 1-8, and rings can have 3-12 carbons, e.g., 3-8.
Suitable alkyl amines include that of formula B9. Suitable
heterocyclic or alkyl heterocyclic amines include that of formula
A9. Suitable pyridines include those of formulas A5 and B5. Any of
the amines can be employed as a quaternary ammonium compound.
Additional suitable quaternary ammonium moieties include trimethyl
alkyl quaternary ammonium moieties, dimethyl ethyl alkyl quaternary
ammonium moieties, dimethyl alkyl quaternary ammonium moieties,
aryl alkyl quaternary ammonium moieties, pyridinium quaternary
ammonium moieties, and the like.
[0162] Recognition elements with a negative charge (e.g., at
neutral pH in aqueous compositions) include carboxylates, phenols
substituted with strongly electron withdrawing groups (e.g.,
substituted tetrachlorophenols), phosphates, phosphonates,
phosphinates, sulphates, sulphonates, thiocarboxylates, and
hydroxamic acids. Suitable carboxylates include alkyl carboxylates,
aryl carboxylates, and aryl alkyl carboxylates. Suitable phosphates
include phosphate mono-, di-, and tri-esters, and phosphate mono-,
di-, and tri-amides. Suitable phosphonates include phosphonate
mono- and di-esters, and phosphonate mono- and di-amides (e.g.,
phosphonamides). Suitable phosphinates include phosphinate esters
and amides.
[0163] Recognition elements with a negative charge and a positive
charge (at neutral pH in aqueous compositions) include sulfoxides,
betaines, and amine oxides.
[0164] Acidic recognition elements can include carboxylates,
phosphates, sulphates, and phenols. Suitable acidic carboxylates
include thiocarboxylates. Suitable acidic phosphates include the
phosphates listed hereinabove.
[0165] Basic recognition elements include amines. Suitable basic
amines include alkyl amines, aryl amines, aryl alkyl amines,
pyridines, heterocyclic amines (saturated or unsaturated, the
nitrogen in the ring or not), amidines, and any additional amines
listed hereinabove. Suitable alkyl amines include that of formula
B9. Suitable heterocyclic or alkyl heterocyclic amines include that
of formula A9. Suitable pyridines include those of formulas A5 and
B5.
[0166] Recognition elements including a hydrogen bond donor include
amines, amides, carboxyls, protonated phosphates, protonated
phosphonates, protonated phosphinates, protonated sulphates,
protonated sulphinates, alcohols, and thiols. Suitable amines
include alkyl amines, aryl amines, aryl alkyl amines, pyridines,
heterocyclic amines (saturated or unsaturated, the nitrogen in the
ring or not), amidines, ureas, and any other amines listed
hereinabove. Suitable alkyl amines include that of formula B9.
Suitable heterocyclic or alkyl heterocyclic amines include that of
formula A9. Suitable pyridines include those of formulas A5 and B5.
Suitable protonated carboxylates, protonated phosphates include
those listed hereinabove. Suitable amides include those of formulas
A8 and B8. Suitable alcohols include primary alcohols, secondary
alcohols, tertiary alcohols, and aromatic alcohols (e.g., phenols).
Suitable alcohols include those of formulas A7 (a primary alcohol)
and B7 (a secondary alcohol).
[0167] Recognition elements including a hydrogen bond acceptor or
one or more free electron pairs include amines, amides,
carboxylates, carboxyl groups, phosphates, phosphonates,
phosphinates, sulphates, sulphonates, alcohols, ethers, thiols, and
thioethers. Suitable amines include alkyl amines, aryl amines, aryl
alkyl amines, pyridines, heterocyclic amines (saturated or
unsaturated, the nitrogen in the ring or not), amidines, ureas, and
amines as listed hereinabove. Suitable alkyl amines include that of
formula B9. Suitable heterocyclic or alkyl heterocyclic amines
include that of formula A9. Suitable pyridines include those of
formulas A5 and B5. Suitable carboxylates include those listed
hereinabove. Suitable amides include those of formulas A8 and B8.
Suitable phosphates, phosphonates and phosphinates include those
listed hereinabove. Suitable alcohols include primary alcohols,
secondary alcohols, tertiary alcohols, aromatic alcohols, and those
listed hereinabove. Suitable alcohols include those of formulas A7
(a primary alcohol) and B7 (a secondary alcohol). Suitable ethers
include alkyl ethers, aryl alkyl ethers. Suitable alkyl ethers
include that of formula A6. Suitable aryl alkyl ethers include that
of formula A4. Suitable thioethers include that of formula B6.
[0168] Recognition elements including uncharged polar or
hydrophilic groups include amides, alcohols, ethers, thiols,
thioethers, esters, thio esters, boranes, borates, and metal
complexes. Suitable amides include those of formulas A8 and B8.
Suitable alcohols include primary alcohols, secondary alcohols,
tertiary alcohols, aromatic alcohols, and those listed hereinabove.
Suitable alcohols include those of formulas A7 (a primary alcohol)
and B7 (a secondary alcohol). Suitable ethers include those listed
hereinabove. Suitable ethers include that of formula A6. Suitable
aryl alkyl ethers include that of formula A4.
[0169] Recognition elements including uncharged hydrophobic groups
include alkyl (substituted and unsubstituted), alkene (conjugated
and unconjugated), alkyne (conjugated and unconjugated), aromatic.
Suitable alkyl groups include lower alkyl, substituted alkyl,
cycloalkyl, aryl alkyl, and heteroaryl alkyl. Suitable lower alkyl
groups include those of formulas A1, A3, A3a, and B1. Suitable aryl
alkyl groups include those of formulas A3, A3a, A4, B3, B3a, and
B4. Suitable alkyl cycloalkyl groups include that of formula B2.
Suitable alkene groups include lower alkene and aryl alkene.
Suitable aryl alkene groups include that of formula B4. Suitable
aromatic groups include unsubstituted aryl, heteroaryl, substituted
aryl, aryl alkyl, heteroaryl alkyl, alkyl substituted aryl, and
polyaromatic hydrocarbons. Suitable aryl alkyl groups include those
of formulas A3, A3a and B4. Suitable alkyl heteroaryl groups
include those of formulas A5 and B5.
[0170] Spacer (e.g., small) recognition elements include hydrogen,
methyl, ethyl, and the like. Bulky recognition elements include 7
or more carbon or hetero atoms.
[0171] Formulas A1-A9 and B1-B9 are:
##STR00003## ##STR00004##
[0172] These A and B recognition elements can be called derivatives
of, according to a standard reference: A1, ethylamine; A2,
isobutylamine; A3, phenethylamine; A4, 4-methoxyphenethylamine; A5,
2-(2-aminoethyl)pyridine; A6, 2-methoxyethylamine; A7,
ethanolamine; A8, N-acetylethylenediamine; A9,
1-(2-aminoethyl)pyrrolidine; B1, acetic acid, B2,
cyclopentylpropionic acid; B3, 3-chlorophenylacetic acid; B4,
cinnamic acid; B5, 3-pyridinepropionic acid; B6, (methylthio)acetic
acid; B7, 3-hydroxybutyric acid; B8, succinamic acid; and B9,
4-(dimethylamino)butyric acid.
[0173] In an embodiment, the recognition elements include one or
more of the structures represented by formulas A1, A2, A3, A3a, A4,
A5, A6, A7, A8, and/or A9 (the A recognition elements) and/or B1,
B2, B3, B3a, B4, B5, B6, B7, B8, and/or B9 (the B recognition
elements). In an embodiment, each building block includes an A
recognition element and a B recognition element. In an embodiment,
a group of 81 such building blocks includes each of the 81 unique
combinations of an A recognition element and a B recognition
element. In an embodiment, the A recognition elements are linked to
a framework at a pendant position. In an embodiment, the B
recognition elements are linked to a framework at an equatorial
position. In an embodiment, the A recognition elements are linked
to a framework at a pendant position and the B recognition elements
are linked to the framework at an equatorial position.
[0174] Although not limiting to the present invention, it is
believed that the A and B recognition elements represent the
assortment of functional groups and geometric configurations
employed by polypeptide receptors. Although not limiting to the
present invention, it is believed that the A recognition elements
represent six advantageous functional groups or configurations and
that the addition of functional groups to several of the aryl
groups increases the range of possible binding interactions.
Although not limiting to the present invention, it is believed that
the B recognition elements represent six advantageous functional
groups, but in different configurations than employed for the A
recognition elements. Although not limiting to the present
invention, it is further believed that this increases the range of
binding interactions and further extends the range of functional
groups and configurations that is explored by molecular
configurations of the building blocks.
[0175] In an embodiment, the building blocks including the A and B
recognition elements can be visualized as occupying a binding space
defined by lipophilicity/hydrophilicity and volume. A volume can be
calculated (using known methods) for each building block including
the various A and B recognition elements. A measure of
lipophilicity/hydrophilicity (logP) can be calculated (using known
methods) for each building block including the various A and B
recognition elements. Negative values of logP show affinity for
water over nonpolar organic solvent and indicate a hydrophilic
nature. A plot of volume versus logP can then show the distribution
of the building blocks through a binding space defined by size and
lipophilicity/hydrophilicity.
[0176] Reagents that form many of the recognition elements are
commercially available. For example, reagents for forming
recognition elements A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9 B1,
B2, B3, B3a, B4, B5, B6, B7, B8, and B9 are commercially
available.
Linkers
[0177] The linker is selected to provide a suitable coupling of the
building block to a support. The framework can interact with the
ligand as part of the artificial receptor. The linker can also
provide bulk, distance from the support, hydrophobicity,
hydrophilicity, and like structural characteristics to the building
block. Coupling building blocks to the support can employ covalent
bonding or noncovalent interactions. Suitable noncovalent
interactions include interactions between ions, hydrogen bonding,
van der Waals interactions, and the like. In an embodiment, the
linker includes moieties that can engage in covalent bonding or
noncovalent interactions. In an embodiment, the linker includes
moieties that can engage in covalent bonding. Suitable groups for
forming covalent and reversible covalent bonds are described
hereinabove.
Linkers for Reversibly Immobilizable Building Blocks
[0178] The linker can be selected to provide suitable reversible
immobilization of the building block on a support or lawn. In an
embodiment, the linker forms a covalent bond with a functional
group on the framework. In an embodiment, the linker also includes
a functional group that can reversibly interact with the support or
lawn, e.g., through reversible covalent bonding or noncovalent
interactions.
[0179] In an embodiment, the linker includes one or more moieties
that can engage in reversible covalent bonding. Suitable groups for
reversible covalent bonding include those described hereinabove. An
artificial receptor can include building blocks reversibly
immobilized on the lawn or support through, for example, imine,
acetal, ketal, disulfide, ester, or like linkages. Such functional
groups can engage in reversible covalent bonding. Such a functional
group can be referred to as a covalent bonding moiety, e.g., a
second covalent bonding moiety.
[0180] In an embodiment, the linker can be functionalized with
moieties that can engage in noncovalent interactions. For example,
the linker can include functional groups such as an ionic group, a
group that can hydrogen bond, or a group that can engage in van der
Waals or other hydrophobic interactions. Such functional groups can
include cationic groups, anionic groups, lipophilic groups,
amphiphilic groups, and the like.
[0181] In an embodiment, the present methods and compositions can
employ a linker including a charged moiety (e.g., a second charged
moiety). Suitable charged moieties include positively charged
moieties and negatively charged moieties. Suitable positively
charged moieties include protonated amines, quaternary ammonium
moieties, sulfonium, sulfoxonium, phosphonium, ferrocene, and the
like. Suitable negatively charged moieties (e.g., at neutral pH in
aqueous compositions) include carboxylates, phenols substituted
with strongly electron withdrawing groups (e.g.,
tetrachlorophenols), phosphates, phosphonates, phosphinates,
sulphates, sulphonates, thiocarboxylates, and hydroxamic acids.
[0182] In an embodiment, the present methods and compositions can
employ a linker including a group that can hydrogen bond, either as
donor or acceptor (e.g., a second hydrogen bonding group). For
example, the linker can include one or more carboxyl groups, amine
groups, hydroxyl groups, carbonyl groups, or the like. Ionic groups
can also participate in hydrogen bonding.
[0183] In an embodiment, the present methods and compositions can
employ a linker including a lipophilic moiety (e.g., a second
lipophilic moiety). Suitable lipophilic moieties include one or
more branched or straight chain C.sub.6-36 alkyl, C.sub.8-24 alkyl,
C.sub.12-24 alkyl, C.sub.12-18 alkyl, or the like; C.sub.6-36
alkenyl, C.sub.8-24 alkenyl, C.sub.12-24 alkenyl, C.sub.12-18
alkenyl, or the like, with, for example, 1 to 4 double bonds;
C.sub.6-36 alkynyl, C.sub.8-24 alkynyl, C.sub.12-24 alkynyl,
C.sub.12-18 alkynyl, or the like, with, for example, 1 to 4 triple
bonds; chains with 1-4 double or triple bonds; chains including
aryl or substituted aryl moieties (e.g., phenyl or naphthyl
moieties at the end or middle of a chain); polyaromatic hydrocarbon
moieties; cycloalkane or substituted alkane moieties with numbers
of carbons as described for chains; combinations or mixtures
thereof; or the like. The alkyl, alkenyl, or alkynyl group can
include branching; within chain functionality like an ether group;
terminal functionality like alcohol, amide, carboxylate or the
like; or the like. In an embodiment the linker includes or is a
lipid, such as a phospholipid. In an embodiment, the lipophilic
moiety includes or is a 12-carbon aliphatic moiety.
[0184] In an embodiment, the linker includes a lipophilic moiety
(e.g., a second lipophilic moiety) and a covalent bonding moiety
(e.g., a second covalent bonding moiety). In an embodiment, the
linker includes a lipophilic moiety (e.g., a second lipophilic
moiety) and a charged moiety (e.g., a second charged moiety).
[0185] In an embodiment, the linker forms or can be visualized as
forming a covalent bond with an alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. Between the bond to the
framework and the group participating in or formed by the
reversible interaction with the support or lawn, the linker can
include an alkyl, substituted alkyl, cycloalkyl, heterocyclic,
substituted heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl
alkyl, ethoxy or propoxy oligomer, a glycoside, or like moiety.
[0186] For example, suitable linkers can include: the functional
group participating in or formed by the bond to the framework, the
functional group or groups participating in or formed by the
reversible interaction with the support or lawn, and a linker
backbone moiety. The linker backbone moiety can include about 4 to
about 48 carbon or heteroatoms, about 8 to about 14 carbon or
heteroatoms, about 12 to about 24 carbon or heteroatoms, about 16
to about 18 carbon or heteroatoms, about 4 to about 12 carbon or
heteroatoms, about 4 to about 8 carbon or heteroatoms, or the like.
The linker backbone can include an alkyl, substituted alkyl,
cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,
aryl, heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a
glycoside, mixtures thereof, or like moiety.
[0187] In an embodiment, the linker includes a lipophilic moiety,
the functional group participating in or formed by the bond to the
framework, and, optionally, one or more moieties for forming a
reversible covalent bond, a hydrogen bond, or an ionic interaction.
In such an embodiment, the lipophilic moiety can have about 4 to
about 48 carbons, about 8 to about 14 carbons, about 12 to about 24
carbons, about 16 to about 18 carbons, or the like. In such an
embodiment, the linker can include about 1 to about 8 reversible
bond/interaction moieties or about 2 to about 4 reversible
bond/interaction moieties. Suitable linkers have structures such as
(CH.sub.2).sub.nCOOH, with n=12-24, n=17-24, or n=16-18.
Additional Embodiments of Linkers
[0188] The linker can be selected to provide a suitable covalent
coupling of the building block to a support. The framework can
interact with the ligand as part of the artificial receptor. The
linker can also provide bulk, distance from the support,
hydrophobicity, hydrophilicity, and like structural characteristics
to the building block. In an embodiment, the linker forms a
covalent bond with a functional group on the framework. In an
embodiment, before attachment to the support the linker also
includes a functional group that can be activated to react with or
that will react with a functional group on the support. In an
embodiment, once attached to the support, the linker forms a
covalent bond with the support and with the framework.
[0189] In an embodiment, the linker forms or can be visualized as
forming a covalent bond with an alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. The linker can include a
carboxyl, alcohol, phenol, thiol, amine, carbonyl, maleimide, or
like group that can react with or be activated to react with the
support. Between the bond to the framework and the group formed by
the attachment to the support, the linker can include an alkyl,
substituted alkyl, cycloalkyl, heterocyclic, substituted
heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl,
ethoxy or propoxy oligomer, a glycoside, or like moiety.
[0190] The linker can include a good leaving group bonded to, for
example, an alkyl or aryl group. The leaving group being "good"
enough to be displaced by the alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. Such a linker can include
a moiety represented by the formula: R--X, in which X is a leaving
group such as halogen (e.g., --Cl, --Br or --I), tosylate,
mesylate, triflate, and R is alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a
glycoside, or like moiety.
[0191] Suitable linker groups include those of formula:
(CH.sub.2).sub.nCOOH, with n=1-16, n=2-8, n=2-6, or n=3. Reagents
that form suitable linkers are commercially available and include
any of a variety of reagents with orthogonal functionality.
Embodiments of Building Blocks
[0192] In an embodiment, building blocks can be represented by
Formula 2:
##STR00005##
in which: RE.sub.1 is recognition element 1, RE.sub.2 is
recognition element 2, T is an optional tether, and L is a linker.
X is absent, C.dbd.O, CH.sub.2, NR, NR.sub.2, NH, NHCONH, SCONH,
CH.dbd.N, or OCH.sub.2NH. In certain embodiments, X is absent or
C.dbd.O. Y is absent, NH, O, CH.sub.2, or NRCO. In certain
embodiments, Y is NH or O. In an embodiment, Y is NH. Z.sub.1 and
Z.sub.2 can independently be CH2, O, NH, S, CO, NR.sup.X, NR.sub.2,
NHCONH, SCONH, CH.dbd.N, or OCH.sub.2NH. In an embodiment, Z.sub.1
and/or Z.sub.2 can independently be 0. Z.sub.2 is optional. R.sub.2
is H, CH.sub.3, or another group that confers chirality on the
building block and has size similar to or smaller than a methyl
group. R.sub.3 is CH.sub.2; CH.sub.2-phenyl; CHCH.sub.3;
(CH.sub.2).sub.n with n=2-3; or cyclic alkyl with 3-8 carbons,
e.g., 5-6 carbons, phenyl, naphthyl. In certain embodiments,
R.sub.3 is CH.sub.2 or CH.sub.2-phenyl.
[0193] RE.sub.1 is B1, B2, B3, B3a, B4, B5, B6, B7, B8, B9, A1, A2,
A3, A3a, A4, A5, A6, A7, A8, or A9. In certain embodiments,
RE.sub.1 is B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9. RE.sub.2 is
A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9, B1, B2, B3, B3a, B4, B5,
B6, B7, B8, or B9. In certain embodiments, RE.sub.2 is A1, A2, A3,
A3a, A4, A5, A6, A7, A8, or A9. In an embodiment, RE.sub.1 can be
B2, B3a, B4, B5, B6, B7, or B8. In an embodiment, RE.sub.2 can be
A2, A3a, A4, A5, A6, A7, or A8.
[0194] T can be any of the tether moieties described
hereinabove.
[0195] In an embodiment, L is the functional group participating in
or formed by the bond to the framework (such groups are described
herein), the functional group or groups participating in or formed
by the reversible interaction with the support or lawn (such groups
are described herein), and a linker backbone moiety. In an
embodiment, the linker backbone moiety is about 4 to about 48
carbon or heteroatom alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a
glycoside, or mixtures thereof; or about 8 to about 14 carbon or
heteroatoms, about 12 to about 24 carbon or heteroatoms, about 16
to about 18 carbon or heteroatoms, about 4 to about 12 carbon or
heteroatoms, about 4 to about 8 carbon or heteroatoms.
[0196] In an embodiment, the L is the functional group
participating in or formed by the bond to the framework (such
groups are described herein) and a lipophilic moiety (such groups
are described herein) of about 4 to about 48 carbons, about 8 to
about 14 carbons, about 12 to about 24 carbons, about 16 to about
18 carbons. In an embodiment, this L also includes about 1 to about
8 reversible bond/interaction moieties (such groups are described
herein) or about 2 to about 4 reversible bond/interaction moieties.
In an embodiment, L is (CH.sub.2).sub.nCOOH, with n=12-24, n=17-24,
or n=16-18.
[0197] In an embodiment, L is (CH.sub.2).sub.nCOOH, with n=1-16,
n=2-8, n=4-6, or n=3.
[0198] Building blocks including an A and/or a B recognition
element, a linker, and an amino acid framework can be made by
methods illustrated in general Scheme 1.
Methods Employing the Artificial Receptors
[0199] Working artificial receptors can be generated to be specific
to a given test ligand or specific to a particular part of a given
test ligand. Heterogeneous and immobilized combinations of building
block molecules form the working artificial receptors. For example,
combinations of 2, 3, 4, or 5 distinct building block molecules
immobilized in proximity to one another on a support provide
molecular structures that serve as candidate and working artificial
receptors. The building blocks can be naive to the test ligand.
Once a plurality of candidate artificial receptors are generated,
they can be tested to determine which are specific or useful for a
given ligand.
[0200] The specific or working artificial receptor or receptor
complex can then be used in a variety of different methods and
systems. For example, the receptors can be employed in methods
and/or devices for binding or detecting a test ligand. By way of
further example, the receptors can be employed in methods and/or
devices for chemical synthesis. Methods and systems for chemical
synthesis can include methods and systems for regiospecific and
stereospecific chemical synthesis. The receptors can also be
employed for developing compounds that disrupt or model binding
interactions. Methods and systems for developing therapeutic agents
can include methods and systems for pharmaceutical and vaccine
development.
