U.S. patent application number 11/375825 was filed with the patent office on 2007-03-22 for use of antibody-surrogate antigen systems for detection of analytes.
This patent application is currently assigned to Applera Corporation. Invention is credited to Joe Y. L. Lam, Zhaochun Ma, Steven M. Menchen, Khairuzzam Bashar Mullah.
Application Number | 20070065948 11/375825 |
Document ID | / |
Family ID | 36589058 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070065948 |
Kind Code |
A1 |
Menchen; Steven M. ; et
al. |
March 22, 2007 |
Use of antibody-surrogate antigen systems for detection of
analytes
Abstract
Compositions, methods and kits for labeling proteins and uses in
reporter systems for detecting, quantifying and/or characterizing
analytes.
Inventors: |
Menchen; Steven M.;
(Fremont, CA) ; Lam; Joe Y. L.; (Castro Valley,
CA) ; Ma; Zhaochun; (San Carlos, CA) ; Mullah;
Khairuzzam Bashar; (Union City, CA) |
Correspondence
Address: |
DECHERT LLP
P.O. BOX 10004
PALO ALTO
CA
94303
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
36589058 |
Appl. No.: |
11/375825 |
Filed: |
March 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60662412 |
Mar 15, 2005 |
|
|
|
Current U.S.
Class: |
436/86 ; 436/518;
530/409; 549/226 |
Current CPC
Class: |
C07D 311/88 20130101;
G01N 33/58 20130101; G01N 33/582 20130101; G01N 33/532
20130101 |
Class at
Publication: |
436/086 ;
530/409; 549/226; 436/518 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/543 20060101 G01N033/543; C07K 14/47 20060101
C07K014/47; C07D 311/88 20060101 C07D311/88 |
Claims
1. A labeling molecule useful for specifically labeling a protein
comprising: (a) a target moiety; (b) a label moiety; (c) an
activatable reactive moiety; and (d) a cleavable tether linking the
target moiety to the molecule; wherein the target moiety, label
moiety, and reactive moiety are connected to one another such that
the target moiety can bind the protein and the reactive moiety can
crosslink the labeling molecule to the protein.
2. The labeling molecule of claim 1 in which the protein is an
antibody and the target moiety is an antigen comprising an epitope
recognized by said antibody.
3. The labeling molecule of claim 2 which has the structure:
TM-(L).sub.N-CT-(L).sub.N-LM-(L).sub.N-RM wherein: TM represents
the target moiety; CT represents the cleavable tether; LM
represents the label moiety; RM represents the reactive moiety; L
represents an optional linker moiety; and n is an integer from 0 to
5.
4. The labeling molecule of claim 3 in which the label moiety
comprises a fluorescent moiety.
5-8. (canceled)
9. The labeling molecule of claim 3 in which the reactive moiety is
specifically activatable.
10-11. (canceled)
12. The labeling molecule of claim 3 in which the reactive moiety
is linked to the label moiety via a linker moiety.
13. (canceled)
14. The labeling molecule of claim 3 in which the cleavable tether
comprises a cleavage moiety linked to one or more linker moieties,
said cleavage moiety comprising a linkage that can be cleaved by a
chemical cleaving agent.
15. (canceled)
16. The labeling molecule of claim 14 in which the cleavable tether
has the structure: (TE).sub.NCM(TE).sub.N wherein: CM represents
the cleavage moiety; TE represents an optional tether element; and
n is an integer from 0 to 5.
17-23. (canceled)
24. The labeling molecule of claim 16 in which the cleavable moiety
comprises the structure: ##STR8## wherein: R.sup.1 and R.sup.2 are
hydrocarbons; TE represents an optional tether element; and n is an
integer from 0 to 5.
25. (canceled)
26. A protein, capable of binding an analyte, that is specifically
labeled in close proximity to the analyte binding site with one or
more label moieties, said protein being substantially unlabeled at
other positions.
27. The protein of claim 26 which is an antibody capable of binding
an antigen comprising an epitope.
28. The antibody of claim 27 in which the protein is labeled with a
fluorescent moiety.
29-32. (canceled)
33. The antibody of claim 27 which is bound to a surrogate antigen
comprising a quenching moiety.
34. (canceled)
35. The antibody of claim 33 which is encapsulated.
36. The antibody of claim 33 which is encapsulated in a
semi-permeable nanocapsule.
37. The antibody of claim 36 in which the nanocapsule is permeable
to an analyte molecule and is impermeable to said antibody.
38. (canceled)
39. The antibody of claim 36 in which the nanocapsule comprises a
cross-linked water soluble vinyl polymer or copolymer.
40. The antibody of claim 36 in which the nanocapsule further
comprises a cell membrane targeting moiety.
41-43. (canceled)
44. A method of making a labeled protein of interest comprising:
(a) contacting the protein with a labeling molecule comprising a
target moiety, a label moiety, a reactive moiety, and a cleavable
tether connected to the target moiety, wherein the target moiety,
label moiety, and reactive moiety are connected to one another such
that the target moiety can bind the protein, and the reactive
moiety can link the molecule to the protein, under conditions such
that the protein binds the target moiety; (b) activating the
reactive moiety so as to form a covalent linkage between the
reactive moiety and the protein; (c) cleaving the cleavable tether;
and (d) removing the cleaved target moiety from the labeled protein
complex.
45-50. (canceled)
51. A method of detecting the presence or absence of a target
analyte molecule in a sample, comprising contacting the sample with
a surrogate analyte-protein complex, said complex comprising a
reporter system capable of generating a detectable fluorescent
signal upon dissociation of the complex that is at least 3-fold
greater than the background signal, wherein the presence of the
detectable fluorescent signal indicates that the target analyte is
present in the sample.
51-62. (canceled)
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(e) to application Ser. No. 60/662,412, filed Mar. 15, 2005, the
contents of which are incorporated herein by reference.
2. BACKGROUND
[0002] Assays using reporter systems are important tools for
studying and detecting analytes in biological and industrial
processes. Although numerous approaches have been developed for
assaying analytes, there is still a need to find new assay designs
that can be used to specifically and conveniently detect and
characterize a wide variety of analytes. The development of protein
based reporter systems has significantly advanced the art of
detection of biological analytes. In spite of these advances,
nonspecific labeling of proteins
3. SUMMARY
[0003] In one aspect, provided herein are labeling molecules useful
for specifically labeling a protein of interest. The proteins are
labeled near the binding site of an analyte of interest. The
labeling molecules typically comprise a reactive moiety, a label
moiety, a target moiety, and a cleavable tether. The reactive
moiety is selected such that it is capable of attaching the
labeling moiety to the protein. In some embodiments, the reactive
moiety comprises a cross-linking group that can form a covalent
linkage with a functional group on the protein that is located near
the binding site of an analyte of interest. In some embodiments,
the reactive moiety comprises a photoreactive group that upon
activation by an external energy source is capable of forming a
covalent bond with a functional group on the protein.
[0004] The label moiety can be any fluorescent entity that is
operative in accordance with the various compositions and methods
described herein. In some embodiments, the fluorescent moiety
comprises at least one fluorescent dye. In some embodiments, the
fluorescent moiety comprises at least one rhodamine dye. In some
embodiments, the fluorescent moiety comprises two or more
fluorescent dyes that can act cooperatively with one another, such
as by, for example, fluorescence resonance energy transfer
("FRET").
[0005] In some embodiments, the label moiety can be a quenching
moiety. The quenching moiety can be any moiety capable of quenching
the fluorescence of the fluorescent moiety of a signal molecule
when it is in close proximity thereto, such as, for example, by
orbital overlap (formation of a ground state dark complex),
collisional quenching, FRET, or another mechanism or combination of
mechanisms. The quenching moiety can itself be fluorescent, or it
can be non-fluorescent. In some embodiments, the quenching moiety
comprises a fluorescent dye that has a structural relationship with
the fluorescent moiety of the signal molecule such that it quenches
the fluorescence of the fluorescent moiety when in close proximity
thereto. In such embodiments, selecting a quenching moiety that
fluoresces at a wavelength resolvable from that of the fluorescent
moiety can provide an internal signal standard to which the
fluorescence signal can be referenced
[0006] The target moiety is capable of binding the labeling
molecule to the protein to be labeled. The target moiety can
comprise any organic or inorganic molecule capable of binding to a
particular position on the protein to be labeled. Non-limiting
examples of molecules that can comprise the target moiety include
antigens, analytes, enzyme substrates, proteins, carbohydrates,
polysaccharides, glycoproteins, hormones, viruses, metabolites,
transition state analogs, cofactors, nucleotides, polynucleotide,
inhibitors, drugs, nutrients, electrolytes, growth factors and
other biomolecules as well as non-biomolecules capable of binding
the protein to be labeled.
[0007] The cleavable tether connects the target moiety to the label
moiety. The cleavable tether comprises at least one cleavable
moiety and one or more optional linker moieties. The cleavable
moiety comprises at least one functional group that can be cleaved
to allow detachment of the target moiety from the protein. The
optional linker moieties can be selected to have specified
properties. For example, the linker moiety can be hydrophobic or
hydrophilic in character, long or short, rigid, semirigid or
flexible, depending upon the application. In some embodiments, the
linker moieties are selected to increase the solubility of the
labeling molecule. In other embodiments, the linker moieties are
selected to provide a cleavable tether of a specified length.