[0201] In an embodiment, methods and systems of the present
invention can be employed for detecting a plurality of ligands of
interest. For example, an unknown biological sample can be
characterized by the presence of a combination of specific ligands.
Such a method can be useful in assays for detecting specific
pathogens or disease states. By way of further example, such an
embodiment can be used for determining the genetic profile of a
subject. For example, cancerous tissue can be detected or a genetic
disposition to cancer can be detected.
[0202] The present artificial receptors can be part of products
used in: analyzing a genome and/or proteome (protein isolation and
characterization); pharmaceutical development (such as
identification of sequence specific small molecule leads,
characterization of protein to protein interactions); detectors for
a test ligand; drug of abuse diagnostics or therapy (such as
clinical or field analysis of cocaine or other drugs of abuse);
hazardous waste analysis or remediation; chemical exposure alert or
intervention; disease diagnostics or therapy; cancer diagnostics or
therapy (such as clinical analysis of prostate specific antigen);
biological agent alert or intervention; food chain contamination
analysis or remediation and clinical analysis of food contaminants;
and the like.
Methods of Binding or Detecting Test Ligands
[0203] In an embodiment, the invention can include methods and/or
devices for binding or detecting a test ligand. For example, the
present artificial receptors can be used for a variety of assays
that presently employ an antibody. The present artificial receptors
can be specific for a given ligand, such as an antigen or an
immunogen. Thus, the present artificial receptors can be used in
formats analogous to enzyme immunoassay, enzyme-linked immunoassay,
immunodiffusion, immunoelectrophoresis, latex agglutination, and
the like. Test ligands that can be detected in such a method
include a drug of abuse, a biological agent (such as a hazardous
agent), a marker for a biological agent, a marker for a disease
state, etc. Methods and systems for detection can include methods
and systems for clinical chemistry, environmental analysis, and
diagnostic assays of all types.
[0204] For example, the artificial receptor can be contacted with a
sample including or suspected of including at least one test
ligand. The building blocks making up the artificial receptors can
be naive to the test ligand. Then, binding of one or more of the
test ligands to the artificial receptors can be detected. Next, the
binding results can be interpreted to provide information about the
sample. In an embodiment, the invention includes a method for
detecting a test ligand in a sample including contacting an
artificial receptor specific to the test ligand with a sample
suspected of containing the test ligand. The method can also
include detecting or quantitating binding of the test ligand to the
artificial receptor. For example, an artificial receptor that binds
(e.g., tightly) the molecule, cell, or microbe under appropriate
conditions can be employed in a format where binding itself is
sufficient to indicate presence of the molecule or organism. Such a
format can also include artificial receptors to be probed with
positive and control samples.
[0205] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand, such as a molecule or cell. The method can include making
an array of candidate artificial receptors. Working artificial
receptors can be identified by contacting the array with test
ligand and identifying which receptors bind the test ligand. The
building blocks making up the artificial receptors can be naive to
the test ligand. Such a method can employ a labeled test ligand.
The method can include producing an array or device including the
working artificial receptor or receptor complex. In an embodiment,
the method can include employing the array or device for detecting
or characterizing the test ligand in a sample, such as a
biological, laboratory, or environmental sample.
[0206] FIG. 9 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
test ligand. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
a test ligand, e.g., a molecule or cell. The building blocks making
up the artificial receptors can be naive to the test ligand. The
molecule or cell can exhibit characteristic binding to one or
several of the candidate artificial receptors from that array. The
one or several artificial receptors can be selected as an
artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample, or characterizing
or detecting the molecule or cell.
[0207] As illustrated in FIG. 9, a test ligand can be identified by
a method employing a single or a plurality of lead or working
artificial receptors. The plurality of lead or working artificial
receptors suitable for identifying a test ligand can be employed in
an array format test. A single lead or working artificial receptor
can be configured on a support as a strip together with positive
and/or negative control receptors, which can also be configured as
strips.
[0208] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single test ligand or working artificial
receptors for a plurality of test ligands. For example, a method
can include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single test
ligand can be employed in a method or system for detecting that
test ligand. Binding to the working artificial receptors indicates
that the sample includes the test ligand. A substrate including
working artificial receptors for a plurality of test ligands can be
employed in a method or system for detecting one, several, or all
of the test ligands. Binding to the working artificial receptors
for a particular test ligand or ligands indicates that the sample
includes such test ligand or ligands.
[0209] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the test ligands. The method can include detecting
the binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 10 schematically illustrates
certain binding patterns on an array of working artificial
receptors. In an embodiment, all artificial receptors for one test
ligand can be arranged in a line across the substrate. Referring to
FIG. 10, receptors that are specific for IL-2 are in a line 12 on
the array 10 of working artificial receptor complexes. Working
artificial receptors that have bound a test ligand (e.g., IL-2) are
indicated as shaded 24. Working artificial receptors that have not
bound a test ligand are illustrated as open circles 26.
[0210] A method employing the illustrated array can include
detecting binding on line 12 of working artificial receptors
through fluorescence or another means described herein. In the
illustrated embodiment, detecting binding on line 12 of working
artificial receptors indicates that the sample contains IL-2.
Further, lack of signal from the other working artificial receptors
in array 10 indicates that the sample does not contain IFN-gamma,
IL-10, TGF-beta, IL-12, or TGF-alpha. Thus, a method employing such
an array can determine whether a sample is a particular type of
biological sample or contains a particular type of molecule or
cell.
[0211] When designed for use with a field assay kit, the device 30
can have spots arranged such that a positive result creates an
easily recognizable pattern 36, such as a plus sign. The readily
recognizable pattern can thus indicate that a particular test
ligand is present in the sample. Alternatively, the artificial
receptors or spots for a particular target 42 can be arranged
randomly on third array 40. In this manner, when the detection
device or array is used, the results of the test may not be
immediately apparent to an observer but will be readily read by a
machine which can be programmed to correlate binding to receptors
or spots in different positions with the identity of a particular
biological sample, molecule, or cell.
[0212] In an embodiment, the invention includes a method for
detecting or characterizing a biological sample, a molecule, or
cell. This embodiment of the method can include selecting an
artificial receptor that binds the biological sample, molecule, or
cell from an array of artificial receptors, contacting the
artificial receptor with a test composition, and detecting binding
of the artificial receptor to the test composition. In such an
embodiment, binding indicates the presence of the biological
sample, molecule, or cell in the test composition. In an
embodiment, the invention includes a method for detecting or
characterizing a biological sample, molecule, or cell. This
embodiment of the method can include contacting an array of
artificial receptors with a test composition and detecting binding
to the artificial receptors. Binding indicates the presence of the
biological sample, molecule, or cell in the test composition.
[0213] The present method can develop or employ a plurality of
working receptors specific for a particular test ligand, e.g.,
biological sample, molecule, or cell. That is, the working
receptors can be specific for a particular test ligand, but
different receptors can interact with different distinct antigens
(e.g., proteins or carbohydrates), ligands, functional groups, or
structural features of the test ligand. Such a method can provide a
robust test for the presence of a test ligand. For example, such a
robust test can reduce the chances of a false-positive or
false-negative result in comparison with an assay that relies upon
a single unique receptor to detect a given test ligand. Further,
this embodiment of the method can develop or employ working
receptors that demonstrate higher binding affinity due to
interaction with multiple antigens or ligands on the same test
ligand (e.g., multivalent binding).
[0214] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a
polynucleotide, e.g., DNA or RNA. The method can include evaluating
an array including a significant number of candidate artificial
receptors for binding to the polynucleotide, e.g., DNA or RNA. The
building blocks making up the artificial receptors can be naive to
the DNA or RNA. The polynucleotide, e.g., DNA or RNA, can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting the
polynucleotide, e.g., DNA or RNA.
[0215] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a polypeptide or
peptide. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the polypeptide or peptide. The building blocks making up the
artificial receptors can be naive to the polypeptide or peptide.
The polypeptide or peptide can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample, or characterizing
or detecting the polypeptide or peptide.
[0216] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a oligo- or
polysaccharide. The method can include evaluating an array
including a significant number of candidate artificial receptors
for binding to the oligo- or polysaccharide. The building blocks
making up the artificial receptors can be naive to the oligo- or
polysaccharide. The oligo- or polysaccharide can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting the oligo- or
polysaccharide.
[0217] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a cell, e.g., a
hepatocyte. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the cell, e.g., a hepatocyte. The building blocks making up the
artificial receptors can be naive to the cell. The cell, e.g., a
hepatocyte, can exhibit characteristic binding to one or several of
the candidate artificial receptors from that array. The one or
several artificial receptors can be selected as an artificial
receptor (e.g., a working artificial receptor or a working
artificial receptor complex) that can be employed in methods for
characterizing a biological sample, or characterizing or detecting
the cell, e.g., a hepatocyte.
Methods of Binding or Detecting Drugs of Abuse
[0218] In an embodiment, the invention can include methods and/or
devices for binding or detecting a drug of abuse. Methods and
systems for detection can include methods and systems for clinical
chemistry, field analysis, and diagnostic assays of all types. For
example, the artificial receptor can be contacted with a sample
including or suspected of including at least one drug of abuse.
Then, binding of one or more of the drugs of abuse to the
artificial receptors can be detected. Next, the binding results can
be interpreted to provide information about the sample. In an
embodiment, the invention includes a method for detecting a drug of
abuse in a sample including contacting an artificial receptor
specific to the drug of abuse with a sample suspected of containing
the drug of abuse. The method can also include detecting or
quantitating binding of the drug of abuse to the artificial
receptor.
[0219] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as a drug of abuse. The method can
include making an array of candidate artificial receptors. The
building blocks making up the artificial receptors can be naive to
the test ligand. Working artificial receptors can be identified by
contacting the array with a drug of abuse and identifying which
receptors bind the drug of abuse. The method can include producing
an array or device including the working artificial receptor or
receptor complex. In an embodiment, the method can include
employing the array or device for detecting or characterizing the
drug of abuse in a sample, such as a biological, laboratory, or
evidence sample.
[0220] FIG. 9 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
drug of abuse. The method can include evaluating an array including
a significant number of candidate artificial receptors for binding
to a drug of abuse. The building blocks making up the artificial
receptors can be naive to the drug of abuse. The drug of abuse can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological or field sample, or characterizing or detecting the drug
of abuse.
[0221] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single drug of abuse or working
artificial receptors for a plurality of drugs of abuse. For
example, a method can include contacting the artificial receptors
with a sample. A substrate including working artificial receptors
for a single drug of abuse can be employed in a method or system
for detecting that drug of abuse. Binding to the working artificial
receptors indicates that the sample includes the drug of abuse. A
substrate including working artificial receptors for a plurality of
drugs of abuse can be employed in a method or system for detecting
one, several, or all of the drugs of abuse. Binding to the working
artificial receptors for a particular drug of abuse or drugs of
abuse indicates that the sample includes such a drug of abuse or
drugs of abuse.
[0222] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the drugs of abuse. The method can include detecting
the binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 10 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including drugs of abuse.
[0223] The present method can develop or employ a plurality of
working receptors specific for a particular drug of abuse or
feature on the drug of abuse. That is, the working receptors can be
specific for a particular drug of abuse, but different receptors
can interact with different distinct ligands, functional groups, or
structural features of the drug of abuse. Such a method can provide
a robust test for the presence of a drug of abuse. For example,
such a robust test can reduce the chances of a false-positive or
false-negative result in comparison with an assay that relies upon
a single unique receptor to detect a given drug of abuse. Further,
this embodiment of the method can develop or employ working
receptors that demonstrate higher binding affinity due to
interaction with multiple ligands or features on the same drug of
abuse (e.g., multivalent binding).
[0224] Suitable drugs of abuse include cannabinoids (e.g., hashish
and marijuana), depressants (e.g., barbiturates, benzodiazepines,
gamma-hydroxy butyrate, methaqualone), dissociative anesthetics
(e.g., ketamine, PCP, and PCP analogs), hallucinogens (e.g., LSD,
mescaline, psilocybin), opiates or opioids (e.g., codeine,
fentanyl, fentanyl analogs, heroin, morphine, opium, oxycodone HCL,
hydrocodone bitartrate), stimulants (e.g., amphetamine, cocaine,
methylenedioxy-methamphetamine, methamphetamine, methylphenidate,
nicotine), inhalants (e.g., solvents), and the like.
[0225] Suitable drugs of abuse include performance enhancing
agents, such as stimulants and beta-blockers, anabolic agents,
oxygen carrier enhancers, masking agents, and inhalants. Suitable
stimulants include caffeine and amphetamines. Suitable
beta-blockers include salbutamol (used in asthma inhalers) and the
like. Suitable anabolic agents include steroids (e.g., anabolic
steroids), steroid analogs, and growth hormone. Suitable oxygen
carrier enhancers include erythropoietin and the like.
Methods of Binding or Detecting Isomers
[0226] In an embodiment, the invention can include methods and/or
devices for binding or detecting an isomer or isomers of a
compound. Methods and systems for detection can include methods and
systems for clinical chemistry, environmental analysis, and
diagnostic assays of all types. For example, the artificial
receptor can be contacted with a sample including or suspected of
including at least one isomer of a compound. Then, binding of one
or more of the isomers of a compound to the artificial receptors
can be detected. Next, the binding results can be interpreted to
provide information about the isomers. In an embodiment, the
invention includes a method for detecting an isomer of a compound
in a sample including contacting an artificial receptor specific to
the isomer with a sample suspected of containing the isomer. The
method can also include detecting or quantitating binding of the
isomer to the artificial receptor.
[0227] The present method can be applied to isomers such as
stereoisomers (e.g., geometric isomers or optical isomers), optical
isomers (e.g., enantiomers and diastereomers), geometric isomers
(e.g., cis- and trans-isomers). The present method can be employed
to develop working or lead artificial receptors or working
artificial complexes that can bind to one or more isomers of a
compound (e.g., enantioselective receptor environments). For
example, the artificial receptor or complex can bind to one
stereoisomer of a compound but bind only weakly or not at all
another stereoisomer of the compound. For example, the artificial
receptor or complex can bind one geometric isomer of a compound but
bind only weakly or not at all another geometric isomer. For
example, the artificial receptor or complex can bind one optical
isomer of a compound but bind only weakly or not at all another
optical isomer. For example, the artificial receptor or complex can
bind one enantiomer of a compound but bind only weakly or not at
all another enantiomer. For example, the artificial receptor or
complex can bind one diastereomer of a compound but bind only
weakly or not at all another diastereomer.
[0228] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as an isomer of a compound. The method
can include making an array of candidate artificial receptors. The
building blocks making up the artificial receptors can be naive to
the test ligand. Working artificial receptors can be identified by
contacting the array with an isomer and identifying which receptors
bind the isomer. The method can include producing an array or
device including the working artificial receptor or receptor
complex. In an embodiment, the method can include employing the
array or device for detecting or characterizing the isomer in a
sample, such as a biological, laboratory, or clinical sample.
[0229] FIG. 9 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting an
isomer. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
an isomer. The building blocks making up the artificial receptors
can be naive to the isomer. The isomer can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological, clinical, or
laboratory sample, or characterizing or detecting the isomer.
[0230] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single isomer or working artificial
receptors for a plurality of isomers. For example, a method can
include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single
isomer can be employed in a method or system for detecting that
isomer. Binding to the working artificial receptors indicates that
the sample includes the isomer. A substrate including working
artificial receptors for a plurality of isomers can be employed in
a method or system for detecting one, several, or all of the
isomers. Binding to the working artificial receptors for a
particular isomer or isomers indicates that the sample includes
such an isomer or isomers.
[0231] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the isomers. The method can include detecting the
binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 10 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including isomers.
[0232] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a stereoisomer.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
stereoisomer. The building blocks making up the artificial
receptors can be naive to the stereoisomer. The stereoisomer can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a lab
or clinical sample or characterizing or detecting the
stereoisomer.
[0233] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a geometric
isomer (e.g., cis- and trans-isomers). The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the geometric isomer (e.g.,
cis- and trans-isomers). The building blocks making up the
artificial receptors can be naive to the geometric isomer. The
geometric isomer can exhibit characteristic binding to one or
several of the candidate artificial receptors from that array. The
one or several artificial receptors can be selected as an
artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a lab or clinical sample or
characterizing or detecting the geometric isomer (e.g., cis- and
trans-isomers).
[0234] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting an optical
isomer. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the optical isomer. The building blocks making up the artificial
receptors can be naive to the optical isomer. The optical isomer
can exhibit characteristic binding to one or several of the
candidate artificial receptors from that array. The one or several
artificial receptors can be selected as an artificial receptor
(e.g., a working artificial receptor or a working artificial
receptor complex) that can be employed in methods for
characterizing a lab or clinical sample or characterizing or
detecting the optical isomer.
[0235] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting an enantiomer.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
enantiomer. The enantiomer can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a lab or clinical sample or
characterizing or detecting the enantiomer.
[0236] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a diastereomer.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
diastereomer. The building blocks making up the artificial
receptors can be naive to the diastereomer. The diastereomer can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a lab
or clinical sample or characterizing or detecting the
diastereomer.
Methods for Binding or Detecting Peptides
[0237] In an embodiment, the invention can include methods and/or
devices for binding or detecting a peptide. Methods and systems for
detection can include methods and systems for clinical chemistry,
environmental analysis, and diagnostic assays of all types. For
example, the artificial receptor can be contacted with a sample
including or suspected of including at least one peptide. Then,
binding of one or more of the peptides to the artificial receptors
can be detected. Next, the binding results can be interpreted to
provide information about the sample. In an embodiment, the
invention includes a method for detecting a peptide in a sample
including contacting an artificial receptor specific to the peptide
with a sample suspected of containing the peptide. The method can
also include detecting or quantitating binding of the peptide to
the artificial receptor.
[0238] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as a peptide. The method can include
making an array of candidate artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. Working artificial receptors can be identified by
contacting the array with a peptide and identifying which receptors
bind the peptide. The method can include producing an array or
device including the working artificial receptor or receptor
complex. In an embodiment, the method can include employing the
array or device for detecting or characterizing the peptide in a
sample, such as a biological, laboratory, or clinical sample.
[0239] FIG. 9 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
peptide. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
a peptide. The building blocks making up the artificial receptors
can be naive to the peptide. The peptide can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological or
environmental sample, or characterizing or detecting the
peptide.
[0240] FIG. 11 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand,
such as a peptide or mixture of peptides. This embodiment of the
present method includes evaluating a plurality (e.g. array) of
candidate artificial receptors for binding to each of a plurality
of peptides. The building blocks making up the artificial receptors
can be naive to one or more of the peptides. The plurality of
peptides can include the peptides found in a cell or organism. The
method can include detecting binding of individual peptides to a
subset of the plurality or array of candidate artificial receptors.
The method can include detecting binding of the peptides found in a
cell or organism to a subset of or all of the plurality or array of
candidate artificial receptors. This can be envisioned as
developing a working artificial receptor or artificial receptor
complex for each peptide or mixture of peptides.
[0241] Thus, each peptide or mixture of peptides can provide a
pattern of bound receptors in the plurality or array. The pattern
of bound receptors can be characteristic of the peptide or mixture
of peptides or a sample including the peptide or mixture of
peptides. The method can include storing a representation of the
binding pattern as an image or a data structure. The representation
of the binding pattern can be evaluated either by an operator or
data processing system. The method can include such evaluating. A
binding pattern from an unknown sample that matches the binding
pattern for a particular peptide then characterizes the unknown
sample as containing that peptide. A binding pattern from an
unknown sample that matches the binding pattern for a particular
mixture of peptides then characterizes the unknown sample as
including or being that mixture of peptides or as including or
being the organism or cell containing that mixture of peptides. A
plurality of binding patterns can be stored as a database.
[0242] An embodiment of the illustrated method can include creating
an array of artificial receptors. This embodiment can also include
compiling a database of the binding patterns of a specific peptide
or mixture of peptides, for example, by probing the array with a
plurality of individual peptides or the peptides found in a cell or
organism. Contacting the array with an unidentified peptide or
mixture of peptides can create a test binding pattern. The method
can then compare the test binding pattern with the binding patterns
of known peptides or mixtures of peptides in the database in order
to characterize or classify the unidentified peptide, mixture of
peptides, or cell or organism. In an embodiment, the database and
the array of receptors has already been constructed and the method
involves probing the array with an unknown peptide or mixture of
peptides to create a test binding pattern and then comparing this
binding pattern with the binding patterns in the database in order
to characterize or classify the unidentified peptide, mixture of
peptides, or cell or organism.
[0243] An array constructed for distinguishing mixtures of peptides
can be contacted with samples from an organism, cell, or tissue of
interest. Peptides that bind to the array can characterize or
detect the organism, cell or tissue; can indicate a disorder caused
by the organism or affecting the cell or tissue; can indicate
successful therapy of a disorder caused by the organism or
affecting the cell or tissue; characterize disease processes;
identify therapeutic leads or strategies; or the like.
[0244] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single peptide or working artificial
receptors for a plurality of peptides. For example, a method can
include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single
peptide can be employed in a method or system for detecting that
peptide. Binding to the working artificial receptors indicates that
the sample includes the peptide. A substrate including working
artificial receptors for a plurality of peptides can be employed in
a method or system for detecting one, several, or all of the
peptides. Binding to the working artificial receptors for a
particular peptide or peptides indicates that the sample includes
such a peptide or peptides.
[0245] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the peptides. The method can include detecting the
binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 10 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including peptides.
[0246] The present method can develop or employ a plurality of
working receptors specific for a particular peptide or feature on
the peptide. That is, the working receptors can be specific for a
particular peptide, but different receptors can interact with
different distinct ligands, functional groups, or structural
features of the peptide. Such a method can provide a robust test
for the presence of a peptide. For example, such a robust test can
reduce the chances of a false-positive or false-negative result in
comparison with an assay that relies upon a single unique receptor
to detect a given peptide. Further, this embodiment of the method
can develop or employ working receptors that demonstrate higher
binding affinity due to interaction with multiple ligands or
features on the same peptide (e.g., multivalent binding).
[0247] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a peptide. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
peptide. The building blocks making up the artificial receptors can
be naive to the test ligand. The peptide can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the peptide.
[0248] The present method can include selecting artificial
receptors that bind a particular peptide and/or the building blocks
making up these receptors (e.g., bound to a scaffold molecule) as
leads for pharmaceutical development or as active agents for
modulating an activity of that peptide. The artificial receptor or
building blocks making up that artificial receptor can be selected
to bind to a portion of a peptide required for its interaction with
an other macromolecule (e.g. carbohydrate, protein, or
polynucleotide), thus disrupting this interaction.
Methods for Binding or Detecting Protein or Proteome
[0249] In an embodiment, the invention can include methods and/or
devices for binding or detecting a protein, one or more of a
plurality of proteins, or a proteome. Methods and systems for
detection can include methods and systems for clinical chemistry,
environmental analysis, diagnostic assays, and for proteome
analysis. For example, the artificial receptor can be contacted
with a sample including at least one protein or one proteome. The
building blocks making up the artificial receptors can be naive to
the test ligand. Then, binding of one or more proteins to the
artificial receptors can be detected. Next, the binding results can
be interpreted to provide information about the sample, e.g., the
proteome. In an embodiment, the invention includes a method for
detecting a protein in a sample including contacting an artificial
receptor specific to the protein with a sample suspected of
containing the protein. The method can also include detecting or
quantitating binding of the protein to the artificial receptor.
[0250] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as one or more proteins. The method
can include making an array of candidate artificial receptors. The
building blocks making up the artificial receptors can be naive to
the test ligand. Working artificial receptors can be identified by
contacting the array with a protein and identifying which receptors
bind the protein. The method can include producing an array or
device including the working artificial receptor or receptor
complex. In an embodiment, the method can include employing the
array or device for detecting or characterizing the protein in a
sample, such as a biological, laboratory, or environmental
sample.
[0251] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single protein or working artificial
receptors for a plurality of proteins. For example, a method can
include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single
protein can be employed in a method or system for detecting that
protein. Binding to the working artificial receptors indicates that
the sample includes the protein. A substrate including working
artificial receptors for a plurality of proteins can be employed in
a method or system for detecting one, several, or all of the
proteins. Binding to the working artificial receptors for a
particular protein or protein indicates that the sample includes
such a protein or protein.
[0252] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the proteins. The method can include detecting the
binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 10 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including proteins.
[0253] FIG. 11 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand,
such as a protein or proteome. This embodiment of the present
method includes evaluating a plurality (e.g. array) of candidate
artificial receptors for binding to each of a plurality of test
ligands. The building blocks making up the artificial receptors can
be naive to the test ligand. The plurality of test ligands can
include a plurality of proteins. The plurality of test ligands can
include the proteins making up the proteome of a cell or organism.
The method can include detecting binding of individual proteins to
a subset of the plurality or array of candidate artificial
receptors. The method can include detecting binding of proteins
making up the proteome to a subset of or all of the plurality or
array of candidate artificial receptors. This can be envisioned as
developing a working artificial receptor or artificial receptor
complex for each protein or for the proteome.
[0254] Thus, each protein or proteome can provide a pattern of
bound receptors in the plurality or array. The pattern of bound
receptors can be characteristic of the protein or proteome or a
sample including the protein or proteome. The method can include
storing a representation of the binding pattern as an image or a
data structure. The representation of the binding pattern can be
evaluated either by an operator or data processing system. The
method can include such evaluating. A binding pattern from an
unknown sample that matches the binding pattern for a particular
protein then characterizes the unknown sample as containing that
protein. A binding pattern from an unknown sample that matches the
binding pattern for a particular proteome then characterizes the
unknown sample as including or being that proteome or as including
or being the organism or cell having that proteome. Similarly, a
binding pattern from an unknown sample can be evaluated against the
patterns of a plurality of particular proteins or proteomes and the
sample can be characterized as containing one or more of the
proteins or proteomes. A plurality of binding patterns can be
stored as a database.
[0255] An embodiment of the illustrated method can include creating
an array of artificial receptors. This embodiment can also include
compiling a database of the binding patterns of specific proteins
or proteomes, for example, by probing the array with a plurality of
individual proteins or proteomes. Contacting the array with
unidentified proteins or proteomes can create a test binding
pattern. The method can then compare the test binding pattern with
the binding patterns of known proteins or proteomes in the database
in order to characterize or classify the unidentified protein,
proteome, or cell or organism. In an embodiment, the database and
the array of receptors has already been constructed and the method
involves probing the array with an unknown protein or proteome to
create a test binding pattern and then comparing this binding
pattern with the binding patterns in the database in order to
characterize or classify the unidentified protein, proteome, or
cell or organism.
[0256] A proteome array can be contacted with samples from an
organism, cell, or tissue of interest. Proteins that bind to the
proteome array can characterize or detect the organism, cell or
tissue; can indicate a disorder caused by the organism or affecting
the cell or tissue; can indicate successful therapy of a disorder
caused by the organism or affecting the cell or tissue;
characterize disease processes; identify therapeutic leads or
strategies; or the like.
[0257] The present method can develop or employ a plurality of
working receptors specific for a particular protein or feature on
the protein. That is, the working receptors can be specific for a
particular protein, but different receptors can interact with
different distinct ligands, functional groups, or structural
features of the protein. Such a method can provide a robust test
for the presence of a protein. For example, such a robust test can
reduce the chances of a false-positive or false-negative result in
comparison with an assay that relies upon a single unique receptor
to detect a given protein. Further, this embodiment of the method
can develop or employ working receptors that demonstrate higher
binding affinity due to interaction with multiple ligands or
features on the same protein (e.g., multivalent binding).
[0258] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a protein. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
protein. The building blocks making up the artificial receptors can
be naive to the test ligand. The protein can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the protein.
[0259] The present method can include selecting artificial
receptors that bind a particular protein and/or the building blocks
making up these receptors (e.g., bound to a scaffold molecule) as
leads for pharmaceutical development or as active agents for
modulating an activity of that protein. The artificial receptor or
building blocks making up that artificial receptor can be selected
to bind to or disrupt the activity of the active site of an enzyme
or the ligand binding site of a receptor. The artificial receptor
or building blocks making up that artificial receptor can be
selected to bind to a portion of a protein required for its
interaction with an other macromolecule (e.g. carbohydrate,
protein, or polynucleotide), thus disrupting this interaction. The
artificial receptor or building blocks making up that artificial
receptor can be selected to bind to the binding site of a receptor
and act as an agonist of that receptor.
[0260] The present method can include selecting working artificial
receptors that bind a preselected protein for use in a system for
proteome analysis. The working artificial receptors for the
preselected protein can be provided on a substrate and the protein
bound to the receptors. In an embodiment, selecting and binding
employ a plurality of different working artificial receptors for
the preselected protein. The plurality of artificial receptors may
bind to different features on the preselected protein and leave
free different features on the preselected protein. This embodiment
of the method includes contacting the working receptors with bound
preselected protein to at least one candidate binding partner for
the preselected protein. The method can include detecting binding
or absence of binding of the candidate binding partner to the
preselected protein. A candidate binding partner that binds to the
preselected protein can be considered a lead binding partner.
[0261] In an embodiment, the method includes contacting the working
receptors with bound preselected protein with a proteome of a cell
or organism serving as the source of candidate binding partners.
The method can then recover from the proteome one or more lead
binding partners. This can then characterize the proteome as
containing or not a binding partner for the preselected
protein.
[0262] In an embodiment, the present artificial receptors can be
employed in studies of proteomics. In such an embodiment, an array
of candidate or working artificial receptors can be contacted with
a mixture of peptides, polypeptides, and/or proteins. Each mixture
can produce a characteristic fingerprint of binding to the array.
In addition, identification of a specific receptor environment for
a target peptide, polypeptide, and/or protein can be utilized for
isolation and analysis of the target. That is, in yet another
embodiment, a particular receptor surface can be employed for
affinity purification methods, e.g. affinity chromatography.
[0263] In an embodiment, the present candidate artificial receptors
can be employed to find receptor surfaces that bind proteins in a
preferred configuration or orientation. Many proteins (e.g.
antibodies, enzymes, receptors) are stable and/or active in
specific environments. Defined receptor surfaces can be used to
produce binding environments that selectively retain or orient the
protein for maximum stability and/or activity. In an embodiment,
the present artificial receptors can be employed to form bioactive
surfaces. For example, receptor surfaces can be used to
specifically bind the active conformation of an antibody or
enzyme.
[0264] In an embodiment, the present method can include labeling a
protein while it remains bound to an artificial receptor. The
resulting protein will be labeled on its portions accessible to the
labeling reagent but not on those portions bound to the artificial
receptor. The method can include releasing the labeled protein from
the artificial receptor. Determining the distribution of labels on
the protein indicates which portion of the protein was bound to the
receptor.
[0265] In certain embodiments, the present artificial receptors can
be employed to distinguish between two conformations of a single
protein. Certain proteins exist in two or more stable
conformations. In an embodiment, the present working artificial
receptor or complex can bind a first conformation of a protein. In
an embodiment, the present working artificial receptor or complex
can bind a second conformation of a protein. In an embodiment, the
present working artificial receptor or complex can bind a first
conformation of a protein, but not a second conformation of the
same protein. In an embodiment, the present working artificial
receptor or complex can bind a second conformation of a protein,
but not a first conformation of the same protein.
[0266] For example, in an embodiment, the present working
artificial receptor or complex can bind a first or non-infectious
conformation of a prion, but not its second or infectious
conformation. For example, in an embodiment, the present working
artificial receptor or complex can bind the second or infectious
conformation of a prion, but not its first or non-infectious
conformation. For example, in an embodiment, the present working
artificial receptor or complex can bind a first or
non-plaque-forming conformation of .beta.-amyloid, but not its
second or plaque-forming conformation. For example, in an
embodiment, the present working artificial receptor or complex can
bind a second or plaque-forming conformation of .beta.-amyloid, but
not its second or non-plaque-forming conformation.
[0267] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a desired
conformation of a protein. The method can include evaluating an
array including a significant number of candidate artificial
receptors for binding to the desired conformation of the protein.
The building blocks making up the artificial receptors can be naive
to the protein or its desired conformation. The desired
conformation of the protein can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample or characterizing or
detecting the desired conformation of the protein.
[0268] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a first or
non-infectious conformation of a prion. The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the first or non-infectious
conformation of a prion. The building blocks making up the
artificial receptors can be naive to the prion. The first or
non-infectious conformation of a prion can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the first or non-infectious
conformation of a prion.
[0269] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a second or
infectious conformation of a prion. The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the second or infectious
conformation of a prion. The building blocks making up the
artificial receptors can be naive to the test ligand. The second or
infectious conformation of a prion can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the second or infectious conformation
of a prion.
[0270] In an embodiment, the present method includes developing
receptors or a receptor system that can distinguish between the
first or non-infectious conformation of a prion and the second or
infectious conformation of the prion. Such a method can include
selecting a working artificial receptor or complex can that bind
the first or non-infectious conformation of a prion, but not the
second or infectious conformation of the prion. This embodiment can
include selecting a working artificial receptor or complex can that
bind the second or infectious conformation of a prion, but not the
first or non-infectious conformation of the prion. Employed
together, these two sets of working artificial receptors or systems
can characterize a biological sample as containing one or both
forms of the prion.
[0271] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a first or
non-plaque-forming conformation of a .beta.-amyloid. The method can
include evaluating an array including a significant number of
candidate artificial receptors for binding to the first or
non-plaque-forming conformation of the .beta.-amyloid. The building
blocks making up the artificial receptors can be naive to the
.beta.-amyloid. The first or non-plaque-forming conformation of the
.beta.-amyloid can exhibit characteristic binding to one or several
of the candidate artificial receptors from that array. The one or
several artificial receptors can be selected as an artificial
receptor (e.g., a working artificial receptor or a working
artificial receptor complex) that can be employed in methods for
characterizing a biological sample or characterizing or detecting
the first or non-plaque-forming conformation of the
.beta.-amyloid.
[0272] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a second or
plaque-forming conformation of a .beta.-amyloid. The method can
include evaluating an array including a significant number of
candidate artificial receptors for binding to the second or
plaque-forming conformation of a .beta.-amyloid. The building
blocks making up the artificial receptors can be naive to the
.beta.-amyloid. The second or plaque-forming conformation of the
.beta.-amyloid can exhibit characteristic binding to one or several
of the candidate artificial receptors from that array. The one or
several artificial receptors can be selected as an artificial
receptor (e.g., a working artificial receptor or a working
artificial receptor complex) that can be employed in methods for
characterizing a biological sample or characterizing or detecting
the second or plaque-forming conformation of the
.beta.-amyloid.
[0273] In an embodiment, the present method includes developing
receptors or a receptor system that can distinguish between the
first or non-plaque-forming conformation of .beta.-amyloid and the
second or plaque-forming conformation of the .beta.-amyloid. Such a
method can include selecting a working artificial receptor or
complex can that bind the first or non-plaque-forming conformation
of .beta.-amyloid, but not the second or plaque-forming
conformation of the .beta.-amyloid. This embodiment can include
selecting a working artificial receptor or complex can that bind
the second or plaque-forming conformation of the .beta.-amyloid,
but not the first or non-plaque-forming conformation of
.beta.-amyloid. Employed together, these two sets of working
artificial receptors or systems can characterize a biological
sample as containing one or both forms of the .beta.-amyloid.
[0274] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting cholera toxin.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the cholera
toxin. The building blocks making up the artificial receptors can
be naive to the test ligand. The cholera toxin can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting cholera
toxin.
[0275] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting at least one
protein of a cancer cell. The method can include evaluating an
array including a significant number of candidate artificial
receptors for binding to the cancer cell protein. The building
blocks making up the artificial receptors can be naive to the test
ligand. The cancer cell protein can exhibit characteristic binding
to one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample or characterizing or
detecting the cancer cell protein.
[0276] In an embodiment, the present method can include contacting
a working artificial receptor or array with a sample from cells or
tissues suspected of being cancerous or including a tumor. The
sample can be serum. Binding of at least one protein to the working
artificial receptor or array can indicate or characterize the
presence of the particular cancer or tumor, such as by
characterizing the pattern of proteins present.
[0277] Cancers that can be detected or characterized by such a
method include, for example, bladder cancer, breast cancer, colon
cancer, kidney cancer, liver cancer, lung cancer, including small
cell lung cancer, esophageal cancer, gall-bladder cancer, ovarian
cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid
cancer, prostate cancer, and skin cancer, including squamous cell
carcinoma; hematopoietic tumors of lymphoid lineage, including
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic
tumors of myeloid lineage, including acute and chronic myelogenous
leukemias, myelodysplastic syndrome and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyosarcoma; tumors of the central and peripheral nervous
system, including astrocytoma, neuroblastoma, glioma and
schwannomas; other tumors, including melanoma, seminoma,
teratocarcinoma, osteosarcoma, xeroderoma pigmentosum,
keratoctanthoma, thyroid follicular cancer, Kaposi's sarcoma, and
the like.
Methods of Binding or Detecting Microbes
[0278] In an embodiment, the invention can include methods and/or
devices for binding or detecting a microbe, e.g., cell or virus.
Methods and systems for detection can include methods and systems
for clinical chemistry, environmental analysis, and diagnostic
assays of all types. For example, the artificial receptor can be
contacted with a sample including or suspected of including at
least one microbe, e.g., cell or virus. The building blocks making
up the artificial receptors can be naive to the test ligand. Then,
binding of one or more of the microbes to the artificial receptors
can be detected. Next, the binding results can be interpreted to
provide information about the sample. In an embodiment, the
invention includes a method for detecting a microbe, e.g., cell or
virus, in a sample including contacting an artificial receptor
specific to the microbe, e.g., cell or virus, with a sample
suspected of containing the microbe, e.g., cell or virus. The
method can also include detecting or quantitating binding of the
microbe, e.g., cell or virus, to the artificial receptor.
[0279] FIG. 8 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as a microbe, e.g., cell or virus. The
method can include making an array of candidate artificial
receptors. The building blocks making up the artificial receptors
can be naive to the test ligand. Working artificial receptors can
be identified by contacting the array with a microbe, e.g., cell or
virus, and identifying which receptors bind the microbe. The method
can include producing an array or device including the working
artificial receptor or receptor complex. In an embodiment, the
method can include employing the array or device for detecting or
characterizing the microbe, e.g., cell or virus, in a sample, such
as a biological, laboratory, or environmental sample.
[0280] FIG. 9 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
microbe, e.g., cell or virus. The method can include evaluating an
array including a significant number of candidate artificial
receptors for binding to a microbe, e.g., cell or virus. The
building blocks making up the artificial receptors can be naive to
the test ligand. The microbe can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample, or characterizing
or detecting the microbe, e.g., cell or virus.
[0281] FIG. 11 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand,
such as a disease causing organism. This embodiment of the present
method includes evaluating a plurality (e.g. array) of candidate
artificial receptors for binding to each of a plurality of test
ligands, such as disease causing organisms. The building blocks
making up the artificial receptors can be naive to the test
ligands. The method can include detecting binding of each test
ligand (e.g., disease causing organism) to a subset of the
plurality or array of candidate artificial receptors. This can be
envisioned as developing a working artificial receptor or
artificial receptor complex for each of the plurality of test
ligands.
[0282] Thus, each test ligand (e.g., disease causing organism) can
provide a pattern of bound receptors in the plurality or array. The
pattern of bound receptors can be characteristic of the test ligand
or a sample including the test ligand. The method can include
storing a representation of the binding pattern as an image or a
data structure. The representation of the binding pattern can be
evaluated either by an operator or data processing system. The
method can include such evaluating. A binding pattern from an
unknown sample that matches the binding pattern for a particular
test ligand (e.g., disease causing organism) then characterizes the
unknown sample as containing that test ligand. Similarly, a binding
pattern from an unknown sample can be evaluated against the
patterns of a plurality of particular test ligands and the sample
can be characterized as containing one or more of the test ligands.
A plurality of binding patterns can be stored as a database.
[0283] An embodiment of the illustrated method can include creating
an array of artificial receptors. This embodiment can also include
compiling a database of the binding patterns of specific disease
causing organisms, for example, by probing the array with a
plurality of individual organisms. Contacting the array with an
unidentified organism can create a test binding pattern. The method
can then compare the test binding pattern with the binding patterns
of known organisms in the database in order to characterize or
classify the unidentified organism. In an embodiment, the database
and the array of receptors has already been constructed and the
method involves probing the array with an unknown organism to
create a test binding pattern and then comparing this binding
pattern with the binding patterns in the database in order to
characterize or classify the unidentified organism.
[0284] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single microbe, e.g., cell or virus, or
working artificial receptors for a plurality of microbes, e.g.,
cells or viruses. For example, a method can include contacting the
artificial receptors with a sample. A substrate including working
artificial receptors for a single microbe, e.g., cell or virus, can
be employed in a method or system for detecting that microbe.
Binding to the working artificial receptors indicates that the
sample includes the microbe. A substrate including working
artificial receptors for a plurality of microbes, e.g., cells or
viruses can be employed in a method or system for detecting one,
several, or all of the microbes. Binding to the working artificial
receptors for a particular microbe or microbes indicates that the
sample includes such a microbe or microbes.
[0285] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the microbes, e.g., cells or viruses. The method can
include detecting the binding pattern of the sample and comparing
it with binding patterns from known samples. FIG. 10 schematically
illustrates binding patterns on an array of working artificial
receptors. Such patterns and schemes can be employed for
identifying a variety of test ligands including microbes.
[0286] The present method can develop or employ a plurality of
working receptors specific for a particular microbe or feature on
the microbe. That is, the working receptors can be specific for a
particular microbe, but different receptors can interact with
different distinct antigens (e.g., proteins or carbohydrates),
ligands, or features of the microbe. Such a method can provide a
robust test for the presence of a microbe. For example, such a
robust test can reduce the chances of a false-positive or
false-negative result in comparison with an assay that relies upon
a single unique receptor to detect a given microbe. Further, this
embodiment of the method can develop or employ working receptors
that demonstrate higher binding affinity due to interaction with
multiple antigens or ligands on the same microbe (e.g., multivalent
binding).
[0287] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a bacterium. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
bacterium. The building blocks making up the artificial receptors
can be naive to the test ligand. The bacterium can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample or characterizing or detecting the bacterium.
[0288] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a virus
particle. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the virus particle. The building blocks making up the artificial
receptors can be naive to the test ligand. The virus particle can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample or for characterizing or detecting the virus
particle.
[0289] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a biohazard. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
biohazard. The building blocks making up the artificial receptors
can be naive to the test ligand. The biohazard can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting the
biohazard.
[0290] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting the Vibrio
cholerae. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
V. cholerae. The building blocks making up the artificial receptors
can be naive to the V. cholerae. The V. cholerae can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting V. cholerae.
[0291] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a microbe. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
microbe. The building blocks making up the artificial receptors can
be naive to the test ligand. One or more artificial receptors that
bind the microbe under appropriate conditions can be selected for
use on an affinity support that can bind that microbe. One or more
artificial receptors that bind the microbe or cell sufficiently
tightly under appropriate conditions can be selected and its
building blocks incorporated onto a scaffold molecule. An
embodiment of the method can employ a support with the one or more
artificial receptors or scaffold-receptors on its surface for
binding or immobilizing the microbe. A support with a plurality of
artificial receptors, each binding to a different portion of the
microbe, on its surface can be employed for multivalent capture or
immobilization of the microbe.