[0008] The protein can be any protein that can be specifically
labeled in close proximity to the analyte binding site and be
substantially unlabeled at other positions. Examples of suitable
proteins include, but are not limited to, antibodies, enzymes,
signal transduction proteins, and receptors.
[0009] Labeled proteins can be made by contacting the protein with
a labeling molecule comprising a target moiety, a label moiety, a
reactive moiety, and a cleavable tether. The target moiety, label
moiety, and reactive moiety are connected to one another such that
the target moiety can bind the protein, and the reactive moiety can
link the labelling molecule to the protein. Agents capable of
activating the reactive moiety are added, such that a covalent
linkage between the reactive moiety and the protein is formed. The
target moiety can be removed by inducing cleavage of the cleavable
tether. The resulting labeled proteins can be used in assays to
detect analytes of interest.
[0010] In another aspect, analyte reporter systems useful for
detecting the presence or absence of analytes of interest, i.e.,
target analytes are provided herein. The analyte reporter systems
comprise a labeled protein and a labeled surrogate analyte. The
labeled protein can be contacted with the labeled surrogate analyte
to form a surrogate analyte-labeled protein complex. In some
embodiments, the protein comprises a fluorescent moiety such that
upon displacement of the surrogate analyte by a target analyte, an
increase in the fluorescence of the fluorescent moiety can be
detected.
[0011] In some embodiments, the fluorescence of the labeled protein
is quenched when the surrogate analyte is bound to the protein.
This quenching may be accomplished by a variety of different
mechanisms. In some embodiments, the protein and surrogate analyte
comprise fluorescent moieties that are capable of "self-quenching"
when in close proximity to each other. In other embodiments,
quenching can be achieved with the aid of a quenching moiety.
[0012] In some embodiments, the surrogate analyte and the labeled
protein can be encapsulated in a nanocapsule, liposome,
microparticle, lipid particle, vesicles and the like. In some
embodiments, the porosity of the membrane comprising the particle
used to encapsulate the surrogate analyte and the labeled protein
is selected to retain the surrogate analyte-labeled protein
complex, and at the same time, allow passage of the target analyte.
Thus, the porosity of the particle membrane is such that it allows
passage of elements that are less than or equal to 0.5 nm to 5.0 nm
in diameter. In some embodiments, the pore diameter of the
particles is less than or equal to 5.0 nm. In some embodiments, the
pore diameter of the particles is less than or equal to 2.0 nm. In
some embodiments, the pore diameter of the particles is less than
or equal to 1.5 nm. In some embodiments, the pore diameter of the
particles is less than or equal to 1.0 nm. In some embodiments, the
pore diameter of the particles is less than or equal to or equal to
0.5 nm.
[0013] Also provided are kits for performing methods of the present
teachings. For example, in some embodiments, the kits comprise a
labeling molecule comprising a target moiety, a label moiety, a
reactive moiety, and a cleavable tether. In other embodiments, the
kits comprise surrogate analyte-protein complexes useful for
detecting the presence of a target analyte. In yet other
embodiments, the kits comprise encapsulated surrogate
analyte-protein complexes. These and other features of the present
teachings are set forth below.
4. BRIEF DESCRIPTION OF THE FIGURES
[0014] FIGS. 1A-E provide an exemplary embodiment of an antibody
labeling scheme utilizing an exemplary labeling molecule comprising
an antigen moiety, a label moiety, a cleavable tether, and a
reactive moiety;
[0015] FIGS. 2A-C provide an exemplary embodiment of an
intracellular encapsulated analyte reporter system; and
[0016] FIG. 3 provides an exemplary embodiment of an analyte
reporter system.
5. DETAILED DESCRIPTION
[0017] It is to be understood that both the foregoing general
description and the following description are exemplary and
explanatory only and are not restrictive of the compositions and
methods described herein. In this application, the use of the
singular includes the plural unless specifically state otherwise.
Also, the use of "or" means "and/or" unless state otherwise.
Similarly, "comprise," "comprises," "comprising," "include,"
"includes" and "including" are not intended to be limiting.
[0018] 5.2 Definitions
[0019] As used herein, the following terms and phrases are intended
to have the following meanings:
[0020] "Antibody" has its standard meaning and is intended to refer
to full-length as well antibody fragments, as are known in the art,
including Fab, Fab.sub.2, single chain antibodies (Fv for example),
monoclonal, polyclonal, chimeric antibodies, etc., either produced
by the modification of whole antibodies or those synthesized de
novo using recombinant DNA technologies.
[0021] "Detect" and "detection" have their standard meaning, and
are intended to encompass detection, measurement, and
characterization of an analyte.
[0022] "Protein" has its standard meaning and is intended to refer
to proteins, oligopeptides and peptides, derivatives and analogs,
including proteins containing non-naturally occurring amino acids
and amino acid analogs, and peptidomimetic structures, and includes
proteins made using recombinant techniques, i.e. through the
expression of a recombinant nucleic acid.
[0023] "Quench" has its standard meaning and is intended to refer
to a reduction in the fluorescence intensity of a fluorescent group
or moiety as measured at a specified wavelength, regardless of the
mechanism by which the reduction is achieved. As specific examples,
the quenching can be due to molecular collision, energy transfer
such as FRET, photoinduced electron transfer such as PET, a change
in the fluorescence spectrum (color) of the fluorescent group or
moiety or any other mechanism (or combination of mechanisms). The
amount of the reduction is not critical and can vary over a broad
range. The only requirement is that the reduction be detectable by
the detection system being used. Thus, a fluorescence signal is
"quenched" if its intensity at a specified wavelength is reduced by
any measurable amount. A fluorescence signal is "substantially
quenched" if its intensity at a specified wavelength is reduced by
at least 50%, for example by 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or even 100%.
[0024] 5.3 Labeling Molecules
[0025] Provided herein are compositions useful for, among other
things, specifically labeling a protein. The compositions generally
comprise a labeling molecule comprising a target moiety, a label
moiety, a reactive moiety, and a cleavable tether. The target
moiety is capable of binding the labeling molecule to a protein of
interest. The reactive moiety is capable of linking the labeling
molecule to the protein. The cleavable tether is capable of being
cleaved such that the target moiety is released from the labeling
molecule. The labeling molecule can be designed to label virtually
any protein of interest, provided that the protein comprises at
least one binding domain capable of binding a target moiety.
Non-limiting examples of suitable proteins that can be labeled
include, but are not limited to, antibodies, enzymes, signal
transduction proteins, receptors, as well as fragments of all of
the foregoing.
[0026] The labeling molecules are useful for specifically labeling
a protein of interest, and typically comprise a target moiety, a
label moiety, a reactive moiety, and a cleavable tether.
[0027] 5.3.1 Target Moiety
[0028] In some embodiments, the labeling molecule comprises one or
more target moiety(ies). The target moiety is capable of binding
the labeling molecule to the protein to be labeled. In embodiments,
using two or more target moieties, each target moiety can be the
same, or some, or all of the target moieties may differ. The target
moiety can comprise any organic or inorganic molecule capable of
binding the protein to be labeled and capable of containing a
cleavable target. Non-limiting examples of molecules that can
comprise the target moiety include antigens, analytes, enzyme
substrates, proteins, carbohydrates, polysaccharides,
glycoproteins, hormones, viruses, metabolites, transition state
analogs, cofactors, nucleotides, polynucleotide, inhibitors, drugs,
nutrients, growth factors and other biomolecules as well as
non-biomolecules capable of binding the protein to be labeled and
capable of comprising a cleavable tether.
[0029] The specificity and strength of binding between the target
moiety and the protein depends in part, on a number of
interactions, including, weak, noncovalent bonds, hydrogen bonds,
ionic interactions, van der Waals attractions and favorable
hydrophobic interactions. The region of the protein that binds with
target moiety, referred to herein as the "target binding site",
usually consists of a cavity in the protein surface formed by a
particular arrangement of amino acids. These amino acids can belong
to different portions of the polypeptide chain that come together
when the protein folds. Separate regions of the protein surface can
provide binding sites for different targets.
[0030] The strength with which any two molecules bind to each other
can be measured directly. For example, the dissociation constant,
K.sub.d, can be used to represent the affinity of the protein for
the target moiety. In some embodiments, the target moiety and the
protein can have a K.sub.d of about 10.sup.-6 or lower. In some
embodiments, the dissociation constant can be about 10.sup.-7 or
lower. In some embodiments, the dissociation constant can be about
10.sup.-8 or lower. In some embodiments, the dissociation constant
can be about 10.sup.-9 or lower. In some embodiments, the
dissociation constant can be about 10.sup.-10 or lower. In some
embodiments, the dissociation constant can be about 10.sup.-11 or
lower.
[0031] The composition of the target moiety can depend, in part, on
the structure of the analyte to be detected, on the protein being
used to detect the target analyte and on the protein to be labeled.
For example, if the protein to be labeled is an antibody, the
target moiety comprises an antigen comprising at least one epitope
recognized by the antibody. As used herein, the term "antigen"
refers to an agent that is capable of eliciting the synthesis of a
specific antibody in a vertebrate. An "epitope" as used herein
refers to an antigenic determinant, e.g., the particular chemical
group or groups within an antigen to which a specific antibody
binds.
[0032] In some embodiments, the target moiety comprises an antigen
comprising an epitope for cAMP.
[0033] In some embodiments, an enzyme can be labeled using, for
example, a target moiety comprising an enzyme substrate, a portion
of an enzyme substrate capable of binding the enzyme, a cofactor, a
transition state analog, or a small molecule such as a drug. The
target moiety can bind the enzyme in any location provided that the
labeling moiety is positioned in close proximity to the active
site, but does not interfere with the binding of the substrate at
the active site, and the analyte binds to the same active site.
[0034] In some embodiments, the protein to be labeled can be a
receptor. In these embodiments, the target moiety can comprise a
ligand, a portion of a ligand capable of binding the receptor, or a
small molecule. The target moiety can bind the receptor in any
location provided that the labeling moiety is positioned in close
proximity to the ligand binding site, but does not interfere with
the binding of the ligand by the receptor.
[0035] In some embodiments, the protein to be labeled can be a
signal transduction protein. In these embodiments, the target
moiety can comprise a ligan, a protein of a ligand capable of
binding the receptor, or a small molecule involved in a signal
transduction pathway. The target moiety can bind the signal
transduction protein in any location provided that the labeling
moiety is positioned in close proximity to the ligand binding site,
but does not interfere with the binding of the ligand by the signal
transduction protein.
[0036] In some embodiments, the target moiety comprises cAMP and
the protein to be labeled comprises a cAMP antibody.
[0037] 5.3.2 Label Moiety
[0038] In some embodiments, the labeling molecule comprises one or
more label moiety(ies). In embodiments employing two or more label
moieties, each label moiety can be the same, or some, or all, of
the label moieties may differ.
[0039] In some embodiments, the label moiety comprises a
fluorescent moiety. The fluorescent moiety can comprise any entity
that provides a fluorescent signal and that can be used in
accordance with the methods and principles described herein.
Typically, the fluorescent moiety of the labeling molecule
comprises a fluorescent dye that in turn comprises a
resonance-delocalized system or aromatic ring system that absorbs
light at a first wavelength and emits fluorescent light at a second
wavelength in response to the absorption event. A wide variety of
such fluorescent dye molecules are known in the art. For example,
fluorescent dyes can be selected from any of a variety of classes
of fluorescent compounds, such as xanthenes, rhodamines,
fluoresceins, cyanines, phthalocyanines, squaraines, bodipy dyes,
coumarins, oxazines, and carbopyronines.
[0040] In some embodiments, the fluorescent moiety comprises a
xanthene dye. Generally, xanthene dyes are characterized by three
main features: (1) a parent xanthene ring; (2) an exocyclic
hydroxyl or amine substituent; and (3) an exocyclic oxo or imminium
substituent. The exocyclic substituents are typically positioned at
the C3 and C6 carbons of the parent xanthene ring, although
"extended" xanthenes in which the parent xanthene ring comprises a
benzo group fused to either or both of the C5/C6 and C3/C4 carbons
are also known. In these extended xanthenes, the characteristic
exocyclic substituents are positioned at the corresponding
positions of the extended xanthene ring. Thus, as used herein, a
"xanthene dye" generally comprises one of the following parent
rings: ##STR1##
[0041] In the parent ring depicted above, A.sup.1 is OH or NH.sub.2
and A.sup.2 is O or NH.sub.2.sup.+. When A.sup.1 is OH and A.sup.2
is O, the parent ring is a fluorescein-type xanthene ring. When
A.sup.1 is NH.sub.2 and A.sup.2 is NH.sub.2.sup.+, the parent ring
is a rhodamine-type xanthene ring. When A.sup.1 is NH.sub.2 and
A.sup.2 O, the parent ring is rhodol-type xanthene ring.
[0042] One or both of nitrogens of A.sup.1 and A.sup.2 (when
present) and/or one or more of the carbon atoms at positions C1,
C2, C2'', C4, C4'', C5, C5'', C7'', C7 and C8 can be independently
substituted with a wide variety of the same or different
substituents. In one embodiment, typical substituents comprises,
but are not limited to, --X, --R.sup.a, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.a, perhalo (C.sub.1-C.sub.6) alkyl, --CX.sub.3,
--CF.sub.3, --CN, --OCN, --SCN, --NCO, --NCS, --NO, --NO.sub.2,
--N.sub.3, --S(O).sub.2O.sup.-, --S(O).sub.2OH,
--S(O).sub.2R.sup.a, --C(O)R, --C(O)X, --C(S)R.sup.a , --C(S)X,
--C(O)OR.sup.a, --C(O)O.sup.-, --C(S)OR.sup.a, --C(O)SR.sup.a,
--C(S)SR.sup.a, --C(O)NR.sup.aR.sup.a, --C(S)NR.sup.aR.sup.a and
--C(NR)NR.sup.aR.sup.a, where each X is independently a halogen
(preferably --F or --Cl) and each R.sup.a is independently
hydrogen, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkanyl,
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl,
(C.sub.5-C.sub.20) aryl, (C.sub.6-C.sub.26) arylalkyl,
(C.sub.5-C.sub.20) arylaryl, 5-20 membered heteroaryl, 6-26
membered heteroarylalkyl, 5-20 membered heteroaryl-heteroaryl,
carboxyl, acetyl, sulfonyl, sulfinyl, sulfone, phosphate, or
phosphonate. Generally, substituents which do not tend to
completely quench the fluorescence of the parent ring are
preferred, but in some embodiments quenching substituents may be
desirable. Substituents that tend to quench fluorescence of parent
xanthene rings comprise heavy atoms, such as --NO.sub.2, --Br and
--I, and/or other functional moieties, such as NO.sub.2.
[0043] The C1 and C2 substituents and/or the C7 and C8 substituents
can be taken together to form substituted or unsubstituted
buta[1,3]dieno or (C.sub.5-C.sub.20) aryleno bridges. For purposes
of illustration, exemplary parent xanthene rings including
unsubstituted benzo bridges fused to the C1/C2 and C7/C8 carbons
are illustrated below: ##STR2##
[0044] The benzo or aryleno bridges may be substituted at one or
more positions with a variety of different substituent groups, such
as the substituent groups previously described above for carbons
C1-C8 in structures (Ia)-(Ic), supra. In embodiments including a
plurality of substituents, the substituents may all be the same, or
some or all of the substituents can differ from one another.
[0045] When A.sup.1 is NH.sub.2 and/or A.sup.2 is NH.sub.2.sup.+,
the nitrogen atoms may be included in one or two bridges adjacent
carbon atom(s). The bridging groups may be the same or different,
and are typically selected from (C.sub.1-C.sub.12) alkyldiyl,
(C.sub.1-C.sub.12) alkyleno, 2-12 membered heteroalkyldiyl and/or
2-12 membered heteroalkyleno bridges. Non-limiting exemplary parent
rings that comprise bridges involving the exocyclic nitrogens are
illustrated below: ##STR3##
[0046] The parent ring may also comprise a substituent at the C9
position. In some embodiments, the C9 substituent is selected from
acetylene, lower (e.g., from 1 to 6 carbon atoms) alkanyl, lower
alkenyl, cyano, aryl, phenyl, heteroaryl, and substituted forms of
any of the preceding groups. In embodiments in which the parent
ring comprises benzo or aryleno bridges fused to the C1/C2 and
C7/C8 positions, such as, for example, rings (Id) (Ie) and (If)
illustrated above, the C9 carbon is preferably unsubstituted.
[0047] In some embodiments, the C9 substituted is a substituted or
unsubstituted phenyl ring such that the xanthene dye comprises one
of the following structures: ##STR4##
[0048] The carbons at positions 3, 4, 5, 6, and 7 may be
substituted with a variety of different substituent groups, such as
the substituent groups previously described for carbons C1-C8. In
some embodiments, the carbon at position C3 is substituted with a
carboxyl (--COOH) or sulfuric acid (--SO.sub.3H) group, or an anion
thereof. Dyes of formulae (IIa) and (IIb) and (IIc) in which
A.sup.1 is OH and A.sup.2 is O are referred to herein as
fluorescein dyes; dyes of formulae (IIa), (IIb) and (IIc) in which
A.sup.1 is NH.sub.2 and A.sup.2 is NH.sub.2.sup.+ are referred to
herein as rhodamine dyes; and dyes of formulae (IIa), (IIb) and
(IIc) in which A.sup.1 is OH and A.sup.2 is NH.sub.2.sup.+ (or in
which A.sup.1 is NH.sub.2 and A.sup.2 is O) are referred to herein
as rhodol dyes.
[0049] As highlighted by the above structures, when xanthene rings
(or extended xanthene rings) are included in fluorescein, rhodamine
and rhodol dyes, their carbon atoms are numbered differently.
Specifically, their carbon atom numberings include primes. Although
the above numbering systems for fluorescein, rhodamine and rhodol
dyes are provided for convenience, it is to be understood that
other numbering systems may be employed, and that they are not
intended to be limiting. It is also to be understood that while one
isomeric form of the dyes are illustrated, they may exist in other
isomeric forms, including, by way of example and not limitation,
other tautomeric forms or geometric forms. As a specific example,
carboxy rhodamine and fluorescein dyes may exist in a lactone
form.