[0292] The present method can include selecting artificial
receptors that bind a particular microbe and/or the building blocks
making up these receptors (e.g., bound to a scaffold molecule) as
leads for pharmaceutical development or as active agents for
modulating an activity of the microbe or as an antibiotic against
that microbe.
[0293] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a microbe of
clinical or environmental interest. The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the microbe of clinical or
environmental interest. The building blocks making up the
artificial receptors can be naive to the test ligand. The microbe
of clinical or environmental interest can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample, or
characterizing or detecting the microbe of clinical or
environmental interest.
[0294] Suitable microbes of clinical or environmental interest
include bacteria, mycoplasma, fungus, rickettsia, or virus.
Suitable bacteria or mycoplasma of clinical or environmental
interest include Escherichia coli, Vibrio cholerae, Acinetobacter
caicoaceticus, Haemophilus influenzae, Actinobacillus actinoides,
Haemophilus parahaemolyticus, Actinobacillus lignieresii,
Haemophilus parainfluenzae, Actinobacillus suis, Legionella
pneumophila, Actinomyces bovis, Leptospira interrogans, Actinomyces
israelli, Mima polymorpha, Aeromonas hydrophila, Moraxella
lacunata, Arachnia propionica, Burkholderia mallei, Burkholderia
pseudomallei, Moraxella osioensis, Arizona hinshawii, Mycobacterium
osioensis, Bacillus cereus, Mycobacterium leprae, Bacteroides spp,
Mycobacterium spp, Bartonella bacilliformis, Plesiomonas
shigelloides, Bordetella bronchiseptica, Proteus spp, Clostridium
difficile, Pseudomonas aeruginosa, Clostridium sordellii,
Salmonella cholerasuis, Clostridium tetani, Salmonella enteritidis,
Corynebacterium diphtheriae, Salmonella typhi, Edwardsiella tarda,
Serratia marcescens, Enterobacter aerogenes, Shigella spp,
Staphylococcus epidermidis, Francisella novicida, Vibrio
parahaemolyticus, Haemophilus ducreyi, Haemophilus gallinarum,
Haemophilus haemolyticus, Bacillus anthracis, Mycobacterium bovis,
Bordetella pertussis, Mycobacterium tuberculosis, Borrella
burgdorfit, Mycoplasma pneumoniae, Borrella spp, Neisseria
gonorrhoeae, Campylobacter, Neisseria meningitides, Chlamydia
psittaci, Nocardia asteroids, Chlamydia trachomatis, Nocardia
brasillensis, Clostridium botulinum, Pasteurella haemolytica,
Clostridium chauvoei, Pasteurelia multocida, Clostridium
haemolyticus, Pasteurella pneumotropica, Clostridium histolyticum,
Pseudomonas pseudomallei, Clostridium novyl, Staphylococcus aureus,
Clostridium perfringens, Streptobacillus moniliformis, Clostridium
septicum, Cyclospora cayatanensis, Streptococcus agalacetiae,
Erysipelothrix insidiosa, Streptococcus pneumoniae, Klebsiella
pneumoniae, Streptococcus pyogenes, Listeria manocytogenes,
Yersinia pestis, Yersinia pseudotuberculosis, Yersinia
enterocolitica, Brucella abortus, Brucella canis, Brucella
melitensis, Brucella suis, and Francisella tularensis.
[0295] Suitable fungus include Absidia, Piedraia hortae,
Aspergillus, Prototheca, Candida, Paecilomyces, Cryptococcus
neoformans, Cryptosporidium parvum, Phialaphora, Dermatophilus
congolensis, Rhizopus, Epidermophyton, Scopulariopsis, Exophiala,
Sporothrix schenkii, Fusarium, Trichophyton, Madurella mycetomi,
Toxoplasma, Trichosporon, Microsporum, Microsporidia, Wangiella
dermatitidis, Mucor, Blastomyces dermatitidis, Giardia lamblia,
Entamoeba histolytica, Coccidioides immitis, and Histoplasma
capsulatum.
[0296] Suitable rickettsia or viruses of clinical or environmental
interest include Coronaviruses, Hepatitis viruses, Hepatitis A
virus, Myxo-Paramyxoviruses (Influenza viruses, Measles virus,
Mumps virus, Newcastle disease virus), Picornavirus (Coxsackie
viruses, Echoviruses, Poliomyelitis virus), Rickettsia akari,
Rochalimaea Quintana, Rochalimaea vinsonii, Norwalk Agent,
Adenoviruses, Arenaviruses (Lymphocytic choriomenigitis,
Viscerotrophic strains), Herpesvirus Group (Herpesvirus hominis,
Cytomegalovirus, Epstein-Barr virus, Caliciviruses, Pseudo-rabies
virus, Varicella virus), Human Immunodeficiency Virus,
Parainfluenza viruses (Respiratory syncytial virus, Subsclerosing
panencephalitis virus), Picornaviruses (Poliomyelitis virus),
Poxviruses Variola, Cowpox virus (Molluscum contagiosum virus,
Monkeypox virus, Orf virus, Paravaccinia virus, Tanapox virus,
Vaccinia virus, Yabapox virus), Papovaviruses (SV 40 virus,
B-K-virus), Spongiform Encephalopathy Viruses (Creutzfeld-Jacob
agent, Kuru agent, BSE), Rhabdoviruses (Rabies virus), Tobaviruses
(Rubella virus), Coxiella burnetii, Rickettsia canada, Rickettsia
prowazekii, Rickettsia rickettsii, Rickettsia Tsutsugamushi,
Rickettsia typhi (R. mooseri), Spotted Fever Group Agents,
Vesicular Stomatis Virus (VSV), and Toga, Arena (e.g., LCM, Junin,
Lassa, Marchupo, Guanarito, etc.), Bunya (e.g., hantavirus, Rift
Valley Fever, etc.), Flaviruses (Dengue), and Filoviruses (e.g.,
Ebola, Marburg, etc.) of all types, Nipah virus, viral encephalitis
agents, LaCrosse, Kyasanur Forest virus, Yellow fever, and West
Nile virus.
[0297] Suitable microbes of clinical or environmental interest
include Variola Viruses, Congo-Crimean hemorrhagic fever,
Tick-borne encephalitis virus complex (Absettarov, Hanzalova, Hypr,
Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and
Russian Spring-Summer Encephalitis), Marburg, Ebola, Junin, Lassa,
Machupo, Herpesvirus simiae, Bluetongue, Louping III, Rift Valley
fever (Zing a), Wesselsbron, Foot and Mouth Disease, Newcastle
Disease, African Swine Fever, Vesicular exanthema, Swine vesicular
disease, Rinderpest, African horse sickness, Avian influenza, and
Sheep pox. Other components of interest include Ricinus
communis.
Methods for Disrupting Binding Interactions
[0298] In an embodiment, the present invention can include methods
and/or devices for detecting agents that disrupt binding of
molecules, for example, macromolecules or a macromolecule and a
small molecule. Methods and systems for detecting such agents can
include methods and systems useful in developing therapeutic
agents, in clinical chemistry, in environmental analysis, and in
diagnostic assays of all types. In an embodiment, such a method
includes decreasing binding of a test ligand to one or more working
artificial receptors with one or more candidate disruptors. In an
embodiment, such a method includes decreasing binding of a binding
partner to a test ligand with one or more candidate disruptors, the
test ligand being bound to one or more working artificial
receptors.
[0299] In an embodiment, the present method includes selecting a
working artificial receptor or receptor complex that binds a target
molecule. This embodiment of the method includes binding the target
molecule to the working receptor(s). The method then includes
contacting the receptor with bound target molecule with one or more
disruptor candidates. Contacting can occur in a high throughput
screening format. The method includes selecting one or more
disruptor candidates that decreases binding of the target molecule
to the working receptor(s) as a lead disruptor(s).
[0300] Any of the general types of test ligands described herein
can be a target molecule. In an embodiment, the target molecule can
be one known to participate in a binding interaction with another
molecule. The target molecule can be on a microbe, and the entire
microbe can be employed as a target molecule. The target molecule
can be a complex of two or more macromolecules, for example, a
complex of two proteins. The target molecule can be a protein. The
target molecule can be a polynucleotide. The target molecule be on
a cell, and the entire cell can be employed as a target molecule.
The target molecule can be a receptor.
[0301] The lead disruptor can be subjected to further evaluation
for example, as a candidate therapeutic agent, candidate vaccine,
or candidate antigen. A lead disruptor that disrupts binding of a
microbe to an artificial receptor of the present invention can be
selected for further evaluation as an antibiotic against that
microbe, as a candidate immunogen against that microbe, or as a
candidate vaccine against that microbe. A lead disruptor that
disrupts binding of a macromolecule to an artificial receptor of
the present invention can be selected for further evaluation as a
therapeutic agent, as a candidate immunogen against that
macromolecule or an organism including that macromolecule, or as a
candidate vaccine against that macromolecule or an organism
including that macromolecule.
[0302] FIG. 12 schematically illustrates an embodiment of a method
for detecting an agent that disrupts a binding interaction of a
target molecule. This embodiment of the present method can be
employed for detecting an agent that disrupts a binding interaction
of a target molecule, such as a macromolecule or a microbe. The
method can include making an array of candidate artificial
receptors. The building blocks making up the artificial receptors
can be naive to the test ligand. Working artificial receptors can
be identified by contacting the array with target molecule and
identifying which receptors bind the target molecule. The method
can include producing an array or device including the working
artificial receptor or receptor complex. This method can include
producing or employing the selected working artificial receptor or
receptor complex on a substrate, such as a slide. The substrate can
include working artificial receptors for a single target molecule
or working artificial receptors for a plurality of target
molecules. The method includes binding the target molecule(s) to
the artificial receptors.
[0303] This illustrated embodiment includes contacting the
artificial receptors with bound target molecule with one or more
candidate disruptors. Release of the target molecule from the
working artificial receptors or decrease in binding of the target
molecule to the artificial receptors indicates that the candidate
disruptor is a working or lead disruptor, and can be selected as
such. A substrate including working artificial receptors for a
single target molecule can be employed in a method or system for
detecting disruptors of binding interactions of that target
molecule. A substrate including working artificial receptors for a
plurality of target molecules can be employed in a method or system
for detecting disruptors of binding interactions for one, several,
or all of the target molecules. The method can include washing
unbound or released target molecule from the support.
[0304] In an embodiment, the present method includes selecting a
working artificial receptor or receptor complex that binds a
protein. This embodiment of the method includes binding the protein
to the working receptor(s). The method then includes contacting the
receptor with bound protein with one or more disrupter candidates.
Contacting can occur in a high throughput screening format. The
method includes selecting one or more disruptor candidates that
decreases binding of the protein to the working receptor(s) as a
lead disruptor(s).
[0305] Such a method can be employed for detecting an agent that
disrupts a binding interaction of a protein. The method can include
making an array of candidate artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. Working artificial receptors can be identified by
contacting the array with protein and identifying which receptors
bind the protein. The method can include producing an array or
device including the working artificial receptor or receptor
complex. This method can include producing or employing the
selected working artificial receptor or receptor complex on a
substrate, such as a slide. The substrate can include working
artificial receptors for a single protein or working artificial
receptors for a plurality of proteins. The method includes binding
the protein(s) to the artificial receptors. This embodiment of the
method includes contacting the artificial receptors with bound
protein with one or more candidate disruptors. Release of the
protein from the working artificial receptors or decrease in
binding of the protein to the artificial receptors indicates that
the candidate disruptor is a working or lead disruptor, and can be
selected as such.
[0306] In an embodiment, the disruptor disrupts a binding
interaction of a protein. Such a disruptor can be envisioned as a
mimic of one or more structural features to which this protein
binds, for example on a microbe, tissue, or cell. The disruptor can
be evaluated for such mimicry. The mimic disruptor can then be used
as an antigen against the structural feature on the microbe,
tissue, or cell. The mimic disruptor can be used as an idiotype or
anti-idiotype against the structural feature on the microbe,
tissue, or cell.
[0307] In an embodiment, the present method includes selecting a
working artificial receptor or receptor complex that binds a
microbe. This embodiment of the method includes binding the microbe
to the working receptor(s). The method then includes contacting the
receptor with bound microbe with one or more disruptor candidates.
Contacting can occur in a high throughput screening format. The
method includes selecting one or more disruptor candidates that
decreases binding of the microbe to the working receptor(s) as a
lead disruptor(s).
[0308] Such a method can be employed for detecting an agent that
disrupts a binding interaction of a microbe. The method can include
making an array of candidate artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. Working artificial receptors can be identified by
contacting the array with microbe and identifying which receptors
bind the microbe. The method can include producing an array or
device including the working artificial receptor or receptor
complex. This method can include producing or employing the
selected working artificial receptor or receptor complex on a
substrate, such as a slide. The substrate can include working
artificial receptors for a single microbe or working artificial
receptors for a plurality of microbes. The method includes binding
the microbe(s) to the artificial receptors. This embodiment of the
method includes contacting the artificial receptors with bound
microbe with one or more candidate disruptors. Release of the
microbe from the working artificial receptors or decrease in
binding of the microbe to the artificial receptors indicates that
the candidate disrupter is a working or lead disruptor, and can be
selected as such.
[0309] In an embodiment, the disruptor disrupts a binding
interaction of a microbe. Such a disruptor can be envisioned as a
mimic of one or more structural features to which this microbe
binds, for example on a protein, another microbe, a tissue, or a
cell. The disruptor can be evaluated for such mimicry. The mimic
disruptor can then be used as an antigen against the structural
feature on the protein, other microbe, tissue, or cell. The mimic
disrupter can be used as an idiotype or anti-idiotype against the
structural feature on the protein, other microbe, tissue, or
cell.
[0310] In an embodiment, the present method includes selecting a
working artificial receptor or receptor complex that binds a cell.
This embodiment of the method includes binding the cell to the
working receptor(s). The method then includes contacting the
receptor with bound cell with one or more disruptor candidates.
Contacting can occur in a high throughput screening format. The
method includes selecting one or more disruptor candidates that
decreases binding of the cell to the working receptor(s) as a lead
disruptor(s).
[0311] Such a method can be employed for detecting an agent that
disrupts a binding interaction of a cell. The method can include
making an array of candidate artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. Working artificial receptors can be identified by
contacting the array with cell and identifying which receptors bind
the cell. The method can include producing an array or device
including the working artificial receptor or receptor complex. This
method can include producing or employing the selected working
artificial receptor or receptor complex on a substrate, such as a
slide. The substrate can include working artificial receptors for a
single cell or working artificial receptors for a plurality of
cells. The method includes binding the cell(s) to the artificial
receptors. This embodiment of the method includes contacting the
artificial receptors with bound cell with one or more candidate
disruptors. Release of the cell from the working artificial
receptors or decrease in binding of the cell to the artificial
receptors indicates that the candidate disrupter is a working or
lead disruptor, and can be selected as such.
[0312] In an embodiment, the disruptor disrupts a binding
interaction of a cell. Such a disruptor can be envisioned as a
mimic of one or more structural features to which this cell binds,
for example on a protein, another cell, a tissue, or a microbe. The
disruptor can be evaluated for such mimicry. The mimic disruptor
can then be used as an antigen against the structural feature on
the protein, microbe, tissue, or other cell. The mimic disruptor
can be used as an idiotype or anti-idiotype against the structural
feature on the protein, microbe, tissue, or other cell.
[0313] Any of a variety of compounds can be employed as a candidate
disrupter. For example, candidate disruptors can include at least
one small molecule. For example, candidate disruptors can include a
library of small molecules. For example, candidate disruptors can
include at least one peptide. For example, candidate disruptors can
include a library of peptides.
Disrupting a Complex
[0314] In an embodiment, the present method includes a method for
detecting a disruptor of binding of a binding partner to a test
ligand (e.g., target molecule), the test ligand being bound to one
or more working artificial receptors. In an embodiment, the present
method includes selecting a working artificial receptor or receptor
complex that binds a complex including a target molecule. Such
selecting can include evaluating artificial receptors for binding
to the target molecule. The building blocks making up the
artificial receptors can be naive to the test ligand. From among
those artificial receptors that bind the target molecule, the
method selects those artificial receptors that can bind the
complex. The complex including the target molecule can also include
one or more binding partners for the target molecule. This
embodiment of the method includes binding the complex to the
selected working receptor(s). The method then includes contacting
the receptor with bound complex with one or more disruptor
candidates. Contacting can occur in a high throughput screening
format. The method includes selecting one or more disruptor
candidates that decrease binding of at least one binding partner to
the complex as a lead disruptor(s).
[0315] FIG. 13 schematically illustrates an embodiment of a method
for detecting an agent that disrupts a binding interaction of a
complex including a target molecule. This embodiment of the present
method can be employed for detecting an agent that disrupts a
binding interaction of a complex, such as a protein:small molecule
complex, a protein:protein complex, a protein:polynucleotide
complex, a protein:polysaccharide complex, a protein:microbe
complex, or a protein:cell complex. The method can include making
an array of candidate artificial receptors. The building blocks
making up the artificial receptors can be naive to the test ligand.
Working artificial receptors can be identified by contacting the
array with target molecule and identifying which receptors bind the
target molecule. The identified working artificial receptors can be
contacted with the complex including the target molecule and the
receptors that bind the complex can be selected. The method can
include producing an array or device including the selected working
artificial receptor or receptor complex. This method can include
producing or employing the selected working artificial receptor or
receptor complex on a substrate, such as a slide. The method
includes binding the complex to the artificial receptors.
[0316] This illustrated embodiment includes contacting the
artificial receptors with bound complex with one or more candidate
disruptors. Release of the binding partner portion of the complex
from the working artificial receptors but retaining the target
molecule on the receptors indicates that the candidate disrupter is
a working or lead complex disruptor, and can be selected as such.
Decrease in binding of the binding partner portion of the complex
to the artificial receptors but retaining the target molecule on
the receptors indicates that the candidate disruptor is a working
or lead complex disruptor, and can be selected as such. The working
or lead complex disruptor can, in an embodiment, be selected as a
lead for developing a therapeutic agent for a disorder mediated by
the complex. The method can include washing unbound or released
binding partner from the support.
[0317] In an embodiment, the complex disruptor disrupts a complex
including at least two proteins, a first protein and a second
protein. FIG. 14 schematically illustrates a candidate disruptor
disrupting a protein:protein complex. In this embodiment, one
protein component remains bound to the receptor and the other
dissociates and leaves the receptor. An embodiment of such a
disrupter can be envisioned as a mimic of one or more structural
features of the binding portion of one of the proteins included in
the complex. The disruptor can be evaluated for such mimicry. For
example, the disrupter can mimic a structural feature of the first
protein that interacts with the second protein. The mimic disruptor
can then be used as an antigen against that feature of the first
protein. The mimic disruptor can be used as an idiotype or
anti-idiotype against the structural feature on the first
protein.
Methods for Making and Using Affinity Supports
[0318] In an embodiment, a working artificial receptor or receptor
complex can be employed to produce or as an affinity support for
any of the test ligands described herein. For example, the present
method can include a method for producing an affinity support for a
test ligand. This method can include selecting a working artificial
receptor or receptor complex that binds to the test ligand. This
method can also include coupling the working artificial receptor or
receptor complex to a support. FIG. 15 schematically illustrates an
embodiment of such a method. The support can be suitable for use as
an affinity support for, for example, chromatography, membrane
filtration, electrophoresis (e.g., 1 or 2 dimensional
electrophoresis), or the like.
[0319] The present method can include selecting artificial
receptors that bind a particular test ligand and/or the building
blocks making up these receptors (e.g., bound to a scaffold
molecule) for isolation or analysis of a particular test ligand.
The building blocks making up the artificial receptors can be naive
to the test ligand. For example, the artificial receptor can be
employed as a receptor surface that can bind the test ligand and
remove (e.g., purify) it from a mixture or biological sample.
[0320] Such a method can include contacting one or more candidate
artificial receptors with a test ligand of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the test ligand as working
artificial receptor(s). The method can then include employing the
working artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a significant quantity of the test
ligand of interest. The support can be a chromatography support or
medium. The support can be a plate, tube, or membrane. In an
embodiment, binding of the test ligand of interest to the support
can be followed by eluting the test ligand of interest from the
support. Eluting can employ a wash with a pH, buffer, solvent, salt
concentration, or ligand concentration effective to elute the test
ligand of interest from the support.
[0321] FIG. 16 schematically illustrates evaluating an array of
candidate artificial receptors for binding of a test ligand and
selecting one or more working artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. FIG. 16 illustrates that a receptor surface employing such
a working artificial receptor can be employed for binding a
protein, immobilizing an antibody, binding a single enantiomer, or
protecting a structural feature (e.g., a functional group) on a
compound. In an embodiment, the receptor surface can bind more than
one structural feature on the protein. In an embodiment, the
working artificial receptor can be selected to bind the constant
portion, rather than the variable portions, of an antibody. In an
embodiment, the receptor surface can include a catalytic moiety
that can catalyze a reaction of a functional group of the bound
test ligand. Such a catalytic moiety can be a building block, for
example, an organometallic building block.
[0322] The present method can include selecting artificial
receptors that bind a particular isomer of a compound and/or the
building blocks making up these receptors (e.g., bound to a
scaffold molecule) for isolation or analysis of a particular
isomer. For example, the artificial receptor can be employed as a
receptor surface that can bind the isomer and remove (e.g., purify)
it from a mixture or biological sample.