[0050] In some embodiments, the fluorescent moiety comprises a
rhodamine dye. Exemplary suitable rhodamine dyes include, but are
not limited to, rhodamine B, 5-carboxyrhodamine, rhodamine X (ROX),
4,7-dichlororhodamine X (dROX), rhodamine 6G (R6G),
4,7-dichlororhodamine 6G, rhodamine 110 (R110),
4,7-dichlororhodamine 110 (dR110), tetramethyl rhodamine (TAMRA)
and 4,7-dichloro-tetramethylrhodamine (dTAMRA). Additional suitable
rhodamine dyes include, for example, those described in U.S. Pat.
Nos. 6,248,884, 6,111,116, 6,080,852, 6,051,719, 6,025,505,
6,017,712, 5,936,087, 5,847,162, 5,840,999, 5,750,409, 5,366,860,
5,231,191, and 5,227,487; PCT Publications WO 97/36960 and WO
99/27020; Lee et al., NUCL. ACIDS RES. 20:2471-2483 (1992),
Arden-Jacob, NEUE LANWELLIGE XANTHEN-FARBSTOFFE FUR
FLUORESZENZSONDEN UND FARBSTOFF LASER, Verlag Shaker, Germany
(1993), Sauer et al., J. FLUORESCENCE 5:247-261 (1995), Lee et al.,
NUCL. ACIDS RES. 25:2816-2822 (1997), and Rosenblum et al., NUCL.
ACIDS RES. 25:4500-4504 (1997). A particularly preferred subset of
rhodamine dyes are 4,7-dichlororhodamines. In one embodiment, the
fluorescent moiety comprises a 4,7-dichloro-orthocarboxyrhodamine
dye.
[0051] In some embodiments, the fluorescent moiety comprises a
fluorescein dye. Exemplary suitable fluorescein include, but are
not limited to, fluorescein dyes described in U.S. Pat. Nos.
6,008,379, 5,840,999, 5,750,409, 5,654,442, 5,188,934, 5,066,580,
4,933,471,4,481,136 and 4,439,356; PCT Publication WO 99/16832, and
EPO Publication 050684. A preferred subset of fluorescein dyes are
4,7-dichlorofluoresceins. Other preferred fluorescein dyes include,
but are not limited to, 5-carboxyfluorescein (5-FAM) and
6-carboxyfluorescein (6-FAM). In one embodiment, the fluorescein
moiety comprises a 4,7-dichloro-orthocarboxyfluorescein dye.
[0052] In some embodiments, the fluorescent moiety can include a
cyanine, a phthalocyanine, a squaraine, or a bodipy dye, such as
those described in the following references and the references
cited therein: U.S. Pat. Nos. 6,080,868, 6,005,113, 5,945,526,
5,863,753, 5,863,727, 5,800,996, and 5,436,134; and PCT Publication
WO 96/04405.
[0053] In some embodiments, the fluorescent moiety can comprise a
network of dyes that operate cooperatively with one another such
as, for example by FRET or another mechanism, to provide large
Stoke's shifts. Such dye networks typically comprise a fluorescence
donor moiety and a fluorescence acceptor moiety, and may comprise
additional moieties that act as both fluorescence acceptors and
donors. The fluorescence donor and acceptor moieties can comprise
any of the previously described dyes, provided that dyes are
selected that can act cooperatively with one another. In a specific
embodiment, the fluorescent moiety comprises a fluorescence donor
moiety which comprises a fluorescein dye and a fluorescence
acceptor moiety which comprises a fluorescein or rhodamine dye.
Non-limiting examples of suitable dye pairs or networks are
described in U.S. Pat. Nos. 6,399,392, 6,232,075, 5,863,727, and
5,800,996.
[0054] In some embodiments, the label moiety comprises a quenching
moiety. The quenching moiety can be any moiety capable of quenching
the fluorescence of a fluorescent moiety when it is in close
proximity thereto, such as, for example, by orbital overlap
(formation of a ground state dark complex), collisional quenching,
FRET, or another mechanism or combination of mechanisms. The
quenching moiety can itself be fluorescent, or it can be
non-fluorescent. In some embodiments, the quenching moiety
comprises a fluorescent dye that has an absorbance spectrum that
sufficiently overlaps the emissions spectrum of a fluorescent
moiety such that it quenches the fluorescence of the fluorescent
moiety when in close proximity thereto.
[0055] 5.3.3 Reactive Moiety
[0056] In some embodiments, the labeling molecule comprises one or
more reactive moiety(ies). The reactive moiety can be used to
attach the label moiety to the protein near a target binding site.
The reactive moiety may be attached directly, or indirectly via one
or more optional linkers to the label moiety. The reactive moiety
can comprise any reactive group that is capable of forming a
covalent linkage with a corresponding reactive group on the
protein. Thus, the reactive moiety can comprise any reactive group
known in the art, so long as it is compatible with the methods and
compositions described herein. In embodiments employing two or more
reactive moieties, each reactive moiety can be the same, or some or
all of the reactive moieties may differ.
[0057] In some embodiments, the reactive moiety comprises a
cross-linker, i.e., a cross-linking group. Cross-linker reactivity,
specificity, and solubility characteristics are well known in the
art. Guidance for selecting appropriate cross-linking agents can be
found in Mattson et al., MOL BIOL REP. Apr; 17(3):167-83(1993),
Double-Agents.TM. Cross-linking reagents, Selection Guide, Pierce
Biotechnology Inc., 2003). See also, Wong, 1993, Chemistry of
Protein Conjugation and Cross-linking, CRC Press, Boca Raton.
[0058] In some embodiments, the reactive moiety comprises a
functional group that can be used to attach the reactive moiety to
the protein by forming a covalent linkage with a complementary
group present on the protein. Pairs of complementary groups capable
of forming covalent linkages are well known. In some embodiments,
the protein comprises a nucleophilic group and the cross-linking
group comprises an electrophilic group. In other embodiments, the
cross-linking group comprises a nucleophilic group and the protein
comprises an electrophilic group. "Complementary" nucleophilic and
electrophilic groups (or precursors thereof that can be suitable
activated) useful for effecting linkages stable to biological and
other assay conditions are well known and can be used. Examples of
suitable complementary nucleophilic and electrophilic groups, as
well as the resultant linkages formed therefrom, are provided in
Table 1. TABLE-US-00001 TABLE 1 Electrophilic Resultant Covalent
Group Nucleophilic Group Linkage activated esters* amines/anilines
Carboxamides acyl azides** amines/anilines Carboxamides acyl
halides amines/anilines Carboxamides acyl halides alcohols/phenols
Esters acyl nitriles alcohols/phenols Esters acyl nitriles
amines/anilines Carboxamides aldehydes amines/anilines Imines
aldehydes or hydrazines Hydrazones ketones aldehydes or
hydroxylamines Oximes ketones Alkyl halides amines/anilines alkyl
amines Alkyl halides carboxylic acids Esters Alkyl halides thiols
Thioethers Alkyl halides alcohols/phenols Ethers Alkyl sulfonates
thiols Thioethers Alkyl sulfonates carboxylic acids Esters Alkyl
sulfonates alcohols/phenols Esters anhydrides alcohols/phenols
Esters anhydrides amines/anilines Caroboxamides aryl halides thiols
Thiophenols aryl halides amines aryl amines aziridines thiols
Thioethers boronates glycols boronate esters carboxylic acids
amines/anilines Carboxamides carboxylic acids alcohols Esters
carboxylic acids hydrazines Hydrazides carbodiimides carboxylic
acids N-acylureas or anhydrides diazoalkanes carboxylic acids
Esters epoxides thiols Thioethers haloacetamides thiols Thioethers
halotriazines amines/anilines Aminotriazines halotriazines
alcohols/phenols triazinyl ethers imido esters amines/anilines
Amidines isocyanates amines/anilines Ureas isocyanates
alcohols/phenols Urethanes isothiocyanates amines/anilines
Thioureas maleimides Thiols Thioethers phosphoramidites Alcohols
Phosphate esters silyl halides Alcohols silyl ethers sulfonate
esters amines/anilines alkyl amines sulfonate esters Thiols
Thioethers sulfonate esters carboxylic acids Esters sulfonate
esters Alcohols Esters sulfonyl halides amines/anilines
Sulfonamides sulfonyl halides phenols/alcohols sulfonate esters
Diazonium salt aryl Azo *Activated esters, as understood in the
art, generally have the formula --C(O)Z, where Z is, a good leaving
group (e.g., oxysuccinimidyl, oxysulfosuccinimidyl,
1-oxybenzotriazolyl, etc.). **Acyl azides can rearrange to
isocyanates.
[0059] In some embodiments, the reactive moiety comprises a
cross-linker comprising a photoreactive group. A photoreactive
group has at least one latent photoreactive group that upon
activation by an external energy source, forms covalent bonds with
other molecules. See, e.g., U.S. Pat. No. 5,002,582, the disclosure
of which is incorporated herein by reference. For example, upon
exposure to a suitable light source, the photoreactive group can be
activated to form a covalent bond with an adjacent chemical
structure e.g. an aliphatic carbon-hydrogen bond.