[0323] Such a method can include contacting one or more candidate
artificial receptors with an isomer of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the isomer as working
artificial receptor(s). The method can then include employing the
working artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a significant quantity of the isomer
of interest. The support can be a chromatography support or medium.
The support can be a plate, tube, or membrane. In an embodiment,
binding of the isomer of interest to the support can be followed by
eluting the isomer of interest from the support. Eluting can employ
a wash with a pH, buffer, solvent, salt concentration, or ligand
concentration effective to elute the isomer of interest from the
support.
[0324] The present method can include selecting artificial
receptors that bind or protect a particular structural feature of a
compound and/or the building blocks making up these receptors
(e.g., bound to a scaffold molecule) for isolation or analysis of
the compound including the structural feature. For example, the
artificial receptor can be employed as a receptor surface that can
bind or protect the structural feature of the compound. Binding of
the structural feature can be determined by lack of binding of an
analogous compound the lacking the structural feature. Protection
of the structural feature can be evaluated by that structural
feature being unavailable to a, for example, solution phase
reactive species when the compound is bound to the receptor
surface.
[0325] Such a method can include contacting one or more candidate
artificial receptors with a compound of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the structural feature of
the compound as lead artificial receptor(s). The lead artificial
receptor can be evaluated for protecting the structural feature.
The method can then include employing the working artificial
receptor(s) to make a receptor surface. Making a receptor surface
can include coupling the building blocks making up the working
artificial receptor(s) to a support. The support can have
sufficient area to bind a significant quantity of the compound of
interest. In an embodiment, binding of the compound of interest to
the support can be followed by reacting a portion of the compound
that is not the bound or protected structure feature.
[0326] The present method can include selecting artificial
receptors that bind a particular peptide or protein and/or the
building blocks making up these receptors (e.g., bound to a
scaffold molecule) for isolation or analysis of a particular
peptide or protein. For example, the artificial receptor can be
employed as a receptor surface that can bind the peptide or protein
and remove (e.g., purify) it from a mixture or biological
sample.
[0327] Such a method can include contacting one or more candidate
artificial receptors with a peptide or protein of interest. The
building blocks making up the artificial receptors can be naive to
the test ligand. The method can include selecting one or more of
the candidate artificial receptors that bind the peptide or protein
as working artificial receptor(s). The method can then include
employing the working artificial receptor(s) to make a receptor
surface. Making a receptor surface can include coupling the
building blocks making up the working artificial receptor(s) to a
support. The support can have sufficient area to bind a significant
quantity of the peptide or protein of interest. The support can be
a chromatography support or medium. The support can be a plate,
tube, or membrane. In an embodiment, binding of the peptide or
protein of interest to the support can be followed by eluting the
peptide or protein of interest from the support. Eluting can employ
a wash with a pH, buffer, salt concentration, or ligand
concentration effective to elute the peptide or protein of interest
from the support.
[0328] In an embodiment, the present artificial receptors can be
employed to form selective membranes. Such a selective membrane can
be based on a molecular gate including an artificial receptor
surface. For example, an artificial receptor surface can line the
walls of pores in the membrane and either allow or block a target
molecule from passing through the pores. For example, an artificial
receptor surface can line the walls of pores in the membrane and
act as "gatekeepers" on e.g. micro cantilevers/molecular
cantilevers to allow gate opening or closing on binding of the
target. The artificial receptors to be used in the selective
membranes can be identified by exposing the target molecule to a
plurality of distinct artificial receptors and then determining
which ones it binds to. For example, the binding can be detected
through any of the techniques described herein, including
fluorescence.
[0329] In certain embodiments, the method can include producing one
or more receptor surfaces, each receptor surface including building
blocks from a working receptor for a particular test ligand. Such a
method can include employing the receptor surface for
chromatography of the test ligand. Chromatographing the test ligand
against a plurality of such receptor surfaces can rank the affinity
of the surfaces for the test ligand. Under a given set of
conditions, the receptor surface that retains the chromatographed
test ligand the longest exhibits the greatest affinity for the test
ligand. The method can include selecting the receptor surface with
suitable (e.g., the greatest) affinity for use as an affinity
support for the test ligand.
[0330] Any of a variety of supports can be employed as the affinity
support. In certain embodiments, the affinity support can be a
dish, a tube, a well, a bead, a chromatography support, a
microchannel, or the like. The artificial receptor affinity support
can be used in various applications, such as chromatography,
microchannel devices, as an immunoassay support, or the like. A
microchannel with the artificial receptor on its surface can be
employed as an analytical device. In an embodiment, the present
artificial receptors can be employed to form bioactive surfaces.
For example, receptor surfaces can be used to specifically bind
antibodies or enzymes.
Methods for Making and Using Reaction Supports
[0331] In an embodiment, a working artificial receptor or receptor
complex can be employed to produce or as a reaction support for any
of the test ligands described herein. For example, the present
method can include a method for producing a reaction support for at
least one test ligand. This method can include selecting a working
artificial receptor or receptor complex that binds to the test
ligand under conditions suitable for a desired reaction with that
test ligand. This method can also include coupling the working
artificial receptor or receptor complex to a support. FIG. 15
schematically illustrates an embodiment of such a method. The
support can be suitable for use as a reaction support for, for
example, oxidation, reduction, substitution, or displacement
reactions.
[0332] The present method can include selecting artificial
receptors that bind a particular test ligand and/or the building
blocks making up these receptors (e.g., bound to a scaffold
molecule) for reaction of a particular test ligand. For example,
the artificial receptor can be employed as a receptor surface that
can bind the test ligand and position it for reaction at a
particular prochiral group, functional group, or orientation.
[0333] Such a method can include contacting one or more candidate
artificial receptors with a test ligand of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the test ligand as working
artificial receptor(s). The method can then include employing the
working artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a desired quantity of the test ligand
of interest. The support can be a chromatography support or medium.
The support can be a plate, bead, tube, or membrane.
[0334] The method also includes contacting the support including
bound test ligand with a reactant for the desired reaction.
Suitable reactants include reducing agent, oxidizing agent,
nucleophile, electrophile, solvent (e.g., aqueous or organic
solvent), or the like. The method can include contacting with one
or more reactants and selecting reactant or reactants suitable for
participating in the desired reaction. This embodiment of the
method includes reacting the test ligand with the reactant. In an
embodiment, reacting can be followed by washing the reactant or
side products from the support. In an embodiment, reacting can be
followed by eluting the product (e.g., reacted test ligand) from
the support. Eluting can employ a wash with a pH, buffer, solvent,
salt concentration, or ligand concentration effective to elute the
product from the support.
[0335] FIG. 16 schematically illustrates evaluating an array of
candidate artificial receptors for binding of a test ligand and
selecting one or more working artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. FIG. 16 illustrates that a receptor surface employing such
a working artificial receptor can be employed for binding a test
ligand. The test ligand can be bound in an orientation that leaves
a reactive moiety available for reaction with a reactant placed
into contact with the receptor surface. In an embodiment, the test
ligand can be bound in an orientation that occludes or protects a
second reactive moiety from reacting with the reactant. This
embodiment includes reacting the test ligand and release of the
reacted test ligand from the receptor surface. Specifically, the
illustration shows the reduction of an aldehyde with sodium
borohydride to produce an alcohol. In an embodiment, the receptor
surface can include a catalytic moiety that can catalyze a reaction
of a functional group of the bound to test ligand the catalytic
reaction can also employ the reactant. Such a catalytic moiety can
be a building block, for example, an organometallic building
block.
[0336] The present method can include selecting artificial
receptors that bind or protect a particular structural feature of a
compound and/or the building blocks making up these receptors
(e.g., bound to a scaffold molecule) for reacting the compound
including the structural feature. For example, the artificial
receptor can be employed as a receptor surface that can bind and
protect the structural feature of the compound while another
feature of the compound reacts with a reactant. Protection of the
structural feature can be evaluated by that structural feature not
reacting with the reactant. For example, a substrate (e.g. a
steroid) can be stereospecifically bound to the artificial receptor
and present a particular moiety/sub-structure/"face" for reaction
with a reagent in solution.
[0337] In an embodiment, a first side of a molecule (or a
functional group) is bound to a receptor surface while a second
side is left exposed. Then a reagent is added that could react with
either side (or group) but is hindered from reacting with the first
side of the molecule since it is bound to the receptor surface,
accordingly the reagent reacts with the second side of the molecule
only.
[0338] Such a method can include contacting one or more candidate
artificial receptors with a compound of interest. The method can
include selecting one or more of the candidate artificial receptors
that bind the structural feature of the compound as lead artificial
receptor(s). The lead artificial receptor can be evaluated for
protecting the structural feature. The method can then include
employing the working artificial receptor(s) to make a receptor
surface. Making a receptor surface can include coupling the
building blocks making up the working artificial receptor(s) to a
support. The support can have sufficient area to bind a desired
quantity of the compound of interest. In an embodiment, binding of
the compound of interest to the support can be followed by reacting
a portion of the compound that is not the bound or protected
structure feature.
[0339] Conventional synthesis of a chiral compound generally
requires complicated procedures. In an embodiment, the present
candidate artificial receptors can be employed to find receptor
surfaces that provide a spatially oriented binding surface for a
stereospecific reaction. For example, an artificial receptor
surface can bind a small molecule so that particular functional
groups are exposed to the environment, and others are obscured by
the receptor. In this manner, the stereospecificity of the reaction
can be controlled. Therefore, an artificial receptor surface can be
employed in synthesis including chiral induction. Similarly,
regiospecificity can also be controlled using receptors of the
present invention.
[0340] The present method can include selecting artificial
receptors that bind a first reaction ligand and a second reaction
ligand or the building blocks making up these receptors (e.g.,
bound to a scaffold molecule) for a reaction including the first
and second reaction ligands. For example, the artificial receptor
can be employed as a receptor surface that can bind the first
reaction ligand and the second reaction ligand at a distance or
orientation at which these ligands can react with one another. The
reaction can optionally include one or more reactants not bound to
the receptor surface.
[0341] Such a method can include contacting one or more candidate
artificial receptors with a first reaction ligand and a second
reaction ligand. The building blocks making up the artificial
receptors can be naive to the ligands. The method can include
selecting one or more of the candidate artificial receptors that
bind both of the reaction ligands as working artificial
receptor(s). The method can then include employing the working
artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a desired quantity of the first and
second reaction ligands. The support can be a chromatography
support or medium. The support can be a plate, bead, tube, or
membrane. The first and second reaction ligands can be bound to the
support at one or several molar ratios, the reaction evaluated, and
a molar ratio selected for conducting the reaction.
[0342] The method can also include contacting the support including
bound reaction ligands with a reactant for the desired reaction.
Suitable reactants include reducing agent, oxidizing agent,
nucleophile, electrophile, or the like. This embodiment of the
method includes reacting the test ligand with the reactant. In an
embodiment, reacting can be followed by washing the reactant or
side products from the support. In an embodiment, the first and
second reaction ligands react without the reactant. In an
embodiment, reacting can be followed by eluting the product (e.g.,
reacted test ligand) from the support. Eluting can employ a wash
with a pH, buffer, solvent, salt concentration, or ligand
concentration effective to elute the product from the support.
[0343] In an embodiment, the present candidate artificial receptors
can be employed to find receptor surfaces that provide a spatially
oriented binding surface for a stereospecific reaction. For
example, an artificial receptor surface can bind a small molecule
with particular functional groups exposed to the environment, and
others obscured by the receptor. Such an artificial receptor
surface can be employed in synthesis including chiral induction.
For example, a substrate (e.g. a steroid) can be stereospecifically
bound to the artificial receptor and present a particular
moiety/sub-structure/"face" for reaction with a reagent in
solution. Similarly, the artificial receptor surface can act as a
protecting group where a reactive moiety of a molecule is
"protected" by binding to the receptor surface so that a different
moiety with similar reactivity can be transformed.
[0344] In an embodiment, the one or more working artificial
receptors that bind a plurality (e.g., 2) of the reactants can be
produced on a substrate and the reactants bound. Each receptor with
a plurality of bound reactants can then be screened against one or
more reagents or conditions (e.g., various molar ratios of the
reactants or various solvents). The artificial receptor allowing or
promoting reaction between the two or more reactants can be
identified. The artificial receptor can then be produced on a
substrate to provide a reactor for the reaction of interest.
[0345] Any of a variety of supports can be employed as the reaction
support. In certain embodiments, the reaction support can be a
dish, a tube, a well, a bead, a chromatography support, a
microchannel, or the like. The artificial receptor reaction support
can be used in various applications, such as a microchannel device.
A microchannel with the artificial receptor on its surface can be
employed as a reactor.
Methods for Making and Using Supported Catalysts
[0346] In an embodiment, a working artificial receptor or receptor
complex can be employed to produce or as a supported catalyst for
any of the test ligands described herein. For example, the present
method can include a method for producing a supported catalyst for
at least one test ligand. This method can include selecting a
working artificial receptor or receptor complex that binds to the
test ligand under conditions suitable for catalyzing a reaction
with that test ligand. This method can also include coupling the
working artificial receptor or receptor complex to a support. FIG.
15 schematically illustrates an embodiment of such a method. The
support can be suitable for use as a supported catalyst for any of
a variety of catalytic moieties or building blocks, such as an
organometallic moiety, a coenzyme, a redox active moiety, a
nucleophilic moiety, an acid moiety, a base moiety, or the
like.
[0347] The present method can include selecting artificial
receptors that bind and catalyze a reaction of a particular test
ligand and/or the building blocks making up these receptors (e.g.,
bound to a scaffold molecule) for catalyzing a reaction of a
particular test ligand. For example, the artificial receptor can be
employed as a receptor surface that can bind the test ligand and
position it for reaction with the catalytic moiety at a particular
prochiral group, functional group, or orientation.
[0348] Such a method can include contacting one or more candidate
artificial receptors with a test ligand of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind and catalyze a desired
reaction of the test ligand as working artificial receptor(s). The
method can then include employing the working artificial
receptor(s) to make a receptor surface. Making a receptor surface
can include coupling the building blocks making up the working
artificial receptor(s) to a support. The support can have
sufficient area to bind a desired quantity of the test ligand of
interest. The support can be a chromatography support or medium.
The support can be a plate, bead, tube, or membrane.
[0349] The method also includes contacting the support including
bound test ligand with a reactant or cofactor for the desired
reaction. Suitable reactants include reducing agent, oxidizing
agent, nucleophile, electrophile, solvent (e.g., aqueous or organic
solvent), or the like. The method can include contacting with one
or more selecting reactants and selecting reactant or reactants
suitable for participating in the desired reaction. This embodiment
of the method includes reacting the test ligand with the reactant.
In an embodiment, reacting can be followed by washing the reactant
or side products from the support. In an embodiment, reacting can
be followed by eluting the product (e.g., reacted test ligand) from
the support. Eluting can employ a wash with a pH, buffer, solvent,
salt concentration, or ligand concentration effective to elute the
product from the support.
[0350] FIG. 16 schematically illustrates evaluating an array of
candidate artificial receptors for binding and catalysis of a
reaction of a test ligand and selecting one or more working
artificial receptors. The building blocks making up the artificial
receptors can be naive to the test ligand. FIG. 16 illustrates that
a receptor surface employing such a working artificial receptor can
be employed for binding and catalysis of a reaction of a test
ligand. The test ligand can be bound in an orientation that leaves
a reactive moiety available for reaction with a the catalytic
moiety of the receptor. In an embodiment, the test ligand can be
bound in an orientation that occludes or protects a second reactive
moiety from reacting with the catalytic moiety. This embodiment
includes reacting the test ligand and release of the reacted test
ligand from the receptor surface. Specifically, the illustration
shows the reduction of an aldehyde with a catalytic moiety (MC) on
the catalytic support to produce an alcohol. In an embodiment, the
receptor surface can include a catalytic moiety that can catalyze a
reaction of a functional group of the bound to test ligand the
catalytic reaction can also employ the reactant. Such a catalytic
moiety can be a building block, for example, an organometallic
building block.
[0351] In an embodiment, the invention includes a method for
identifying a catalyst including binding a first reaction ligand to
an artificial receptor array, contacting the array with a reactant,
and identifying those artificial receptors that have promoted the
reaction. The array can be screened for those artificial receptors
that produce (e.g., catalyze conversion of the reactant to) a
desired product.
[0352] Any of a variety of supports can be employed as the
catalytic support. In certain embodiments, the catalytic support
can be a dish, a tube, a well, a bead, a chromatography support, a
microchannel, or the like. The artificial receptor reaction support
can be used in various applications, such as a microchannel device.
A microchannel with the artificial receptor on its surface can be
employed as a reactor.
Methods for Making or Detecting Non-Binding Surfaces or
Substances
[0353] In an embodiment, the invention can include methods and/or
devices for not binding a test ligand. Methods and systems for not
binding a test ligand can be employed in systems useful in clinical
chemistry, environmental analysis, and diagnostic assays of all
types. In an embodiment, the invention includes a method for making
a substrate that does not bind a test ligand. The method can
include contacting at least one candidate artificial receptor with
the test ligand, detecting binding or lack of binding of the test
ligand to one or more of the artificial receptors, and selecting an
artificial receptor that does not bind the test ligand as a working
non-binding surface. The artificial receptor or receptors that do
not bind a first test ligand can be tested against one or more
additional test ligands. In such a manner, the non-binding
artificial receptors can be screened for those artificial receptors
that do not bind any of a plurality of test ligands. A surface
covered with the building blocks making up such non-binding
artificial receptors can then be employed as a working non-binding
surface.
[0354] In an embodiment, the present method can employ an array of
candidate artificial receptors. This embodiment of the method can
employ an array including a significant number of the present
artificial receptors to produce one or more working artificial
receptors or non-binding surfaces for one or more test ligands. The
method can include evaluating an array including a significant
number of candidate artificial receptors for not binding to at
least one test ligand. Those candidate artificial receptors that do
not bind to any of one or more test ligands can be selected as a
non-binding surface for those one or more test ligands.
[0355] In an embodiment, the present method can evaluate an array
of candidate artificial receptors to develop a surface that does
not bind one or more proteins (e.g., plasma proteins). This
embodiment of the method can employ an array including a
significant number of the present artificial receptors to produce
one or more working artificial receptors or non-binding surfaces
for one or more proteins (e.g., plasma proteins). The method can
include evaluating an array including a significant number of
candidate artificial receptors for not binding to at least one
protein (e.g., plasma protein). Those candidate artificial
receptors that do not bind to any of one or more proteins (e.g.,
plasma proteins) can be selected as a non-binding surface for those
proteins. Such a surface can be employed on an implantable medical
device.
[0356] In an embodiment, the present method can evaluate an array
of candidate artificial receptors to develop a surface that does
not bind one or more cells. This embodiment of the method can
employ an array including a significant number of the present
artificial receptors to produce one or more working artificial
receptors or non-binding surfaces for one or more cells. The method
can include evaluating an array including a significant number of
candidate artificial receptors for not binding to at least one
cell. Those candidate artificial receptors that do not bind to any
of one or more cells can be selected as a non-binding surface for
those cells. Such a surface can be employed on an implantable
medical device.
[0357] In an embodiment, the present method can evaluate an array
of candidate artificial receptors to develop a surface that does
not bind one or more microbes. This embodiment of the method can
employ an array including a significant number of the present
artificial receptors to produce one or more working artificial
receptors or non-binding surfaces for one or more microbes. The
method can include evaluating an array including a significant
number of candidate artificial receptors for not binding to at
least one microbe. Those candidate artificial receptors that do not
bind to any of one or more microbes can be selected as a
non-binding surface for those microbes. Such a surface can be
employed on an implantable medical device. Such a surface can be
employed in a system otherwise subject to biofouling, such as water
piping, reservoirs or flumes in a food processing plant, cooling
towers, ship bottoms.
Methods for Detecting
[0358] Contacting an array including a plurality of artificial
receptors with a test ligand can identify one or more lead or
working artificial receptors. Binding of the test ligand to the
lead or working artificial receptor or complex can produce a
detectable signal. The detectable signal can be produced, for
example, through mechanisms and properties such as scattering,
absorbing or emitting light, producing or quenching fluorescence or
luminescence, producing or quenching an electrical signal, and the
like. Spectroscopic detection methods include use of labels or
enzymes to produce light for detection by optical sensors or
optical sensor arrays. The light can be ultraviolet, visible, or
infrared light, which can be produced and/or detected through
fluorescence, fluorescence polarization, chemiluminescence,
bioluminescence, or chemibioluminescence.
[0359] Systems and methods for detecting electrical conduction, and
changes in electrical conduction, include ellipsometry, surface
plasmon resonance, capacitance, conductometry, surface acoustic
wave, quartz crystal microbalance, Love-wave, infrared evanescent
wave, enzyme labels with electrochemical detection, nanowire field
effect transistors, MOSFETS--metal oxide semiconductor field effect
transistors, CHEMFETS--organic membrane metal oxide semiconductor
field effect transistors, ICP--intrinsically conducting polymers,
FRET--fluorescence resonance energy transfer.
[0360] In an embodiment, the working artificial receptor or complex
can be configured on the surface of an optical fiber, for example,
as a series of discrete areas, spots, zones, or the like. The
working artificial receptor or complex can be contacted with the
test ligand or sample suspected of containing the test ligand. The
test ligand sample can be in the form of a stream of air, an
aerosol, or liquid (e.g., a solution or suspension). A detectable
calorimetric, fluorometric, or like signal can be produced by a
label incorporated into the optic fiber surface. The colorimetric
or fluorogenic signal can be intrinsic to the ligand or can be
produced upon binding of the ligand to the working artificial
receptors.
[0361] Apparatus that can detect such binding to or signal from a
working artificial receptor or complex includes UV, visible, or
infrared spectrometer, fluorescence or luminescence spectrometer,
surface plasmon resonance, surface acoustic wave or quartz crystal
microbalance detectors, pH, voltammetry or amperometry meters,
radioisotope detector, or the like.
[0362] In such an apparatus, a working artificial receptor or
complex can be positioned on an optic fiber to provide a detectable
signal, such as an increase or decrease in transmitted light,
reflected light, fluorescence, luminescence, or the like. The
detectable signal can originate from, for example, a signaling
moiety incorporated into the working artificial receptor or complex
or a signaling moiety added to the working artificial receptor. The
signal can also be intrinsic to the working artificial receptor or
to the test ligand. The signal can come from, for example, the
interaction of the test ligand with the working artificial
receptor, the interaction of the test ligand with a signaling
moiety that has been incorporated into the working artificial
receptor, into the optic fiber or onto the optic fiber. In an
embodiment, the present method can include selecting an artificial
receptor for which binding induces a change in the signal from the
signaling moiety, e.g., a fluorescent moiety. Such a change can
signal binding to the artificial receptor.
[0363] In an embodiment, the working artificial receptor can be on
a support such as a surface of a test tube, microwell, capillary,
microchannel, or the like. The test ligand or a sample suspected of
containing the test ligand can be contacted with the working
artificial receptor or complex by addition of a solution containing
the test ligand or a sample suspected of containing the test
ligand. A detectable signal can be produced by a labeled compound
or conjugate of the test ligand. This labeled moiety can be reacted
with the working artificial receptor or complex in competition with
the solution containing the test ligand or the sample suspected of
containing the test ligand.
[0364] In an embodiment of the system, the working artificial
receptor is on a support such as the surface of a surface acoustic
wave or quartz crystal microbalance or surface plasmon resonance
detector. The test ligand or a sample suspected of containing the
test ligand can be brought into contact with the working artificial
receptor or complex by exposure to a stream of air, to an aerosol,
or to a solution containing the test ligand or a sample suspected
of containing the test ligand. A detectable electrical signal can
be produced by the interaction of the test ligand with the working
artificial receptor or complex on the active surface of the surface
acoustic wave or quartz crystal microbalance or surface plasmon
resonance detector.
[0365] In an embodiment of the system, more than one working
artificial receptor, arranged as regions or spots in an array, on a
support, such as a glass or plastic surface can be incorporated
onto the signaling surfaces of one or more surface plasmon
resonance detectors. The ligands of interest or a sample suspected
of containing the ligands of interest (e.g., a sample containing a
mixture of DNA segments or fragments, proteins or protein
fragments, carbohydrates or carbohydrate fragments, or the like) is
brought into contact with the working artificial receptors or
array. Contacting can be accomplished by addition of a solution of
the ligands of interest or a sample suspected of containing the
ligands of interest. Detectable electrical signals can be produced
by binding of the ligands of interest to the working artificial
receptors array on the surface of the surface plasmon resonance
detectors. Such detectors produce a signal for each working
artificial receptor in the array, which produces a pattern of
signal response, which is characteristic of the composition of the
sample of interest.
[0366] The present artificial receptors can be part of products
used in: analyzing a genome and/or proteome; pharmaceutical
development; detectors for any of the test ligands; drug of abuse
diagnostics or therapy; hazardous waste analysis or remediation;
chemical warfare alert or intervention; disease diagnostics or
therapy; cancer diagnostics or therapy; biowarfare alert or
intervention; food chain contamination analysis or remediation; and
the like.
[0367] More specifically, the present artificial receptors can be
used in products for identification of sequence specific small
molecule leads; protein isolation and identification;
identification of protein to protein interactions; detecting
contaminants in food or food products; clinical analysis of food
contaminants; clinical analysis of prostate specific antigen;
clinical and field or clinical analysis of cocaine; clinical and
field or clinical analysis of other drugs of abuse; other clinical
analysis systems, home test systems, or field analysis systems;
monitors or alert systems for bioterrorism or chemical warfare
agents; and the like.
Artificial Receptors
[0368] A candidate artificial receptor, a lead artificial receptor,
or a working artificial receptor includes combination of building
blocks immobilized (e.g., reversibly) on, for example, a support.
An individual artificial receptor can be a heterogeneous building
block spot on a slide or a plurality of building blocks coated on a
slide, tube, or well. In the present embodiment, at least one of
the building blocks includes a tether moiety. The building blocks
can be immobilized through any of a variety of interactions, such
as covalent, electrostatic, or hydrophobic interactions. For
example, the building block and support or lawn can each include
one or more functional groups or moieties that can form covalent,
electrostatic, hydrogen bonding, van der Waals, or like
interactions.
[0369] An array of candidate artificial receptors can be a
commercial product sold to parties interested in using the
candidate artificial receptors as implements in developing
receptors for test ligands of interest. In an embodiment, a useful
array of candidate artificial receptors includes at least one glass
slide, the at least one glass slide including spots of a
predetermined number of combinations of members of a set of
building blocks, each combination including a predetermined number
of building blocks. In an embodiment, at least one of the building
blocks includes a tether moiety.
[0370] One or more lead artificial receptors can be developed from
a plurality of candidate artificial receptors. In an embodiment, a
lead artificial receptor includes a combination of building blocks
and binds detectable quantities of test ligand upon exposure to,
for example, several picomoles of test ligand at a concentration of
1, 0.1, or 0.01 .mu.g/ml, or at 1, 0.1, or 0.01 ng/ml test ligand;
at a concentration of 0.01 .mu.g/ml, or at 1, 0.1, or 0.01 ng/ml
test ligand; or a concentration of 1, 0.1, or 0.01 ng/ml test
ligand. In an embodiment, at least one of the building blocks
includes a tether moiety.
[0371] Artificial receptors, particularly candidate or lead
artificial receptors, can be in the form of an array of artificial
receptors. Such an array can include, for example, 1.66 million
spots, each spot including one combination of 4 building blocks
from a set of 81 building blocks. In an embodiment, at least one of
the building blocks includes a tether moiety. Such an array can
include, for example, 28,000 spots, each spot including one
combination of 2 or 3 building blocks from a set of 19 building
blocks. In an embodiment, at least one of the building blocks
includes a tether moiety. Each spot is a candidate artificial
receptor and a combination of building blocks. The array can also
be constructed to include lead artificial receptors. For example,
the array of artificial receptors can include combinations of fewer
building blocks and/or a subset of the building blocks.
[0372] In an embodiment, an array of candidate artificial receptors
includes building blocks of general Formula 2 (shown hereinabove),
with RE.sub.1 being B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9
(shown hereinabove) and with RE.sub.2 being A1, A2, A3, A3a, A4,
A5, A6, A7, A8, or A9 (shown hereinabove). In an embodiment, the
framework is tyrosine.
[0373] One or more working artificial receptors can be developed
from one or more lead artificial receptors. In an embodiment, a
working artificial receptor includes a combination of building
blocks and binds categorizing or identifying quantities of test
ligand upon exposure to, for example, several picomoles of test
ligand at a concentration of 100, 10, 1, 0.1, 0.01, or 0.001 ng/ml
test ligand; at a concentration of 10, 1, 0.1, 0.01, or 0.001 ng/ml
test ligand; or a concentration of 1, 0.1, 0.01, or 0.001 ng/ml
test ligand. In an embodiment, at least one of the building blocks
includes a tether moiety.
[0374] In an embodiment, the artificial receptor of the invention
includes a plurality of building blocks coupled to a support. In an
embodiment, the plurality of building blocks can include or be
building blocks of Formula 2 (shown hereinabove). An abbreviation
for the building block including a linker, a tether, a tyrosine
framework, and recognition elements AxBy is tether-TyrAxBy. In an
embodiment, a candidate artificial receptor can include
combinations of building blocks of formula tether-TyrA1B1,
tether-TyrA2B2, tether-TyrA2B4, tether-TyrA2B6, tether-TyrA2B8,
tether-TyrA3B3, tether-TyrA4B2, tether-TyrA4B4, tether-TyrA4B6,
tether-TyrA4B8, tether-TyrA5B5, tether-TyrA6B2, tether-TyrA6B4,
tether-TyrA6B6, tether-TyrA6B8, tether-TyrA7B7, tether-TyrA8B2,
tether-TyrA8B4, tether-TyrA8B6, or tether-TyrA8B8.
Techniques for Using Artificial Receptors
[0375] The present invention includes a method of using artificial
receptors. The present invention includes a method of screening
candidate artificial receptors to find lead artificial receptors
that bind a particular test ligand. Detecting test ligand bound to
a candidate artificial receptor can be accomplished using known
methods for detecting binding to arrays on a slide or to coated
tubes or wells. For example, the method can employ test ligand
labeled with a detectable label, such as a fluorophore or an enzyme
that produces a detectable product. Alternatively, the method can
employ an antibody (or other binding agent) specific for the test
ligand and including a detectable label. One or more of the spots
that are labeled by the test ligand or that are more or most
intensely labeled with the test ligand are selected as lead
artificial receptors. The degree of labeling can be evaluated by
evaluating the signal strength from the label. The amount of signal
can be directly proportional to the amount of label and binding.
FIG. 17 provides a schematic illustration of an embodiment of this
process.
[0376] According to the present method, screening candidate
artificial receptors against a test ligand can yield one or more
lead artificial receptors. One or more lead artificial receptors
can be a working artificial receptor. That is, the one or more lead
artificial receptors can be useful for detecting the ligand of
interest as is. The method can then employ the one or more
artificial receptors as a working artificial receptor for
monitoring or detecting the test ligand. Alternatively, the one or
more lead artificial receptors can be employed in the method for
developing a working artificial receptor. For example, the one or
more lead artificial receptors can provide structural or other
information useful for designing or screening for an improved lead
artificial receptor or a working artificial receptor. Such
designing or screening can include making and testing additional
candidate artificial receptors including combinations of a subset
of building blocks, a different set of building blocks, or a
different number of building blocks.
[0377] The present invention includes a method of screening
candidate artificial receptors to find lead artificial receptors
that bind a particular test ligand. The method can include allowing
movement of the building blocks that make up the artificial
receptors. Movement of building blocks can include mobilizing the
building block to move along or on the support and/or to leave the
support and enter a fluid (e.g., liquid) phase separate from the
support or lawn.
[0378] In an embodiment, building blocks can be mobilized to move
along or on the support (translate or shuffle). Such translation
can be employed, for example, to allow building blocks already
bound to a test ligand to rearrange into a lower energy or tighter
binding configuration still bound to the test ligand. Such
translation can be employed, for example, to allow the ligand
access to building blocks that are on the support but not bound to
the ligand. These building blocks can translate into proximity with
and bind to a test ligand.
[0379] Building blocks can be induced to move along or on the
support or to be reversibly immobilized on the support through any
of a variety of mechanisms. For example, inducing mobility of
building blocks can include altering the conditions of the support
or lawn. That is, altering the conditions can reverse the
immobilization of the building blocks, thus mobilizing them.
Reversibly immobilizing the building blocks after they have moved
can include, for example, returning to the previous conditions.
Suitable alterations of conditions include changing pH, changing
temperature, changing polarity or hydrophobicity, changing ionic
strength, changing nucleophilicity or electrophilicity (e.g. of
solvent or solute), and the like.
[0380] A building block reversibly immobilized by hydrophobic
interactions can be mobilized by increasing the temperature, by
exposing the surface, lawn, or building block to a more hydrophobic
solvent (e.g., an organic solvent or a surfactant), or by reducing
ionic strength around the building block. In an embodiment, the
organic solvent includes acetonitrile, acetic acid, an alcohol,
tetrahydrofuran (THF), dimethylformamide (DMF), hydrocarbons such
as hexane or octane, acetone, chloroform, methylene chloride, or
the like, or mixture thereof. In an embodiment, the surfactant
includes a nonionic surfactant, such as a nonylphenol ethoxylate,
or the like. A building block that is mobile on a support can be
reversibly immobilized by hydrophobic interactions, for example, by
decreasing the temperature, exposing the surface, lawn, or building
block to a more hydrophilic solvent (e.g., an aqueous solvent) or
increased ionic strength.
[0381] A building block reversibly immobilized by hydrogen bonding
can be mobilized by increasing the ionic strength, concentration of
hydrophilic solvent, or concentration of a competing hydrogen
bonder in the environs of the building block. A building block that
is mobile on a support can be reversibly immobilized through an
electrostatic interaction by decreasing ionic strength of the
hydrophilic solvent, or the like.
[0382] A building block reversibly immobilized by an electrostatic
interaction can be mobilized by increasing the ionic strength in
the environs of the building block. Increasing ionic strength can
disrupt electrostatic interactions. A building block that is mobile
on a support can be reversibly immobilized through an electrostatic
interaction by decreasing ionic strength.
[0383] A building block reversibly immobilized by an imine, acetal,
or ketal bond can be mobilized by decreasing the pH or increasing
concentration of a nucleophilic catalyst in the environs of the
building block. In an embodiment, the pH is about 1 to about 4.
Imines, acetals, and ketals undergo acid catalyzed hydrolysis. A
building block that is mobile on a support can be reversibly
immobilized by a reversible covalent interaction, such as by
forming an imine, acetal, or ketal bond, by increasing the pH.
[0384] In an embodiment, building blocks can be mobilized to leave
the support and enter a fluid (e.g., liquid) phase separate from
the support or lawn (exchange). For example, building blocks can be
exchanged onto and/or off of the support. Exchange can be employed,
for example, to allow building blocks on a support but not bound to
a test ligand to be removed from the support. Exchange can be
employed, for example, to add additional building blocks to the
support. The added building blocks can have structures selected
based on knowledge of the structures of the building blocks in
artificial receptors that bind the test ligand. The added building
blocks can have structures selected to provide additional
structural diversity. The added building blocks can include all of
the building blocks.
[0385] A building block reversibly immobilized by hydrophobic
interactions can be released from the support by, for example,
raising the temperature, e.g., of the support and/or artificial
receptor. For example, the hydrophobic interactions (e.g., the
hydrophobic group on the support or lawn and on the building block)
can be selected to provide immobilized building block at about room
temperature or below and release can be accomplished at a
temperature above room temperature. For example, the hydrophobic
interactions can be selected to provide immobilized building block
at about refrigerator temperature (e.g., 4.degree. C.) or below and
release can be accomplished at a temperature of, for example, room
temperature or above. By way of further example, a building block
can be reversibly immobilized by hydrophobic interactions, for
example, by contacting the surface or artificial receptor with a
fluid containing the building block and that is at or below room
temperature.
[0386] A building block reversibly immobilized by hydrophobic
interactions can be released from the support by, for example,
contacting the artificial receptor with a sufficiently hydrophobic
fluid (e.g., an organic solvent or a surfactant). In an embodiment,
the organic solvent includes acetonitrile, acetic acid, an alcohol,
tetrahydrofuran (THF), dimethylformamide (DMF), hydrocarbons such
as hexane or octane, acetone, chloroform, methylene chloride, or
the like, or mixture thereof. In an embodiment, the surfactant
includes a nonionic surfactant, such as a nonylphenol ethoxylate,
or the like. Such reversible immobilization can also be effected by
contacting the surface or artificial receptor with a hydrophilic
solvent and allowing the somewhat lipophilic building block to
partition on to the hydrophobic surface or lawn.
[0387] A building block reversibly immobilized by an imine, acetal,
or ketal bond can be released from the support by, for example,
contacting the artificial receptor with fluid having an acid pH or
including a nucleophilic catalyst. In an embodiment, the pH is
about 1 to about 4. A building block can be reversibly immobilized
by a reversible covalent interaction, such as by forming an imine,
acetal, or ketal bond, by contacting the surface or artificial
receptor with fluid having a neutral or basic pH.
[0388] A building block reversibly immobilized by an electrostatic
interaction can be released by, for example, contacting the
artificial receptor with fluid having sufficiently high ionic
strength to disrupt the electrostatic interaction. A building block
can be reversibly immobilized through an electrostatic interaction
by contacting the surface or artificial receptor with fluid having
ionic strength that promotes electrostatic interaction between the
building block and the support and/or lawn.
Test Ligands
[0389] The test ligand can be any ligand for which binding to an
array or surface can be detected. The test ligand can be a pure
compound, a mixture, or a "dirty" mixture containing a natural
product or pollutant. Such dirty mixtures can be tissue homogenate,
biological fluid, soil sample, water sample, or the like.
[0390] Test ligands include prostate specific antigen, other cancer
markers, insulin, warfarin, other anti-coagulants, cocaine, other
drugs-of-abuse, markers for E. coli, markers for Salmonella sp.,
markers for other food-borne toxins, food-borne toxins, markers for
Smallpox virus, markers for anthrax, markers for other possible
toxic biological agents, pharmaceuticals and medicines, pollutants
and chemicals in hazardous waste, toxic chemical agents, markers of
disease, pharmaceuticals, pollutants, biologically important
cations (e.g., potassium or calcium ion), peptides, carbohydrates,
enzymes, bacteria, viruses, mixtures thereof, and the like. In
certain embodiments, the test ligand can be at least one of small
organic molecules, inorganic/organic complexes, metal ion, mixture
of proteins, protein, nucleic acid, mixture of nucleic acids,
mixtures thereof, and the like.
[0391] Suitable test ligands include any compound or category of
compounds described elsewhere in this document as being a test
ligand, including, for example, the microbes, proteins, cancer
cells, drugs of abuse, and the like described above.
[0392] The present invention may be better understood with
reference to the following examples. These examples are intended to
be representative of specific embodiments of the invention, and are
not intended as limiting the scope of the invention.
EXAMPLES
Example 1
Synthesis of Building Blocks
[0393] Selected building blocks representative of the
alkyl-aromatic-polar span of the an embodiment of the building
blocks were synthesized and demonstrated effectiveness of these
building blocks for making candidate artificial receptors. These
building blocks were made on a framework that can be represented by
tyrosine and included numerous recognition element pairs. These
recognition element pairs include enough of the range from alkyl,
to aromatic, to polar to represent a significant degree of the
interactions and functional groups of the full set of 81 such
building blocks.
Synthesis
[0394] Building block synthesis employed a general procedure
outlined in Scheme 7, which specifically illustrates synthesis of a
building block on a tyrosine framework with recognition element
pair A4B4. This general procedure was employed for synthesis of
building blocks including TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6,
TyrA2B8, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA6B2, TyrA6B4,
TyrA6B6, TyrA6B8, TyrA8B2, TyrA8B4, TyrA8B6, TyrA8B8, and TyrA9B9,
respectively.
##STR00006##
Results
[0395] Synthesis of the desired building blocks proved to be
generally straightforward. These syntheses illustrate the relative
simplicity of preparing the building blocks with 2 recognition
elements having different structural characteristics or structures
(e.g. A4B2, A6B3, etc.) once the building blocks with corresponding
recognition elements (e.g. A2B2, A4B4, etc) have been prepared via
their X BOC intermediate.
[0396] The conversion of one of these building blocks to a building
block with a lipophilic linker can be accomplished by reacting the
activated building block with, for example, dodecyl amine.
Example 2
Preparation and Evaluation of Microarrays of Candidate Artificial
Receptors
[0397] Microarrays of candidate artificial receptors were made and
evaluated for binding several protein ligands. The results obtained
demonstrate the 1) the simplicity with which microarrays of
candidate artificial receptors can be prepared, 2) binding affinity
and binding pattern reproducibility, 3) significantly improved
binding for building block heterogeneous receptor environments when
compared to the respective homogeneous controls, and 4) ligand
distinctive binding patterns (e.g., working receptor
complexes).
Materials and Methods
[0398] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4,
TyrA4B6, TyrA6B2, TyrA6B4, and TyrA6B6. The abbreviation for the
building block including a linker, a tyrosine framework, and
recognition elements AxBy is TyrAxBy.
[0399] Microarrays for the evaluation of the 130 n=2 and n=3, and
for evaluation of the 273 n=2, n=3, and n=4, candidate receptor
environments were prepared as follows by modifications of known
methods. As used herein, "n" is the number of different building
blocks employed in a receptor environment. Briefly: Amine modified
(amine "lawn"; SuperAmine Microarray plates) microarray plates were
purchased from Telechem Inc., Sunnyvale, Calif. (www.arrayit.com).
These plates were manufactured specifically for microarray
preparation and had a nominal amine load of 2-4 amines per square
nm according to the manufacturer. The CAM microarrays were prepared
using a pin microarray spotter instrument from Telechem Inc.
(SpotBot.TM. Arrayer) typically with 200 um diameter spotting pins
from Telechem Inc. (Stealth Micro Spotting Pins, SMP6) and 400-420
um spot spacing.
[0400] The 9 building blocks were activated in aqueous
dimethylformamide (DMF) solution as described above. For preparing
the 384-well feed plate, the activated building block solutions
were diluted 10-fold with a solution of DMF/H.sub.2O/PEG400
(90/10/10, v/v/v; PEG400 is polyethylene glycol nominal 400 FW,
Aldrich Chemical Co., Milwaukee, Wis.). These stock solutions were
aliquotted (10 .mu.l per aliquot) into the wells of a 384-well
microwell plate (Telechem Inc.). A separate series of controls were
prepared by aliquotting 10 .mu.l of building block with either 10
.mu.l or 20 .mu.l of the activated [1-1] solution. The plate was
covered with aluminum foil and placed on the bed of a rotary shaker
for 15 minutes at 1,000 RPM. This master plate was stored covered
with aluminum foil at -20.degree. C. when not in use.