[0060] Suitable photoreactive groups include, for example, azides,
diazos, diazirines, aromatic ketones, and quinones. Exemplary
photoreactive groups, and their residues upon activation, are shown
in Table 2. TABLE-US-00002 TABLE 2 Photoreactive Group Residue
Functionality aryl azides amine R--NH--R' acyl azides amide
R--CO--NH--R' azidoformates carbamate WO--CO--NH--R' sulfonyl
azides sulfonamide R-SO.sub.2--NH--R' phosphoryl azides
phosphoramide (RO).sub.2PO--NH--R' diazoalkanes new C--C bond
diazoketones new C--C bond and ketone diazoacetates new C--C bond
and ester beta-keto-alpha-diazoacetates new C--C bond and
beta-ketoester aliphatic azo new C--C bond diazirines new C--C bond
ketenes new C--C bond photoactivated ketones new C--C bond and
alcohol
[0061] In some embodiments, the photoreactive group comprises an
aryl ketone, such as, acetophenone, anthraquinone, anthrone and
anthrone-like heterocycles (i.e., heterocyclic analogues of
anthrone such as those having N, O, or S in the 10-position), or
their substituted (e.g., ring substituted) derivatives,
benzophenone, benzophenone maleimido, succinimidyl ester of
4-benzoylbenzoic acid.
[0062] The functional groups of some aryl ketones can undergo
multiple activation/inactivation/reactivation cycles. For example,
benzophenone is capable of photochemical excitation with the
initial formation of an excited singlet state that undergoes
intersystem crossing to the triplet state. The excited triplet
state can insert into carbon-hydrogen bonds by abstraction of a
hydrogen atom (from a polymeric coating layer, for example), thus
creating a radical pair. Subsequent collapse of the radical pair
leads to formation of a new carbon-carbon bond. If a reactive bond
(e.g., carbon/hydrogen) is not available for bonding, the
ultraviolet light-induced excitation of the benzophenone group is
reversible and the molecule returns to ground state energy level
upon removal of the energy source. Photoreactive aryl ketones such
as benzophenone and acetophenone can undergo multiple reactivations
in water and hence can provide increased linking efficiency.
[0063] In some embodiments the photoreactive group comprises an
azide, and includes arylazides (C.sub.6 R.sub.5N.sub.3) such as
phenyl azide, 4-fluoro-3-nitrophenyl azide, acyl azides
(--CO--N.sub.3) such as ethyl azidofomiate, phenyl azidoformate,
sulfonyl azides (--SO.sub.2--N.sub.3) such as benzenesulfonyl
azide, and phosphoryl azides (RO).sub.2PON.sub.3 such as diphenyl
phosphoryl azide and diethyl phosphoryl azide.
[0064] In some embodiments, the photoreactive group comprises a
diazo compounds, and includes diazoalkanes (--CHN.sub.2) such as
diazomethane and diphenyldiazomethane, diazoketones
(--CO--CHN.sub.2) such as diazoacetophenone and
1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates
(--CO--CN.sub.2--CO--O--) such as t-butyl alpha
diazoacetoacetate.
[0065] Other suitable photoreactive groups include aliphatic azo
compounds such as azobisisobutyronitrile, diazirines (--CHN.sub.2)
such as 3-trifluoromethyl-3-phenyldiazirine and ketenes
(--CH.dbd.C.dbd.O) such as ketene and diphenylketene, and
photoreactive groups including quinone such as, for example
anthraquinone.
[0066] Additional examples of suitable activatable reactive
moieties, includes, but are not limited to, molecules that can be
activated by pH, heat, or electrochemically such as peroxide.
[0067] In some embodiments, the reactive moiety comprises a
chemical group activated by a change in pH. Chemical groups
activated by a change in pH are well known and can be used in
forming covalent bonds. (see, e.g., Double-Agents.TM. Cross-linking
reagents, Selection Guide, Pierce Biotechnology Inc., 2003).
Suitable non-limiting examples include imidoesters, maleimide
groups, iodoacetyl group, and glyoxals.
[0068] Imidoesters can react with primary amines to form amidine
bonds at about pH 8-10. N-Hydroxysuccinimide-Esters (NHS-Esters)
can react with primary amines at physiological pH. The accessible
.alpha.-amine groups present on the N-termini of proteins and
.epsilon.-amines on lysine residues react with NHS-esters and form
amide bonds. Maleimide groups can react specifically with
sulfhydryl groups when the pH of the reaction mixture is between pH
6.5 and 7.5 and forms a stable thioether linkage. At neutral pH,
maleimides can react with sulfhydryls 1,000-fold faster than with
amines, but at pH>8.5, the reaction favors primary amines.
Iodoacetyl group can react with sulfhydryl groups at physiological
pH. The reaction of the iodoacetyl group with a sulfhydryl proceeds
by nucleophilic substitution of iodine with a thiol producing a
stable thioether linkage. Iodoacetyl groups can react with
imidazoles at a pH of about pH 6.9-7.0. Glyoxals can target
arginines at mildly alkaline pH. Glyoxals are useful compounds for
targeting the guanidinyl portion of arginine residues. There can be
cross-reactivity with lysines at about pH 9.
[0069] In some embodiments, the reactive moiety comprises a
chemical group that can be activated by heat. Chemical groups that
can be activated by heat are well known by those skilled in the
art. Suitable non-limiting examples of chemical groups that can be
activated by heat include peroxides, e.g., benzoyl peroxide, and
azo groups, e.g., azobisisobutryronitriles.
[0070] In some embodiments, the reactive moiety comprises a
chemical group that can be activated electrochemically. Chemical
groups that can be activated electrochemically are well known by
those skilled in the art. Suitable non-limiting examples of
chemical groups that can be activated electrochemically include
halobenzyl compounds, such as .alpha.-bromotoluene, and
organometallic compounds, such as phenyl mercury compounds.
[0071] In some embodiments, a linker moiety can be inserted between
the reactive groups to minimize steric interference with the
activity of the protein. The chemical composition of the linker
moiety is not critical. Any type of linker that permits the
resultant labeled protein to function as described herein can be
used. Suitable linker moieties are described below.
[0072] 5.3.4 Cleavable Tether
[0073] In some embodiments, the labeling molecule comprises a
cleavable tether. Generally, the cleavable tether connects the
target moiety to the label moiety. The cleavable tether comprises
at least one cleavable moiety and one or more optional linker
moieties. The cleavable moiety comprises at least one functional
group that can be cleaved to allow detachment of the target moiety
from the protein. The optional linker moieties typically comprise
one or more linkage groups that can be used to affect the
solubility of the labeling molecule and/or that function to link
the cleavable tether to the target moiety and the label moiety.
[0074] The cleavable moiety can comprise any number of functional
groups. For example, the cleavable moiety can comprise a functional
group that can be cleaved by a selected cleaving agent when the
cleavable tether is bound to, or interacting with, the labeled
protein. As another example, the cleavable moiety can comprises a
functional group that can be cleaved under selected cleaving
conditions, or by a selected chemical reaction. Thus, cleavable
moieties can include functional groups that can be photolytically,
chemically, thermally or enzymatically cleaved. See, e.g., U.S.
Pat. No. 5,721,099, U.S. Pat. Pub. No. 2004/0166529, and Greene et
al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 2nd ed. Wiley, 1991,
and U.S. patent application Ser. No. 10/828,647.
[0075] In some embodiments, the cleavable moiety comprises silyl
groups that can be cleaved with halogens, such as fluoride, bromine
or chlorine, by oxidation or acid.
[0076] In other embodiments, the cleavable moiety can comprise
photolabile linkages, such as o-nitrobenzyl, 7-nitroindanyl,
2-nitrobenzhydryl ethers or esters, etc., that can be cleaved with
electromagnetic radiation.
[0077] Other examples of cleavable moieties are known to those
skilled in the art. For example, catechols, which can be cleaved
with cerium salts, can be used as cleavable moieties. Olefins,
which can be cleaved with ozone, permanganate or osmium tetroxide,
can be used as cleavable moieties. Sulfides, which can be cleaved
with singlet oxygen or by enzyme catalyzed oxidative cleavage with
hydrogen peroxide, where the resulting sulfone can undergo
elimination, can be used as cleavable moieties. Furans, which can
be cleaved with oxygen or bromine in methanol, can be used as
cleavable moieties. Tertiary alcohols ketals and acetals, which can
be cleaved with acid, can be used as cleavable moieties. Alpha- and
beta-substituted ethers and esters, which can be cleaved with base,
where the substituent is an electron withdrawing group, e.g.,
sulfone, sulfoxide, ketone, etc., and the like, can be used as
cleavable moieties. Substituted benzyl ether or derivatives
thereof, e.g. benzhydryl ether, indanyl ether, etc., which may be
cleaved by acidic or mild reductive conditions, can be used as
cleavable moieties.
[0078] In some embodiments, two or more cleavable moieties are
used. In these embodiments, each cleavable moiety can be the same,
or some, or all, of the cleavable moieties may differ.
[0079] In some embodiments, the cleavable tether comprises one or
more optional linker moieties. The linker moieties can comprise any
linkage group capable of connecting the cleavable moiety to another
moiety in the labeling molecule. In embodiments employing two or
more linker moieties, each of the linker moieties can be the same,
or some, or all, of the linker moieties may differ.
[0080] In some embodiments, the linker moiety comprises one or more
(bis)ethylene glycol group(s). As will be appreciated by a person
skilled in the art, the number of oxyethylene units comprising the
linker moiety can be selectively varied. For example, one, two,
three or more oxyethylene units may be used to form a linker
moiety. Virtually any combination of the same or different
oxyethylene units that permits the cleavable tether to function as
described herein may be used. In a specific example, the linker
moiety may comprise from 1 to about 5 of the same or different
lower oxyethylene units (e.g., --(CH.sub.2).sub.xCH.sub.2--), where
x is an integer ranging from 0 to 6). The chemical composition of
the linker moiety is not critical. Any type of linker moiety that
permits the resultant labeling molecule to function as described
herein can be used.