[0401] For preparing the 384-well SpotBot.TM. plate, a well-to-well
transfer (e.g. A-1 to A-1, A-2 to A-2, etc.) from the feed plate to
a second 384-well plate was performed using a 4 .mu.l transfer
pipette. This plate was stored tightly covered with aluminum foil
at -20.degree. C. when not in use. The SpotBot.TM. was used to
prepare up to 13 microarray plates per run using the 4 .mu.l
microwell plate. The SpotBot.TM. was programmed to spot from each
microwell in quadruplicate. The wash station on the SpotBot.TM.
used a wash solution of EtOH/H.sub.2O (20/80, v/v). This wash
solution was also used to rinse the microarrays on completion of
the SpotBot.TM. printing run. The plates were given a final rinse
with deionized (DI) water, dried using a stream of compressed air,
and stored at room temperature.
[0402] Certain of the microarrays were further modified by reacting
the remaining amines with succinic anhydride to form a carboxylate
lawn in place of the amine lawn.
[0403] The following test ligands and labels were used in these
experiments:
[0404] 1) r-Phycoerythrin, a commercially available and
intrinsically fluorescent protein with a FW of 2,000,000.
[0405] 2) Ovalbumin labeled with the Alexa.TM. fluorophore
(Molecular Probes Inc., Eugene, Oreg.).
[0406] 3) BSA, bovine serum albumin, labeled with activated
Rhodamine (Pierce Chemical, Rockford, Ill.) using the known
activated carboxyl protocol. BSA has a FW of 68,000; the material
used for this study had ca. 1.0 rhodamine per BSA.
[0407] 4) Horseradish peroxidase (HRP) modified with extra amines
and labeled as the acetamide derivative or with a
2,3,7,8-tetrachlorodibenzodixoin derivative were available through
known methods. Fluorescence detection of these BRP conjugates was
based on the Alexa 647-tyramide kit available from Molecular
Probes, Eugene, Oreg.
[0408] 5) Cholera toxin labeled with the Alexa.TM. fluorophore
(Molecular Probes Inc., Eugene, Oreg.).
[0409] Microarray incubation and analysis was conducted as follows:
For test ligand incubation with the microarrays, solutions (e.g.
500 .mu.l) of the target proteins in PBS-T (PBS with 20 .mu.l/L of
Tween-20) at typical concentrations of 10, 1.0 and 0.1 .mu.g/ml
were placed onto the surface of a microarray and allowed to react
for, e.g., 30 minutes. The microarray was rinsed with PBS-T and DI
water and dried using a stream of compressed air.
[0410] The incubated microarray was scanned using an Axon Model
4200A Fluorescence Microarray Scanner (Axon Instruments, Union
City, Calif.). The Axon scanner and its associated software produce
a false color 16-bit image of the fluorescence intensity of the
plate. This 16-bit data is integrated using the Axon software to
give a Fluorescence Units value (range 0-65,536) for each spot on
the microarray. This data is then exported into an Excel file
(Microsoft) for further analysis including mean, standard deviation
and coefficient of variation calculations.
Results
[0411] The CARA.TM.: Combinatorial Artificial Receptor Array.TM.
concept has been demonstrated using a microarray format. A CARA
microarray based on N=9 building blocks was prepared and evaluated
for binding to several protein and substituted protein ligands.
This microarray included 144 candidate receptors (18 n=1 controls
plus 6 blanks; 36 n=2 candidate receptors; 84 n=3 candidate
receptors). This microarray demonstrated: 1) the simplicity of CARA
microarray preparation, 2) binding affinity and binding pattern
reproducibility, 3) significantly improved binding for building
block heterogeneous receptor environments when compared to the
respective homogeneous controls, and 4) ligand distinctive binding
patterns.
Reading the Arrays
[0412] A typical false color/gray scale image of a microarray that
was incubated with 2.0 .mu.g/ml r-phycoerythrin is shown in FIG.
18. This image illustrates that the processes of both preparing the
microarray and probing it with a protein test ligand produced the
expected range of binding as seen in the visual range of relative
fluorescence from dark to bright spots.
[0413] The starting point in analysis of the data was to take the
integrated fluorescence units data for the array of spots and
normalize to the observed value for the [1-1] building block
control. Subsequent analysis included mean, standard deviation and
coefficient of variation calculations. Additionally, control values
for homogeneous building blocks were obtained from the building
block plus [1-1] data.
First Set of Experiments
[0414] The following protein ligands were evaluated for binding to
the candidate artificial receptors in the microarray. The resulting
Fluorescence Units versus candidate receptor environment data is
presented in both a 2D format where the candidate receptors are
placed along the X-axis and the Fluorescence Units are shown on the
Y-axis and a 3D format where the Candidate Receptors are placed in
an X-Y format and the Fluorescence Units are shown on the Z-axis. A
key for the composition of each spot was developed (not shown). A
key for the building blocks in each of the 2D and 3D
representations of the results was also developed (not shown). The
data presented are for 1-2 .mu.g/ml protein concentrations.
[0415] FIGS. 19 and 20 illustrate binding data for r-phycoerythrin
(intrinsic fluorescence). FIGS. 21 and 22 illustrate binding data
for ovalbumin (commercially available with fluorescence label).
FIGS. 23 and 24 illustrate binding data for bovine serum albumin
(labeled with rhodamine). FIGS. 25 and 26 illustrate binding data
for HRP-NH-Ac (fluorescent tyramide read-out). FIGS. 27 and 28
illustrate binding data for HRP-NH-TCDD (fluorescent tyramide
read-out).
[0416] These results demonstrate not only the application of the
CARA microarray to candidate artificial receptor evaluation but
also a few of the many read-out methods (e.g. intrinsic
fluorescence, fluorescently labeled, in situ fluorescence labeling)
which can be utilized for high throughput candidate receptor
evaluation.
[0417] The evaluation of candidate receptors benefits from
reproducibility. The following results demonstrate that the present
microarrays provided reproducible ligand binding.
[0418] The microarrays were printed with each combination of
building blocks spotted in quadruplicate. Visual inspection of a
direct plot (FIG. 29) of the raw fluorescence data (from the run
illustrated in FIG. 18) for one block of binding data obtained for
r-phycoerythrin demonstrates that the candidate receptor
environment "spots" showed reproducible binding to the test ligand.
Further analysis of the r-phycoerythrin data (FIG. 18) led to only
9 out of 768 spots (1.2%) being deleted as outliers. Analysis of
the r-phycoerythrin quadruplicate data for the entire array gives a
mean standard deviation for each experimental quadruplicate set of
938 fluorescence units, with a mean coefficient of variation of
19.8%.
[0419] Although these values are acceptable, a more realistic
comparison employed the standard deviation and coefficient of
variation of the more strongly bound, more fluorescent receptors.
The overall mean standard deviation unrealistically inflates the
coefficient of variation for the weakly bound, less fluorescent
receptors. The coefficient of variation for the 19 receptors with
greater than 10,000 Fluorescent Units of bound target is 11.1%,
which is well within the range required to produce meaningful
binding data.
[0420] One goal of the CARA approach is the facile preparation of a
significant number of candidate receptors through combinations of
structurally simple building blocks. The following results
establish that both the individual building blocks and combinations
of building blocks have a significant, positive effect on test
ligand binding.
[0421] The binding data illustrated in FIGS. 27-28 demonstrate that
heterogeneous combinations of building blocks (n=2, n=3) are
dramatically superior candidate receptors made from a single
building block (n=1). For example, FIG. 20 illustrates both the
diversity of binding observed for n=2, n=3 candidate receptors with
fluorescent units ranging from 0 to ca. 40,000. These data also
illustrate and the ca. 10-fold improvement in binding affinity
obtained upon going from the homogeneous (n=1) to heterogeneous
(n=2, n=3) receptor environments.
[0422] The effect of heterogeneous building blocks is most easily
observed by comparing selected n=3 receptor environments candidate
receptors including 1 or 2 of those building blocks (their n=2 and
n=1 subsets). FIGS. 30 and 31 illustrate this comparison for two
different n=3 receptor environments using the r-phycoerythrin data.
In these examples, it is clear that progression from the
homogeneous system (n=1) to the heterogeneous systems (n=2, n=3)
produces significantly enhanced binding.
[0423] Although van der Waals interactions are an important part of
molecular recognition, it is important to establish that the
observed binding is not a simple case of hydrophobic/hydrophilic
partitioning. That is, that the observed binding was the result of
specific interactions between the individual building blocks and
the target The simplest way to evaluate the effects of
hydrophobicity and hydrophilicity is to compare building block logP
value with observed binding. LogP is a known and accepted measure
of lipophilicity, which can be measured or calculated by known
methods for each of the building blocks. FIGS. 32 and 33 establish
that the observed target binding, as measured by fluorescence
units, is not directly proportional to building block logP. The
plots in FIGS. 32 and 33 illustrate a non-linear relationship
between binding (fluorescence units) and building block logP.
[0424] One advantage of the present methods and arrays is that the
ability to screen large numbers of candidate receptor environments
will lead to a combination of useful target affinities and to
significant target binding diversity. High target affinity is
useful for specific target binding, isolation, etc. while binding
diversity can provide multiplexed target detection systems. This
example employed a relatively small number of building blocks to
produce ca. 120 binding environments. The following analysis of the
present data clearly demonstrates that even a relatively small
number of binding environments can produce diverse and useful
artificial receptors.
[0425] The target binding experiments performed for this study used
protein concentrations including 0.1 to 10 .mu.g/ml. Considering
the BSA data as representative, it is clear that some of the
receptor environments readily bound 1.0 ug/ml BSA concentrations
near the saturation values for fluorescence units (see, e.g., FIG.
24). Based on these data and the formula weight of 68,000 for BSA,
several of the receptor environments readily bind BSA at ca. 15
picomole/ml or 15 nanomolar concentrations. Additional experiments
using lower concentrations of protein (data not shown) indicate
that, even with a small selection of candidate receptor
environments, femptomole/ml or picomolar detection limits have been
attained.
[0426] One goal of artificial receptor development is the specific
recognition of a particular target. FIG. 34 compares the observed
binding for r-phycoerythrin and BSA. Comparison of the overall
binding pattern indicates some general similarities. However,
comparison of specific features of binding for each receptor
environment demonstrates that the two targets have distinctive
recognition features as indicated by the (*) in FIG. 34.
[0427] One goal of artificial receptor development is to develop
receptors which can be used for the multiplexed detection of
specific targets. Comparison of the r-phycoerythrin, BSA and
ovalbumin data from this study (FIGS. 20, 22, and 24) were used to
select representative artificial receptors for each target. FIGS.
35, 36, and 37 employ data obtained in the present example to
illustrate identification of each of these three targets by their
distinctive binding patterns.
Conclusions
[0428] The optimum receptor for a particular target requires
molecular recognition which is greater than the expected sum of the
individual hydrophilic, hydrophobic, ionic, etc. interactions.
Thus, the identification of an optimum (specific, sensitive)
artificial receptor from the limited pool of candidate receptors
explored in this prototype study, was not expected and not likely.
Rather, the goal was to demonstrate that all of the key components
of the CARA: Combinatorial Artificial Receptor Array concept could
be assembled to form a functional receptor microarray. This goal
has been successfully demonstrated.
[0429] This study has conclusively established that CARA
microarrays can be readily prepared and that target binding to the
candidate receptor environments can be used to identify artificial
receptors and test ligands. In addition, these results demonstrate
that there is significant binding enhancement for the building
block heterogeneous (n=2, n=3, or n=4) candidate receptors when
compared to their homogeneous (n=1) counterparts. When combined
with the binding pattern recognition results and the demonstrated
importance of both the heterogeneous receptor elements and
heterogeneous building blocks, these results clearly demonstrate
the significance of the CARA Candidate Artificial Receptor->Lead
Artificial Receptor->Working Artificial Receptor strategy.
Example 3
Preparation and Evaluation of Microarrays of Candidate Artificial
Receptors Including Reversibly Immobilized Building Blocks
[0430] Microarrays of candidate artificial receptors including
building blocks immobilized through van der Waals interactions were
made and evaluated for binding of a protein ligand. The evaluation
was conducted at several temperatures, above and below a phase
transition temperature for the lawn (vide infra).
Materials and Methods
[0431] Building blocks 2-2, 2-4, 2-6, 4-2, 4-4, 4-6, 6-2, 6-4, 6-6
where prepared as described in Example 1. The C12 amide was
prepared using the previously described carbodiimide activation of
the carboxyl followed by addition of dodecylamine. This produced a
building block with a 12 carbon alkyl chain linker for reversible
immobilization in the C18 lawn.
[0432] Amino lawn microarray plates (Telechem) were modified to
produce the C18 lawn by reaction of stearoyl chloride (Aldrich
Chemical Co.) in A) dimethylformamide/PEG 400 solution (90:10, v/v,
PEG 400 is polyethylene glycol average MW 400 (Aldrich Chemical
Co.) or B) methylene chloride/TEA solution (100 ml methylene
chloride, 200 .mu.l triethylamine) using the lawn modification
procedures generally described in Example 2.
[0433] The C18 lawn plates where printed using the SpotBot standard
procedure as described in Example 2. The building blocks were in
printing solutions prepared by solution of ca. 10 mg of each
building block in 300 .mu.l of methylene chloride and 100 .mu.l
methanol. To this stock was added 900 .mu.l of dimethylformamide
and 100 .mu.l of PEG 400. The 36 combinations of the 9 building
blocks taken two at a time (N9:n2, 36 combinations) where prepared
in a 384-well microwell plate which was then used in the SpotBot to
print the microarray in quadruplicate. A random selection of the
print positions contained only print solution.
[0434] The selected microarray was incubated with a 1.0 .mu.g/ml
solution of the test ligand, cholera toxin subunit B labeled with
the Alexa.TM. fluorophore (Molecular Probes Inc., Eugene, Oreg.),
using the following variables: 1) the microarray was washed with
methylene chloride, ethanol and water to create a control plate;
and 2) the microarray was incubated at 4.degree. C., 23.degree. C.,
or 44.degree. C. After incubation, the plate(s) were rinsed with
water, dried and scanned (AXON 4100A). Data analysis was as
described in Example 2.
Results
[0435] A control array from which the building blocks had been
removed by washing with organic solvent did not bind cholera toxin
(FIG. 38). FIGS. 39-41 illustrate fluorescence signals from arrays
printed identically, but incubated with cholera toxin at 4.degree.
C., 23.degree. C., or 44.degree. C., respectively. Spots of
fluorescence can be seen in each array, with very pronounced spots
produced by incubation at 44.degree. C. The fluorescence values for
the spots in each of these three arrays are shown in FIGS. 42-44.
Fluorescence signal generally increases with temperature, with many
nearly equally large signals observed after incubation at
44.degree. C. Linear increases with temperature can reflect
expected improvements in binding with temperature. Nonlinear
increases reflect rearrangement of the building blocks on the
surface to achieve improved binding, which occurred above the phase
transition for the lipid surface (vide infra).
[0436] FIG. 45 can be compared to FIG. 51. The fluorescence signals
plotted in FIG. 51 resulted from binding to reversibly immobilized
building blocks on a support at 23.degree. C. The fluorescence
signals plotted in FIG. 45 resulted from binding to covalently
immobilized building blocks on a support at 23.degree. C. These
figures compare the same combinations of building blocks in the
same relative positions, but immobilized in two different ways.
[0437] The binding to covalently immobilized building blocks was
also evaluated at 4.degree. C., 23.degree. C., or 44.degree. C.
FIG. 46 illustrates the changes in fluorescence signal from
individual combinations of covalently immobilized building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C. Binding increased
modestly with temperature. The mean increase in binding was
1.3-fold. A plot of the fluorescence signal for each of the
covalently immobilized artificial receptors at 23.degree. C.
against its signal at 44.degree. C. (not shown) yields a linear
correlation with a correlation coefficient of 0.75. This linear
correlation indicates that the mean 1.3-fold increase in binding is
a thermodynamic effect and not optimization of binding.
[0438] FIG. 47 illustrates the changes in fluorescence signal from
individual combinations of reversibly immobilized building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C. This graph
illustrates that at least one combination of building blocks
(candidate artificial receptor) exhibited a signal that remained
constant as temperature increased. At least one candidate
artificial receptor exhibited an approximately linear increase in
signal as temperature increased. Such a linear increase indicates
normal temperature effects on binding. The candidate artificial
receptor with the lowest binding signal at 4.degree. C. became one
of the best binders at 44.degree. C. This indicates that
rearrangement of the building blocks of this receptor above the
phase transition for the lawn, which increases the building blocks'
mobility, produced increased binding. Other receptors characterized
by greater changes in binding between 23.degree. C. and 44.degree.
C. (compared to between 4.degree. C. and 23.degree. C.) also
underwent dynamic affinity optimization.
[0439] FIG. 48 illustrates the data presented in FIG. 46 (lines
marked A) and the data presented in FIG. 47 (lines marked B). The
increases in binding observed with the reversibly immobilized
building blocks are significantly greater than the increases
observed with covalently bound building blocks. Binding to
reversibly immobilized building blocks increased from 23.degree. C.
and 44.degree. C. by a median value of 6.1-fold and a mean value of
24-fold. This confirms that movement of the reversibly immobilized
building blocks within the receptors increased binding (i.e., the
receptor underwent dynamic affinity optimization).
[0440] A plot of the fluorescence signal for each of the reversibly
immobilized artificial receptors at 23.degree. C. against its
signal at 44.degree. C. (not shown) yields no correlation
(correlation coefficient of 0.004). A plot of the fluorescence
signal for each of the reversibly immobilized artificial receptors
at 44.degree. C. against the signal for the corresponding
covalently immobilized receptor (not shown) also yields no
correlation (correlation coefficient 0.004). This lack of
correlation provides further evidence that movement of the
reversibly immobilized building blocks within the receptors
increased binding.
[0441] FIG. 49 illustrates a graph of the fluorescence signal at
44.degree. C. divided by the signal at 23.degree. C. against the
fluorescence signal obtained from binding at 23.degree. C. for the
artificial receptors with reversibly immobilized receptors. This
comparison indicates that the binding enhancement is independent of
the initial affinity of the receptor for the test ligand.
[0442] Table 1 identifies the reversibly immobilized building
blocks making up each of the artificial receptors, lists the
fluorescence signal (binding strength) at 44.degree. C. and
23.degree. C., and the ratios of the observed binding at these two
temperatures. These data illustrate that each artificial receptor
reflects a unique attribute for each combination of building blocks
relative to the role of each individual building block.
TABLE-US-00001 TABLE 1 Building Blocks Ratio Making Up of Signals,
Receptor Signal at 44.degree. C. Signal at 23.degree. C. 44.degree.
C./23.degree. C. 22 24 24136 4611 5.23 22 26 16660 43 387.44 22 42
17287 -167 -103.51 22 44 16726 275 60.82 22 46 25016 3903 6.41 22
62 13990 3068 4.56 22 64 15294 3062 4.99 22 66 11980 3627 3.30 24
26 22688 1291 17.57 24 42 26808 -662 -40.50 24 44 23154 904 25.61
24 46 42197 2814 15.00 24 62 19374 2567 7.55 24 64 27599 262 105.34
24 66 16238 5334 3.04 26 42 22282 4974 4.48 26 44 26240 530 49.51
26 46 23144 4273 5.42 26 62 29022 4920 5.90 26 64 23416 5551 4.22
26 66 19553 5353 3.65 42 44 29093 6555 4.44 42 46 18637 3039 6.13
42 62 22643 4853 4.67 42 64 20836 6343 3.28 42 66 14391 9220 1.56
44 46 25600 3266 7.84 44 62 15544 4771 3.26 44 64 25842 3073 8.41
44 66 22471 5142 4.37 46 62 32764 8522 3.84 46 64 21901 3343 6.55
46 66 23516 3742 6.28 62 64 24069 7149 3.37 62 66 15831 2424 6.53
64 66 21310 2746 7.76
Conclusions
[0443] This experiment demonstrated that an array including
reversibly immobilized building blocks binds a protein substrate,
like an array with covalently immobilized building blocks. The
binding increased nonlinearly as temperature increased, indicating
that movement of the building blocks increased binding. Many of the
candidate artificial receptors demonstrated improved binding upon
mobilization of the building blocks.
Example 4
The Oligosaccharide Portion of GM1 Competes with Artificial
Receptors for Binding to Cholera Toxin
[0444] Microarrays of candidate artificial receptors were made and
evaluated for binding of cholera toxin. The arrays were also
evaluated for disrupting that binding. Disrupting of binding
employed a compound that binds to cholera toxin, the
oligosaccharide moiety from GM1 (GM1 OS). The results obtained
demonstrate that a ligand of a protein specifically disrupted
binding of the protein to the microarray.
Materials and Methods
[0445] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3,
TyrA3B5, TyrA3B7, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA5B3,
TyrA5B5, TyrA5B7, TyrA6B2, TyrA6B4, TyrA6B6, TyrA6B8, TyrA7B3,
TyrA7B5, TyrA7B7, TyrA8B2, TyrA8B4, TyrA8B6, and TyrA8B8. The
abbreviation for the building block including a linker, a tyrosine
framework, and recognition elements AxBy is TyrAxBy.
[0446] Microarrays for the evaluation of the 171 n=2 candidate
receptor environments were prepared as follows by modifications of
known methods. An "n=2" receptor environment includes two different
building blocks. Briefly: Amine modified (amine "lawn"; SuperAmine
Microarray plates) microarray plates were purchased from Telechem
Inc., Sunnyvale, Calif. These plates were manufactured specifically
for microarray preparation and had a nominal amine load of 2-4
amines per square nm according to the manufacturer. The microarrays
were prepared using a pin microarray spotter instrument from
Telechem Inc. (SpotBot.TM. Arrayer) typically with 200 .mu.m
diameter spotting pins from Telechem Inc. (Stealth Micro Spotting
Pins, SMP6) and 400-420 .mu.m spot spacing.