[0081] A linker moiety can be selected to have specified
properties. For example, the linker moiety can be hydrophobic in
character, hydrophilic in character, long or short, rigid,
semirigid or flexible, depending upon the particular application.
The linker moiety can be optionally substituted with one or more
substituents or one or more linking groups for the attachment of
additional substituents, which may be the same or different,
thereby providing a "polyvalent" linking moiety capable of
conjugating or linking additional molecules or substances to the
labeling molecule. In certain embodiments, however, the linker
moiety does not comprise such additional substituents or linking
groups.
[0082] A wide variety of linker moieties comprised of stable bonds
are known in the art, and include by way of example and not
limitation, alkyldiyls, substituted alkyldiyls, alkylenos (e.g.,
alkanos), substituted alkylenos, heteroalkyldiyls, substituted
heteroalkyldiyls, heteroalkylenos, substituted heteroalkylenos,
acyclic heteroatomic bridges, aryldiyls, substituted aryldiyls,
arylaryldiyls, substituted arylaryldiyls, arylalkyldiyls,
substituted arylalkyldiyls, heteroaryldiyls, substituted
heteroaryldiyls, heteroaryl-heteroaryl diyls, substituted
heteroaryl-heteroaryl diyls, heteroarylalkyldiyls, substituted
heteroarylalkyldiyls, heteroaryl-heteroalkyldiyls, substituted
heteroaryl-heteroalkyldiyls, and the like. Thus, a linker moiety
can include single, double, triple or aromatic carbon-carbon bonds,
nitrogen-nitrogen bonds, carbon-nitrogen bonds, carbon-oxygen
bonds, carbon-sulfur bonds and combinations of such bonds, and may
therefore include functionalities such as carbonyls, ethers,
thioethers, carboxamides, sulfonamides, ureas, urethanes,
hydrazines, etc. In some embodiments, the linker moiety has from
1-20 non-hydrogen atoms selected from the group consisting of C, N,
O, P, and S and is composed of any combination of ether, thioether,
amine, ester, carboxamide, sulfonamides, hydrazide, aromatic and
heteroaromatic groups.
[0083] Choosing a linker moiety having properties suitable for a
particular application is within the capabilities of those having
skill in the art. For example, where a rigid linker moiety is
desired, the linker moiety may comprise a rigid polypeptide such as
polyproline, a rigid polyunsaturated alkyldiyl or an aryldiyl,
biaryldiyl, arylarydiyl, arylalkyldiyl, heteroaryldiyl,
biheteroaryldiyl, heteroarylalkyldiyl, heteroaryl-heteroaryldiyl,
etc. Where a flexible linker moiety is desired, the linker moiety
may comprise a flexible polypeptide such as polyglycine or a
flexible saturated alkanyldiyl or heteroalkanyldiyl. Hydrophilic
linker moieties may comprise, for example, polyalcohols,
polyethers, such as polyalkyleneglycols, or polyelectroyles, such
as polyquaternary amines. Hydrophobic linker moieties may comprise,
for example, alkyldiyls or aryldiyls.
[0084] In some embodiments, the linker moiety comprises a peptide
bond. Skilled artisans will appreciate that while using peptide
bonds may be convenient, the various moieties comprising the
labeling molecule can be linked to one another via any linkage that
is stable to the conditions under which the labeling molecule will
be used.
[0085] In some embodiments, the cleavable tether comprises the
structure: ##STR5##
[0086] wherein: [0087] Y and/or X is an oxygen atom, [0088] R.sup.1
and R.sup.2 are hydrocarbons; [0089] L represents optional linker
moieties; and [0090] n is an integer from 0 to 5.
[0091] In the embodiment illustrated above, when Y or X is an
oxygen atom, the other moiety can comprise an oxygen atom or a
carbon atom. For example, when Y is an oxygen atom, X can be a
carbon atom. As another example, when X is an oxygen atom, Y can be
a carbon atom. In yet another example, both Y and X can be oxygen
atoms.
[0092] In some embodiments, the cleavable tether comprises the
structure: ##STR6##
[0093] wherein R.sup.1 and R.sup.2 are hydrocarbons.
[0094] The cleavable tether can be of any length provided, that
upon cleavage of the cleavable moiety, the labeling moiety is
positioned in close proximity to the analyte binding site at a
location that does not interfere with target binding. Choosing a
cleavable tether having properties suitable for a particular
application is within the capabilities of those having skill in the
art. For example, competitive binding assays can be performed with
proteins labeled with labeling molecules comprising cleavable
tethers of various lengths to determine the optimal length of the
cleavable tether such that the label moiety does not substantially
interfere with target binding.
[0095] In some embodiments the cleavable tether can be from 5 to
about 20 Angstroms.
[0096] The various moieties comprising the labeling molecule can be
connected to each other in any way that permits them to perform
their respective functions. In some, embodiments the target moiety,
label moiety, reactive moiety, and cleavable tether can be directly
linked to each other i.e., covalently linked to each other. In
some, embodiments, one, some, or all of the moieties can be
connected indirectly to one another, i.e., via one or more optional
linkers. Any of the linkers previously described can be used to
link the various moieties to one another.
[0097] In some embodiments, the cleavable tether can be linked
directly to the target moiety. In some embodiments, the cleavable
tether can be linked indirectly to the target moiety via an
optional linker. In some embodiments, the cleavable tether is
linked directly to the label moiety. In some embodiments, the
cleavable tether is linked indirectly to the label moiety via an
optional linker. In some embodiments, the cleavable tether is
linked directly to the reactive moiety. In some embodiments, the
cleavable tether is linked indirectly to the reactive moiety via an
optional linker.
[0098] In some embodiments, the target moiety, label moiety,
reactive moiety, and cleavable tether can be linked to one another
via a multivalent linker. Multivalent linkers can be any molecule
having two, three, four, or more attachment sites suitable for
attaching other molecules and moieties thereto, or that can be
appropriately activated to attach other molecules and moieties
thereto. For example, the backbone of the linker to which the
reactive (or activatable) linking groups are attached could be a
linear, branched or cyclic saturated or unsaturated alkyl, a mono
or polycyclic aryl or an arylalkyl. The linker need not be limited
to carbon and hydrogen atoms. Indeed, the linker can comprise
single, double, triple or aromatic carbon-carbon bonds,
carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen
bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and
combinations thereof, and therefore can comprise functionalities
such as carbonyls, ethers, thioethers, carboxamides, sulfonamides,
ureas, urethanes, hydrazines, etc.
[0099] The functional groups on the multivalent linker can be any
member of a pair of complementary reactive groups capable of
forming covalent linkages. In some embodiments, each reactive group
comprising the multifunctional linker is an electrophilic group or
a nucleophilic group that is capable of reacting with a
complementary nucleophilic group or electrophilic group to form a
covalent linkage stable to biological assay conditions
[0100] The reactive groups on the multivalent linker may all be the
same, or some or all of them may be different. In some embodiments,
reactive groups are selected that have different chemical
reactivities to facilitate the selective attachment of the various
moieties described herein, to the linker.
[0101] In some embodiments the multivalent linker can be a
trifunctional linker. In some embodiments, the trifunctional linker
is an amino acid, which may be an alpha amino acid, a beta amino
acid, a gamma amino or other type of amino acid that comprises a
side chain having a suitable reactive functional group. Specific
examples of suitable amino acids comprise, but are not limited to,
lysine, glutamate, cysteine, serine, homoserine and
1,3-diaminobutyric acid. These amino acids may be in either the D-
or L-configuration, or may constitute racemic or other mixtures
thereof.
[0102] In some embodiments, the labeling molecule comprises the
structure:
TM-(L.sup.1).sub.n-CT-(L.sup.2).sub.n-LM-(L.sup.3).sub.n-RM
[0103] wherein:
[0104] TM represents the target moiety;
[0105] CT represents the cleavable tether;
[0106] LM represents the label moiety;
[0107] RM represents the reactive moiety;
[0108] L.sup.1-L.sup.3 represents optional linkers; and
[0109] n is an integer form 0 to 5
[0110] L.sup.1, L.sup.2, and L.sup.3 can comprise any of the
optional linkers discussed above. In some embodiments, the linkers
can be the same. In some embodiments, the linkers can be different.
In some embodiments, some of the linkers can be the same and others
different.
[0111] In some embodiments, the labeling molecule comprises the
structure: ##STR7##
[0112] wherein:
[0113] TM represents the target moiety, comprising a cAMP
antigen;
[0114] CT represents the cleavable tether, comprising silyl
cleavable moiety and two
[0115] optional alkyl linkers;
[0116] LM represents the label moiety comprising a
carboxyfluorescein dye (FAM);
[0117] RM represents a photoactive reactive moiety; and
[0118] L.sup.1-L.sup.3 represents optional linkers.
[0119] 5.3.5 Specifically Labeled Proteins
[0120] Also, provided herein are proteins, capable of binding a
target analyte of interest, that can be specifically labeled in
close proximity to the target analyte binding site, and are
substantially unlabeled at other positions. The protein can be any
protein, or fragment thereof, that is operative in accordance with
the various compositions and methods described herein. For example,
in some embodiments, the protein can be an antibody capable of
binding a target antigen comprising an epitope. In other
embodiments, the protein can be an enzyme capable of binding a
target substrate. In yet other embodiments, the protein can be a
receptor capable of binding a target ligand. In still other
embodiments, the protein can be a signal transduction protein
capable of binding a target ligand.
[0121] The proteins can be specifically labeled with one or more
label moiety(ies). Any of the label moieties previously described
can be included on the protein. In embodiments employing two or
more label moieties, each label moiety can be the same, or some or
all of the label moieties can differ. For example, in some
embodiments the protein can comprise a fluorescent moiety. The
fluorescent moiety can comprise any entity that provides a
fluorescent signal and that can be used in accordance with the
methods and principles described herein. In some embodiments, the
protein comprises a quenching moiety capable of quenching the
fluorescence of a fluorescent moiety.
[0122] The exact distance between the label moiety and the target
binding site is not critical and can vary over a broad range. The
only requirement is that the label moiety be sufficiently distant
from the binding site of the target, so as to not substantially
block the analyte and surrogate analyte from binding to the binding
site on the protein. In some embodiments, the label can be about 3
to 50 Angstroms from the binding site. In some embodiments, the
label can be about 5 to 30 Angstroms from the binding site. In some
embodiments, the label can be about 5 to 20 Angstroms from the
binding site.
[0123] Methods for making labeled proteins generally comprise
contacting the protein with a labeling molecule comprising a target
moiety, a label moiety, a reactive moiety, and a cleavable tether.
The target moiety, label moiety, and reactive moiety are connected
to one another such that the target moiety can bind the protein,
and the reactive moiety can link the label moiety to the protein.
Agents capable of activating the reactive moiety are added to form
a covalent linkage between the reactive moiety and the protein. The
target moiety can be detached from the protein by a) inducing
cleavage of the cleavable tether by a suitable agent or condition
as described above, and b) providing conditions that effectively
remove the target moiety from the vicinity of the protein, such as
by dilution, or by the addition of a surrogate analyte or
analyte.
[0124] FIG. 1 illustrates an exemplary embodiment of a method of
making a functionally labeled antibody 100 with a labeling molecule
110. FIG. 1A illustrates contacting the antibody 100 with a
labeling molecule 110 comprising an antigen moiety AgM, a label
moiety LM, a reactive moiety RM, and a cleavable tether CT
connected to the antigen moiety AgM. As will be appreciated by a
person skilled in the art, the arrangement of various moieties
illustrated in label molecule 110 provided in FIG. 1 is merely
exemplary, and the various moieties can be connected to each other
in any way that permits them to perform their respective functions.
FIG. 1B illustrates contacting the antibody 100 with a labeling
molecule 110. FIG. 1C illustrates activating the reactive moiety
*RM so as to form a covalent linkage between the activated reactive
moiety *RM and the antibody 100. FIG. 1D. illustrates cleaving the
cleavable tether *CT. FIG. 1E illustrates the cleavage products
following cleavage of the cleavable tether, e.g., the labeled
antibody and the antigen moiety AgM.
[0125] In some embodiments, the method further comprises isolating
the labeled protein from a cleavage product comprising the target
moiety. Methods for isolating proteins from cleavage products are
known by those skilled in the art.
[0126] Typically, proteins suitable for labeling comprise at least
one binding domain for a target analyte and a reactive group
capable of reacting with the reactive moiety. Non-limiting examples
of suitable proteins, label moieties, reactive moieties, and
cleavable tethers are described above.
[0127] 5.3.6 Surrogate Analyte-Labeled Protein Complex
[0128] Also provided herein are surrogate analyte-protein complexes
useful for, among other things, as reporter systems for analyte
detection. The surrogate analyte-protein complex comprises a
labeled protein and labeled surrogate analyte. FIG. 2A illustrates
an exemplary surrogate analyte-protein complex wherein the
surrogate analyte is a surrogate antigen complexed with a labeled
protein, i.e., antibody. In FIG. 2A the antibody is labeled with a
fluorescent moiety F and the surrogate antigen sA is labeled with a
quenching moiety Q. The labeled F antibody and surrogate antigen sA
form a surrogate antigen-antibody complex wherein the quenching
moiety Q is positioned so that it is able to quench the
fluorescence of the fluorescent moiety F on the antibody. The
surrogate antigen-antibody complex is capable of producing a
detectable increase in fluorescence upon dissociation of the
complex caused by the competitive binding of antigen A to the
antibody and the displacement of surrogate antigen sA.
[0129] The surrogate analyte can be any labeled organic or
inorganic molecule capable of competing with the target of interest
(i.e. analyte) for binding the labeled protein and capable of
comprising a linker that can be optionally attached to a quenching
moiety pr a fluorescent moiety. Non-limiting examples of suitable
molecules that can comprise the surrogate analyte include antigens,
enzyme substrates, proteins, peptides, carbohydrates,
polysaccharides, glycoproteins, hormones, receptors, virus,
metabolites, transition state analogs, cofactors, inhibitors,
drugs, nutrients, electrolytes growth factors and other
biomolecules as well as non-biomolecules capable of binding the
labeled protein.
[0130] In some embodiments the surrogate analyte comprises
cAMP.
[0131] The surrogate analyte comprises one or more label
moiety(ies). Any of the label moieties previously described can be
used to label the surrogate analyte. In embodiments employing two
or more label moieties, each label moiety can be the same, or some
or all of the label moieties can differ.
[0132] In some embodiments, the labeled protein comprises a
fluorescent moiety and the surrogate analyte comprises a quenching
moiety. The fluorescence signal of the fluorescent moiety can be
quenched when the surrogate analyte is bound to the labeled
protein. The binding of a target analyte to the labeled protein can
displace the surrogate analyte and reduce or eliminate the
quenching effect, thereby producing a detectable increase in
fluorescence.
[0133] In some embodiments, the labeled protein comprises a
quenching moiety and the surrogate analyte comprises a fluorescent
moiety. The fluorescence signal of the fluorescent moiety can
quenched when the surrogate analyte is bound to the labeled
protein. The binding of a target analyte to the labeled protein can
displace the surrogate analyte and reduce or eliminate the
quenching effect, thereby producing a detectable increase in
fluorescence.
[0134] In some embodiments, surrogate analyte-protein complexes are
encapsulated in a particle. Encapsulated surrogate analyte-protein
complexes can be used for the detection of intracellular analyte
molecules, to increase the local concentration of the
surrogate-analyte-protein complexes and/or to sequester the
surrogate analyte-protein complexes from agents that would
otherwise degrade or inactivate the surrogate analyte-protein
complexes.
[0135] The surrogate analyte-protein complexes can be encapsulated
in nanocapsules, liposomes, microparticles, lipid particles,
vesicles, and the like. The particles used to encapsulate the
surrogate analyte-protein complexes can be permeable,
semi-permeable or impermeable. In some embodiments, the surrogate
analyte-protein complex can be encapsulated in semi-permeable
nanocapsule having a diameter from 10 to 1000 nanometers. In some
embodiments, the porosity of the membrane comprising the particle
used to encapsulate the surrogate analyte and the labeled protein
is selected to retain the surrogate analyte-labeled protein
complex, and at the same time, allow passage of the target analyte.
Thus, the porosity of the particle membrane is such that it allows
passage of elements that are less than or equal to 0.5 nm to 5.0 nm
in diameter. In some embodiments, the pore diameter of the
particles is less than or equal to 5.0 nm. In some embodiments, the
pore diameter of the particles is less than or equal to 2.0 nm. In
some embodiments, the pore diameter of the particles is less than
or equal to 1.5 nm. In some embodiments, the pore diameter of the
particles is less than or equal to 1.0 nm. In some embodiments, the
pore diameter of the particles is less than or equal to or equal to
0.5 nm.
[0136] In some embodiments, the nanocapsule membrane comprises a
cross-linked water soluble vinyl polymer or copolymer. Suitable
types of vinyl polymer or copolymer include, but are not limited
to, polyvinyl pyrrolidone, polyacrylic acid, polyacrylamide,
poly-N,N-dimethylacrylamide, and polyhydroxyethylmethacryate.
[0137] The pore diameter of the nanocapsule can be designed to
retain the surrogate analyte-protein complex, the labeled protein,
and/or the labeled protein-target analyte complex and allow the
surrogate analyte or the target analyte to pass through the
nanocapsule. For example, in some, embodiments, the nanocapsule is
permeable to a target analyte and/or a surrogate analyte and is
impermeable to a labeled antibody or fragment thereof. In some
embodiments, the nanocapsule is permeable to a target analyte
and/or a surrogate analyte and is impermeable to a labeled enzyme
or fragment thereof. In some, embodiments, the nanocapsule is
permeable to a target analyte and/or a surrogate analyte and is
impermeable to a receptor or fragment thereof.
[0138] In some embodiments, a targeting moiety can be attached to
the nanocapsule and used, for example, to target the nanocapsule to
a particular cell or collection of cells. As used herein,
"targeting moiety" includes any chemical moiety capable of binding
to, or otherwise transporting through, a particular type of
membrane and/or organelle in a cell, tissue, or organ. A variety of
agents that direct compositions to particular cells are known in
the art (see, for example, Cotten et al., Methods Enzym, 217: 618,
1993), and U.S. Pat. Nos. 6,692,911, 6,835,393). Suitable
non-limiting examples of targeting moieties include proteins (such
as insulin, EGF, or transferrin), lectins, antibodies and
fragments, carbohydrates, lipids, oligonucleotides, DNA, RNA, or
small molecules and drugs. Additional examples, of useful targeting
moieties include, but are in no way limited to, transfection agents
such as Pro-Ject (Pierce Biotechnology), viral peptide fragments
such as transportans, pore forming toxins such as streptolysin-O,
hydrophobic esters, polycations such as polylysine,
asiaglycoproteins, and diphtheria toxin.
[0139] In some embodiments, the surrogate analyte-protein complexes
or the labeled proteins are immobilized on, or attached to, a
substrate, such as a solid support or a solid surface. The solid
support can be any material known to those of ordinary skill in the
art that can be utilized in the assays systems described herein. In
general, the support will be amenable to the detection system of
choice (e.g. fluorescence when fluors are used as the label.).
Suitable solid support include metal surfaces such as gold, glass
and modified or functionalized glass, fiberglass, teflon, ceramics,
mica, plastic (including acrylics, polystyrene and copolymers of
styrene and other materials, polypropylene, polyethylene,
polybutylene, polyimide, polycarbonate, polyurethanes, Teflon.TM.,
and derivatives thereof, etc.), GETEK (a blend of polypropylene
oxide and fiberglass), etc, polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses and
a variety of other polymers. In some embodiments, the solid support
can allow high throughput screening, such as microtiter plates and
beads (sometimes referred to herein as microspheres). The
composition of the beads will vary, depending on the use. Suitable
bead compositions include those used in peptide, nucleic acid and
organic moiety synthesis, including, but not limited to, plastics,
ceramics, glass, polystyrene, methylstyrene, acrylic polymers,
paramagnetic materials, thoria sol, carbon graphite, titanium
dioxide, latex or cross-linked dextrans such as Sepharose,
cellulose, nylon, cross-linked micelles and Teflon may all be
used.
[0140] Methods for coupling molecules to a solid support are well
known in the art and have been widely used in the making of
affinity columns, ELISA assay plates, support-bound peptide and
drug candidate libraries and polynucleotide arrays. See, for
example, Sigel et al., FEBS LETT. 147: 45-48 (1982). Any of the
various chemistries and methodologies can be used to immobilize the
surrogate analyte or labeled protein. The surrogate analyte or
labeled protein can be stably attached to a solid substrate by
covalent and/or non-covalent interactions. For instance, the
surrogate analyte or labeled protein can be covalently deposited to
the surface of a solid support via cross-linking agents, such as
glutaraldehyde, borohydride, or other bifunctional agents. The
surrogate analyte or labeled protein may also be covalently linked
to the substrate via an alkylamino-linker group or a polymer
linker. The coupling methods should not substantially affect the
binding specificity and/or affinity between the target and the
surrogate analyte or labeled protein.
[0141] 5.3.7 Assays
[0142] Also provided herein are assays for detecting the presence
or absence of a target analyte in a sample. The sample to be tested
can be any suitable sample selected by the user. The sample can be
naturally occurring or man-made. For example, the sample can be a
blood sample, tissue sample, cell sample, buccal sample, skin
sample, urine sample, water sample, or soil sample. The sample can
be from a living organism, such as a eukaryote, prokaryote, mammal,
human, yeast, or bacterium. The sample can be a cell, tissue, or
organ. The sample can be processed prior to contact with a
surrogate analyte-protein complex or labeled protein of the present
teachings by any method known in the art. For example, the sample
can be subjected to a lysing step, precipitation step, column
chromatography step, heat step, etc.
[0143] The assays comprise contacting a sample with a "reporter
system" comprising a surrogate analyte-protein complex or a labeled
protein as described herein. In some embodiments, the reporter
system can be encapsulated. In other embodiments, the reporter
system can be attached to a solid support.
[0144] FIG. 3 illustrates an exemplary reporter system comprising
one or more surrogate analyte-protein complexes, each comprising a
labeled protein ("reporter labeled antibody") and a surrogate
analyte ("quencher labeled antigen"), encapsulated in a
semipermeable capsule ("hollow shell") to which can be attached
targeting moieties ("cell membrane transporting agent"). The
targeting moieties can be used to introduce the capsules comprising
the reporter system into a cell of interest. As illustrated in FIG.
3, binding of the surrogate analyte to the labeled antibody
quenches the signal from the "reporter". The "reporter" depicted in
FIG. 3 can comprise any of the label moieties described herein.
Passage of one or more target analytes into the capsule can
displace one or more surrogate analytes, generating a measurable
increase in fluorescence and indicating the presence of the target
analyte.
[0145] The assays typically comprise contacting a reporter system
with a sample comprising one or more target analytes of interest.
In embodiments employing two or more target analytes, each labeled
protein comprising the reporter system can be the same, or some, or
all of the labeled proteins can differ.
[0146] The assays taught herein typically comprise the use of a
buffer, such as a buffer described in the "Biological Buffers"
section of the 2003 Sigma-Aldrich Catalog. Exemplary buffers
include sodium phosphate, sodium acetate, PBS, MES, MOPS, HEPES,
Tris (Trizma), bicine, TAPS, CAPS, and the like. The buffer is
present in an amount sufficient to generate and maintain a desired
pH and/or ionic strength. The pH of the binding buffer can be
selected according to the pH dependency of the binding activity.
For example, the pH can be from 2 to 12, from 4 to 11, or from 6 to
10. The buffer may also contain any necessary cofactors or agents
required for binding. The identities and concentration of such
cofactors and/or agents will depend upon the particular assay
system and will be apparent to those of skill in the art. The
concentration of the labeled proteins present in a reporter system
may vary substantially. For example, the assay buffer can comprise
from about 10.sup.-10 to 10.sup.-3 labeled proteins. In some
embodiments, the assay buffer comprises from about 1 pM to 1 .mu.M
labeled proteins. If a plurality of different types of labeled
proteins are used, each may comprise in the assay buffer in the
above concentration ranges.
[0147] The assays typically do not require the presence of
detergents or other components. In general, it is desirable to
avoid high concentrations of components in the reaction mixture
that can adversely affect the fluorescence properties of the
reaction product, or that can interfere with the detection of
target analytes.
[0148] The fluorescence signal can be monitored using conventional
methods and instruments. For example, the surrogate analyte-protein
complexes of the present teachings can be used in a continuous
monitoring phase, in real time, to allow the user to rapidly
determine whether an analyte is present in the sample, and
optionally, the amount or activity of the analyte. In some
embodiments, the fluorescence signal can be measured from at least
two different time points. In some embodiments, the signal can be
monitored continuously or at several selected time points.
Alternatively, the fluorescence signal can be measured in an
end-point embodiment in which a signal is measured after a certain
amount of time, and the signal is compared against a control signal
(sample without analyte), threshold signal, or standard curve.
[0149] The amount of the fluorescence signal generated is not
critical and can vary over a broad range. The only requirement is
that the fluorescence be measurable by the detection system being
used. In some embodiments, a fluorescence signal that is at least
2-fold greater than the background signal can be generated upon
dissociation of the surrogate analyte-protein complex. In some
embodiments, a fluorescence signal that is at least 3-fold greater
than the background signal can be generated upon dissociation of
the surrogate analyte-protein complex. In some embodiments, a
fluorescence signal that is at least 4-fold greater than the
background signal can be generated upon dissociation of the
surrogate analyte-protein complex. In some embodiments, a
fluorescence signal that is at least 5-fold greater than the
background signal can be generated upon dissociation of the
surrogate analyte-protein complex. In some embodiments, a
fluorescence signal between 2 to 10-fold greater than the
background signal can be generated upon dissociation of the
surrogate analyte-protein complex.
[0150] 5.3.8 Kits
[0151] Also provided are kits for performing the methods of the
present teachings. In some embodiments, the kits comprise a
labeling molecule comprising a target moiety, a label moiety, a
reactive moiety, and a cleavable tether. In some embodiments, the
kits can contain a buffer for preparing a reaction mixture that
facilitates the protein labeling. In some embodiments, the kit can
further comprise chemicals for the activation of the reactive
moiety and/or cleaving of the cleavable tether. These other
components can be provided separately from each other, or mixed
together in dry or liquid form.
[0152] In some embodiments, the kit comprises surrogate
analyte-protein complex useful for detecting the presence or
absence of a target analyte. In some embodiments, the kit comprises
an encapsulated surrogate analyte-protein complex. In some
embodiments, the kit comprises a surrogate analyte-protein complex
that is attached to a solid support. In some embodiments, the kit
comprises a surrogate analyte that is attached to a solid support.
In some embodiments, the kit comprises a labeled protein that is
attached to a solid support.
[0153] All publications and patent applications mentioned herein
are hereby incorporated by reference as if each publication or
patent application was specifically and individually indicated to
be incorporated by reference. The section headings used herein are
for organizational purposes only and are not to be construed as
limiting the subject matter described in any way. While the present
teachings are described in conjunction with various embodiments, it
is not intended that the present teachings be limited to such
embodiments. On the contrary, the present teachings encompass
various alternatives, modifications, and equivalents, as will be
appreciated by those skilled in the art.
* * * * *