[0447] The 19 building blocks were activated in aqueous
dimethylformamide (DMF) solution as described above. For preparing
the 384-well feed plate, the activated building block solutions
were diluted 10-fold with a solution of DMF/H.sub.2O/PEG400
(90/10/10, v/v/v; PEG400 is polyethylene glycol nominal 400 FW,
Aldrich Chemical Co., Milwaukee, Wis.). These stock solutions were
aliquotted (10 .mu.l per aliquot) into the wells of a 384-well
microwell plate (Telechem Inc.). Control spots included the
building block [1-1]. The plate was covered with aluminum foil and
placed on the bed of a rotary shaker for 15 minutes at 1,000 RPM.
This master plate was stored covered with aluminum foil at
-20.degree. C. when not in use.
[0448] For preparing the 384-well SpotBot.TM. plate, a well-to-well
transfer (e.g. A-1 to A-1, A-2 to A-2, etc.) from the feed plate to
a second 384-well plate was performed using a 4 .mu.l transfer
pipette. This plate was stored tightly covered with aluminum foil
at -20.degree. C. when not in use. The SpotBot.TM. was used to
prepare up to 13 microarray plates per run using the 4 .mu.l
microwell plate. The SpotBot.TM. was programmed to spot from each
microwell in quadruplicate. The wash station on the SpotBot.TM.
used a wash solution of EtOH/H.sub.2O (20/80, v/v). This wash
solution was adjusted to pH 4 with 1 M HCl and used to rinse the
microarrays on completion of the SpotBot.TM. printing run. The
plates were given a final rinse with deionized (DI) water, dried
using a stream of compressed air, and stored at room temperature.
The microarrays were further modified by reacting the remaining
amines with acetic anhydride to form an acetamide lawn in place of
the amine lawn.
[0449] The test ligand employed in these experiments was cholera
toxin labeled with the Alexa.TM. fluorophore (Molecular Probes
Inc., Eugene, Oreg.). The candidate disruptor employed in these
experiments was GM1 OS (GM1 oligosaccharide), a known ligand for
cholera toxin.
[0450] Microarray incubation and analysis was conducted as follows:
For control incubations with the microarrays, solutions (e.g. 500
.mu.l) of the cholera toxin in PBS-T (PBS with 20 .mu.l/L of
Tween-20) at a concentrations of 1.7 pmol/ml (0.1 .mu.g/ml) was
placed onto the surface of a microarray and allowed to react for 30
minutes. For disruptor incubations with the microarrays, solutions
(e.g. 500 .mu.l) of the cholera toxin (1.7 pmol/ml, 0.1 .mu.g/ml)
and the desired concentration of GM1 OS in PBS-T (PBS with 20
.mu.l/L of Tween-20) was placed onto the surface of a microarray
and allowed to react for 30 minutes. GM1 OS was added at 0.34 and
at 5.1 .mu.M in separate experiments. After either of these
incubations, the microarray was rinsed with PBS-T and DI water and
dried using a stream of compressed air.
[0451] The incubated microarray was scanned using an Axon Model
4200A Fluorescence Microarray Scanner (Axon Instruments, Union
City, Calif.). The Axon scanner and its associated software produce
a false color 16-bit image of the fluorescence intensity of the
plate. This 16-bit data is integrated using the Axon software to
give a Fluorescence Units value (range 0-65,536) for each spot on
the microarray. This data is then exported into an Excel file
(Microsoft) for further analysis including mean, standard deviation
and coefficient of variation calculations.
[0452] Table 2 identifies the building blocks in each of the first
150 receptor environments.
TABLE-US-00002 TABLE 2 Building Blocks 1 22 24 2 22 28 3 22 42 4 22
46 5 22 55 6 22 64 7 22 68 8 22 82 9 22 86 10 24 26 11 24 33 12 24
44 13 26 77 14 26 84 15 26 88 16 28 42 17 22 26 18 22 33 19 22 44
20 22 48 21 22 62 22 22 66 23 22 77 24 22 84 25 22 88 26 24 28 27
24 42 28 26 82 29 26 85 30 28 33 31 28 44 32 28 46 33 28 55 34 28
64 35 28 68 36 28 82 37 28 86 38 33 42 39 33 46 40 42 88 41 44 48
42 44 62 43 44 66 44 44 77 45 44 84 46 44 88 47 46 55 48 28 48 49
28 62 50 28 66 51 28 77 52 28 84 53 28 88 54 33 44 55 44 46 56 44
55 57 44 64 58 44 68 59 44 82 60 44 86 61 46 48 62 46 62 63 24 46
64 24 55 65 24 64 66 24 68 67 24 82 68 24 86 69 26 28 70 26 42 71
26 46 72 26 55 73 26 64 74 26 68 75 33 48 76 33 63 77 33 66 78 33
77 79 24 48 80 24 62 81 24 66 82 24 77 83 24 84 84 24 88 85 26 33
86 26 44 87 26 48 88 26 62 89 26 66 90 33 55 91 33 64 92 33 68 93
33 82 94 33 84 95 33 88 96 42 46 97 42 55 98 42 64 99 42 68 100 42
82 101 42 86 102 46 88 103 48 62 104 48 66 105 46 77 106 48 84 107
48 88 108 55 64 109 55 68 110 33 86 111 42 44 112 42 48 113 42 62
114 42 66 115 42 77 116 42 84 117 48 55 118 48 64 119 48 68 120 48
82 121 48 86 122 55 62 123 55 66 124 55 77 125 46 64 126 46 68 127
46 82 128 46 86 129 62 77 130 62 84 131 62 88 132 64 68 133 64 82
134 64 86 135 66 68 136 66 82 137 66 86 138 68 77 139 68 84 140 68
88 141 46 66 142 46 77 143 46 84 144 62 82 145 62 86 146 64 66 147
64 77 148 64 84 149 64 88 150 66 77
Results
Low Concentration of GM1 OS
[0453] FIG. 50 illustrates binding of cholera toxin to the
microarray of candidate artificial receptors followed by washing
with buffer produced fluorescence signals. These fluorescence
signals demonstrate that the cholera toxin bound strongly to
certain receptor environments, weakly to others, and undetectably
to some. Comparison to experiments including those reported in
Example 2 indicates that cholera toxin binding was reproducible
from array to array and from month to month.
[0454] Binding of cholera toxin was also conducted with competition
from GM1 OS (0.34 .mu.M). FIG. 51 illustrates the fluorescence
signals due to cholera toxin binding that were detected after this
competition. Notably, many of the signals illustrated in FIG. 51
are significantly smaller than the corresponding signals recorded
in FIG. 50. The small signals observed in FIG. 51 represent less
cholera toxin bound to the array. GM1 OS significantly disrupted
binding of cholera toxin to many of the receptor environments.
[0455] The disruption in cholera toxin binding caused by GM1 OS can
be visualized as the ratio of the amount bound in the absence of
GM1 OS to the amount bound in competition with GM1 OS. This ratio
is illustrated in FIG. 52. The larger the ratio, the less cholera
toxin remained bound to the artificial receptor after competition
with GM1 OS. The ratio can be as large as about 30. The ratios are
independent of the quantity bound in the control.
High Concentration of GM1 OS
[0456] Binding of cholera toxin to the microarray of candidate
artificial receptors followed by washing with buffer produced
fluorescence signals illustrated in FIG. 53. As before, cholera
toxin was reproducible and it bound strongly to certain receptor
environments, weakly to others, and undetectably to some. FIG. 54
illustrates the fluorescence signals detected due to cholera toxin
binding that were detected upon competition with GM1 OS at 5.1
.mu.M. Again, GM1 OS significantly disrupted binding of cholera
toxin to many of the receptor environments.
[0457] This disruption is presented as the ratio of the amount
bound in the absence of GM1 OS to the amount bound after contacting
with GM1 OS in FIG. 56. The ratios range up to about 18 and are
independent of the quantity bound in the control.
Conclusions
[0458] This experiment demonstrated that binding of a test ligand
to an artificial receptor of the present invention can be
diminished (e.g., competed) by a candidate disruptor molecule. In
this case the test ligand was the protein cholera toxin and the
candidate disruptor was a compound known to bind to cholera toxin,
GM1 OS. The degree to which binding of the test ligand was
disrupted was independent of the degree to which the test ligand
bound to the artificial receptor.
Example 5
GM1 Competes with Artificial Receptors for Binding to Cholera
Toxin
[0459] Microarrays of candidate artificial receptors were made and
evaluated for binding of cholera toxin. The arrays were also
evaluated for disrupting that binding. Disrupting of binding
employed a compound that binds to cholera toxin, the liposaccharide
GM1. The results obtained demonstrate that a ligand of a protein
specifically disrupts binding of the protein to the microarray.
Materials and Methods
[0460] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4,
TyrA4B6, TyrA6B2, TyrA6B4, and TyrA6B6 in groups of 4 building
blocks per artificial receptor. The abbreviation for the building
block including a linker, a tyrosine framework, and recognition
elements AxBy is TyrAxBy.
[0461] Microarrays for the evaluation of the 126 n=4 candidate
receptor environments were prepared as described above for Example
4. The test ligand employed in these experiments was cholera toxin
labeled with the Alexa.TM. fluorophore (Molecular Probes Inc.,
Eugene, Oreg.). Cholera toxin was employed at 5.3 nM in both the
control and the competition experiments. The candidate disrupter
employed in these experiments was GM1, a known ligand for cholera
toxin, which competed at concentrations of 0.042, 0.42, and 8.4
.mu.M. Microarray incubation and analysis was conducted as
described for Example 4.
[0462] Table 3 identifies the building blocks in each receptor
environment.
TABLE-US-00003 TABLE 3 Building Blocks 1 22 24 26 42 2 22 24 26 44
3 22 24 26 46 4 22 24 26 61 5 22 24 26 64 6 22 24 26 66 7 22 24 42
44 8 22 24 42 46 9 22 24 42 62 10 22 24 42 46 11 22 24 42 66 12 22
24 44 46 13 22 24 44 62 14 22 24 44 64 15 22 24 44 66 16 22 24 46
62 17 22 24 46 64 18 22 24 46 66 19 22 24 62 64 20 22 24 62 66 21
22 24 64 66 22 22 26 42 44 23 22 26 42 46 24 22 26 42 62 25 22 26
42 64 26 22 26 42 66 27 22 26 44 46 28 22 26 44 62 29 22 26 44 64
30 22 26 44 66 31 22 26 46 62 32 22 26 46 64 33 22 26 46 66 34 22
26 62 64 35 22 26 62 66 36 22 26 64 66 37 22 42 44 46 38 22 42 44
62 39 22 42 44 64 40 22 42 44 66 41 22 42 46 62 42 22 42 46 64 43
22 42 46 66 44 22 42 62 64 45 22 42 62 66 46 22 42 64 66 47 22 44
46 62 48 22 44 46 64 49 22 44 46 66 50 22 44 62 64 51 22 44 62 66
52 22 44 64 66 53 22 46 62 64 54 22 46 62 66 55 22 46 64 66 56 22
62 64 66 57 24 26 42 44 58 24 26 42 46 59 24 26 42 62 60 24 26 42
64 61 24 26 42 66 62 24 26 44 46 63 24 26 44 62 64 24 26 44 64 65
24 26 44 66 66 24 26 46 62 67 24 26 46 64 68 24 26 46 66 69 24 26
62 64 70 24 26 62 66 71 24 26 64 66 72 24 42 44 46 73 24 42 44 62
74 24 42 44 64 75 24 42 44 66 76 24 42 46 62 77 24 42 46 64 78 24
42 46 66 79 24 42 62 64 80 24 42 62 66 81 24 42 64 66 82 24 44 46
62 83 24 44 46 64 84 24 44 46 66 85 24 44 62 64 86 24 44 62 66 87
24 44 64 66 88 24 46 62 64 89 24 46 62 66 90 24 46 64 66 91 24 62
64 66 92 26 42 44 46 93 26 42 44 62 94 26 42 44 64 95 26 42 44 66
96 26 42 46 62 97 26 42 46 64 98 26 42 46 66 99 26 42 62 64 100 26
42 62 66 101 26 42 64 66 102 26 44 46 62 103 26 44 46 64 104 26 44
46 66 105 26 44 62 64 106 26 44 62 66 107 26 44 64 66 108 26 46 62
64 109 26 46 62 66 110 26 46 64 66 111 26 62 64 66 112 42 44 46 62
113 42 44 46 64 114 42 44 46 66 115 42 44 62 64 116 42 44 62 66 117
42 44 64 66 118 42 46 62 64 119 42 46 62 66 120 42 46 64 66 121 42
62 64 66 122 44 46 62 64 123 44 46 62 66 124 44 46 64 66 125 44 62
64 66 126 46 62 64 66
Results
[0463] FIG. 56 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors alone and in competition with each of the three
concentrations of GM1. The magnitude of the fluorescence signal
decreases steadily with increasing concentration of GM1. The amount
of decrease is not quantitatively identical for all of the
receptors, but each receptor experienced decreased binding of
cholera toxin. These decreases indicate that GM1 competed with the
artificial receptor for binding to the cholera toxin.
[0464] The decreases show a pattern of relative competition for the
binding site on cholera toxin. This can be demonstrated through
graphs of fluorescence signal obtained at a particular
concentration of GM1 against fluorescence signal in the absence of
GM1 (not shown). Certain of the receptors appear at similar
relative positions on these plots as concentration of GM1
increases.
[0465] The disruption in cholera toxin binding caused by GM1 can be
visualized as the ratio of the amount bound in the absence of GM1
OS to the amount bound upon competition with GM1. This ratio is
illustrated in FIG. 57. The larger the ratio, the more cholera
toxin remained bound to the artificial receptor upon competition
with GM1. The ratio can be as large as about 14. The ratios are
independent of the quantity bound in the control.
[0466] Interestingly, in several instances minor changes in
structure to the artificial receptor caused significant changes in
the ratio. For example, the artificial receptor including building
blocks 24, 26, 46, and 66 differs from that including 24, 42, 46,
and 66 by only substitution of a single building block. (xy
indicates building block TyrAxBy.) The substitution of building
block 42 for 26 increased binding in the presence of GM1 by about
14-fold.
[0467] By way of further example, the artificial receptor including
building blocks 22, 24, 46, and 64 differs from that including 22,
46, 62, and 64 by only substitution of a single building block. The
substitution of building block 24 for 62 increased binding in the
presence of GM1 by about 3-fold.
[0468] Even substitution of a single recognition element affected
binding. The artificial receptor including building blocks 22, 24,
42, and 44 differs from that including 22, 24, 42, and 46 by only
substitution of a single recognition element. The substitution of
building block 44 for 46 (a change of recognition element B6 to B4)
increased binding in the presence of GM1 by about 3-fold.
Conclusions
[0469] This experiment demonstrated that binding of a test ligand
to an artificial receptor of the present invention can be
diminished (e.g., competed) by a candidate disruptor molecule. In
this case the test ligand was the protein cholera toxin and the
candidate disrupter was a compound known to bind to cholera toxin,
GM1. Minor changes in structure of the building blocks making up
the artificial receptor caused significant changes in the
competition.
Example 6
GM1 Employed as a Building Block Alters Binding of Cholera Toxin to
the Present Artificial Receptors
[0470] Microarrays of candidate artificial receptors were made, GM1
was bound to the arrays, and they were evaluated for binding of
cholera toxin. The results obtained demonstrate that adding GM1 as
a building block in an array of artificial receptors can increase
binding to certain of the receptors.
Materials and Methods
[0471] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
those described in Example 4. Microarrays for the evaluation of the
171 n=2 candidate receptor environments were prepared as described
above for Example 4. The test ligand employed in these experiments
was cholera toxin labeled with the Alexa.TM. fluorophore (Molecular
Probes Inc., Eugene, Oreg.). Cholera toxin was employed at 0.01
ug/ml (0.17 pM) or 0.1 ug/ml (1.7 pM) in both the control and the
competition experiments. GM1 was employed as a test ligand for the
artificial receptors and became a building block for receptors used
to bind cholera toxin. The arrays were contacted with GM1 at either
100 .mu.g/ml, 10 .mu.g/ml, or 1 .mu.g/ml as described above for
cholera toxin and then rinsed with deionized water. The arrays were
then contacted with cholera toxin under the conditions described
above. Microarray analysis was conducted as described for Example
4. Table 2 identifies the building blocks in each receptor
environment.
Results
[0472] FIG. 58 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors without pretreatment with GM1. Binding of GM1 to the
microarray of candidate artificial receptors followed by binding of
cholera toxin produced fluorescence signals illustrated in FIGS.
59, 60, and 61 (100 .mu.g/ml, 10 .mu.g/ml, and 1 .mu.g/ml GM1,
respectively).
[0473] The enhancement of cholera toxin binding caused by
pretreatment with GM1 can be visualized as the ratio of the amount
bound in the presence of GM1 to the amount bound in the absence of
GM1. This ratio is illustrated in FIG. 62 for 1 .mu.g/ml GM1. The
larger the ratio, the more cholera toxin bound to the artificial
receptor after pretreatment with GM1. The ratio can be as large as
about 16.
[0474] In several instances minor changes in structure to the
artificial receptor caused significant changes in the ratio. For
example, the artificial receptor including building blocks 46 and
48 differs from that including 46 and 88 by only substitution of a
single recognition element on a single building block. (xy
indicates building block TyrAxBy.) The substitution of building
block 48 for 88 (a change of recognition element A8 to A4)
increased the ratio representing increased binding the presence of
GM1 building block from about 0.5 to about 16. Similarly, the
artificial receptor including building blocks 42 and 77 differs
from that including 24 and 77 by only substitution of a single
building block. The substitution of building block 42 for 24
increased the ratio representing increased binding the presence of
GM1 building block from about 2 to about 14.
[0475] Interestingly, several building blocks that exhibited high
levels of binding of cholera toxin (signals of 45,000 to 65,000
fluorescence units) and that include the building block 33 were not
strongly affected by the presence of GM1 as a building block.
Conclusions
[0476] This experiment demonstrated that binding of GM1 to an
artificial receptor of the present invention can significantly
increase binding by cholera toxin. Minor changes in structure of
the building blocks making up the artificial receptor caused
significant changes in the degree to which GM1 enhanced binding of
cholera toxin.
Discussion of Examples 4-6
[0477] We have previously demonstrated that an array of working
artificial receptors bind to a protein target in a manner which is
complementary to the specific environment presented by each region
of the proteins surface topology. Thus the pattern of binding of a
protein target to an array of working artificial receptors
describes the proteins surface topology; including surface
structures which participate in e.g., protein.about.small molecule,
protein.about.peptide, protein-protein, protein.about.carbohydrate,
protein.about.DNA, etc. interactions. It is thus possible to use
the binding of a selected protein to a working artificial receptor
array to characterize these protein.about.small molecule,
protein.about.peptide, protein-protein, protein.about.carbohydrate,
protein.about.DNA, etc. interactions. Moreover, it is possible to
utilize the protein to array interactions to define "leads" for the
disruption of these interactions.
[0478] Cholera Toxin B sub-unit binds to GM1 on the cell surface
(structure of GM1). Studies to identify competitors to this binding
event have shown that competitors to the cholera toxin: GM1 binding
interaction (binding site) can utilize both a sugar and an
alkyl/aromatic functionality (Pickens, et al., Chemistry and
Biology, vol. 9, pp 215-224 (2002)). We have previously
demonstrated that fluorescently labeled Cholera Toxin B sub-unit
binds to arrays of working artificial receptors to give a defined
binding pattern which (vida infra) reflects cholera toxin B's
surface topology. For this study, we sought to demonstrate that the
binding of the cholera toxin to at least some members of the array
could be disrupted using cholera toxins natural ligand, GM1.
[0479] The results presented in the figures clearly demonstrate
that these goals have been achieved. Specifically, competition
between the GM1 OS pentasaccharide or GM1 and a working artificial
receptor array for cholera binding clearly gave a binding pattern
which was distinct from the cholera binding pattern control.
Moreover, these results demonstrated the complementarity between
several of the working artificial receptors which contained a
naphthyl moiety when compared to working artificial receptors which
only contained phenyl functionality. These results are in keeping
with the active site competition studies in Pickens, et al. and
indicate that the naphthyl and phenyl derivatives represent good
mimics/probes for the cholera to GM1 interaction. The specificity
of these interactions was particularly demonstrated by the
observation that the change of a single building block out of 4 in
a combination of 4 building blocks system changed a non-competitive
to a significantly competitive environment. These results also
indicated that selected working artificial receptors can be used to
develop a high-throughput screen for the further evaluation of the
cholera: GM1 interaction.
[0480] Additionally, we sought to demonstrate that an affinity
support/membrane mimic could be prepared by pre-incubating an array
of artificial receptors with GM1 which would then bind/capture
cholera toxin in a binding pattern which could be used to select a
working artificial receptor(s) for, for example, the
high-throughput screen of lead compounds which will disrupt the
"cholera: membrane.about.GM1 mimic". The GM1 pre-incubation studies
clearly demonstrated that several of the working artificial
receptors which were poor cholera binders significantly increased
their cholera binding, presumably through an affinity interaction
between the cholera toxin and both the immobilized GM1
pentasaccharide moiety and the working artificial receptor building
block environment.
[0481] It should be noted that, as used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition containing
"a compound" includes a mixture of two or more compounds. It should
also be noted that the term "or" is generally employed in its sense
including "and/or" unless the content clearly dictates
otherwise.
[0482] It should also be noted that, as used in this specification
and the appended claims, the phrase "adapted and configured"
describes a system, apparatus, or other structure that is
constructed or configured to perform a particular task or adopt a
particular configuration to. The phrase "adapted and configured"
can be used interchangeably with other similar phrases such as
arranged and configured, constructed and arranged, adapted,
constructed, manufactured and arranged, and the like.
[0483] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains.
[0484] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *