U.S. patent application number 10/092068 was filed with the patent office on 2002-09-12 for methods and systems for monitoring intracellular binding reactions.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Farinas, Javier A., Nikiforov, Theo T., Wada, H. Garrett.
Application Number | 20020127591 10/092068 |
Document ID | / |
Family ID | 22638302 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127591 |
Kind Code |
A1 |
Wada, H. Garrett ; et
al. |
September 12, 2002 |
Methods and systems for monitoring intracellular binding
reactions
Abstract
Intracellular binding reactions, and particularly DNA/DNA
binding protein reactions are detected in situ, using intracellular
fluorescence polarization detection. The methods comprise providing
a biological cell having at least a first component of a binding
reaction disposed therein. The cell is contacted with a second
component of the binding reaction whereby the second component is
internalized within the biological cell. At least one of the first
and second components has a fluorescent label. The amount of
binding between the first and second components within the cell is
determined by measuring a level of polarized and/or depolarized
fluorescence emitted from within the biological cell.
Inventors: |
Wada, H. Garrett; (Atherton,
CA) ; Farinas, Javier A.; (Los Altos, CA) ;
Nikiforov, Theo T.; (San Jose, CA) |
Correspondence
Address: |
CALIPER TECHNOLOGIES CORP
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043
US
|
Assignee: |
Caliper Technologies Corp.
Mountain View
CA
|
Family ID: |
22638302 |
Appl. No.: |
10/092068 |
Filed: |
March 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10092068 |
Mar 5, 2002 |
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09750638 |
Dec 28, 2000 |
|
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6379884 |
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60174976 |
Jan 6, 2000 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/7.1 |
Current CPC
Class: |
G01N 33/5041 20130101;
G01N 21/6458 20130101; C12Q 2561/119 20130101; G01N 33/542
20130101; G01N 33/6872 20130101; C12Q 1/6841 20130101; G01N 33/5008
20130101; G01N 21/6428 20130101; C12Q 1/6841 20130101; G01N 33/582
20130101; G01N 21/6445 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 435/7.1 |
International
Class: |
C12Q 001/68; G01N
033/53; C12M 001/34 |
Claims
What is claimed is:
1. A system for monitoring intracellular binding interactions,
comprising: a reaction vessel having disposed therein a cell
suspension comprising biological cells having at least a first
component of a binding reaction disposed within the cells, and a
second component of the binding reaction comprising a non-protein
molecule and having a fluorescent label associated therewith; and a
detector in sensory communication with contents of the reaction
vessel, the detector being configured to detect an amount of
polarized fluorescence emitted from the reaction vessel.
2. The system of claim 1, wherein the reaction vessel comprises a
well in a multiwell plate.
3. The system of claim 1, wherein the reaction vessel comprises a
microfluidic channel.
4. The system of claim 1, wherein the second component of the
binding reaction comprises a binding fragment of a full length
protein that is capable of binding the first component.
5. The system of claim 4, wherein the second component is between
about 4 and 100 amino acid residues in length.
6. The system of claim 4, wherein the second component is between
about 4 and about 50 residues in length.
7. The system of claim 4, wherein the second component comprises a
molecular weight that is less than about 10 kD.
8. The system of claim 4, wherein the second component comprises a
molecular weight that is less than about 5 kD.
9. The system of claim 4, wherein the second component comprises a
carbohydrate, a lipid, cAMP, cGMP or diacylglycerol.
10. The system of claim 1, wherein the first component of the
binding reaction comprises an intracellular nucleic acid binding
protein and the second component comprises a nucleic acid
probe.
11. The system of claim 10, wherein the nucleic acid probe is from
about 5 to about 100 bases in length.
12. The system of claim 10, wherein the nucleic acid probe is from
about 10 to about 50 bases in length.
13. The system of claim 10, wherein the first component comprises a
DNA binding protein and the second component comprises a
fluorescently labeled DNA probe.
14. The system of claim 10, wherein the nucleic acid probe
comprises a translocation functionality.
15. The system of claim 14, wherein the translocation functionality
comprises a translocating peptide.
16. The system of claim 15, wherein the translocating peptide
comprises Antp-HD or a fragment thereof.
17. The system of claim 15, wherein the translocating peptide
comprises a polypeptide that includes a sequence homologous to
residues 48-60 of an HIV-1 tat protein (SEQ ID NO:1).
18. The system of claim 10, wherein the nucleic acid binding
protein is a component of a cell signaling pathway, activation of
the pathway activating or deactivating the nucleic acid binding
protein.
19. The system of claim 1, wherein the cell is selected from a
mammalian cell, bacterial cell, fungal cell, yeast cell, insect
cell, and a plant cell.
20. The system of claim 19, wherein the cell is a mammalian cell
that is selected from a CHO cell, a HEK-293 cell, a L-cell, a 3T3
cell, a COS cell, a THP-1 cell, a RBL-1 cell, a YB-1 cell, a Jurkat
cell and a U937 cell.
21. The system of claim 1, wherein the cell is disposed in a
suspension of cells.
22. The system of claim 1, wherein the reaction vessel comprises a
window providing optical access.
23. The system of claim 22, wherein the reaction vessel comprises a
test tube.
24. The system of claim 22, wherein the reaction vessel comprises a
cuvette.
25. The system of claim 22, wherein the reaction vessel comprises a
well in a multiwell plate.
26. The system of claim 22, wherein the reaction vessel comprises
at least a first fluidic channel.
27. The system of claim 26, wherein the first fluidic channel
comprises at least a first microscale fluidic channel disposed
within a body structure.
28. The system of claim 27, wherein the microscale fluidic channel
comprises a first of at least two intersecting microscale channels
disposed in the body structure.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
No. 09/750,638 filed Dec. 28, 2000, which claims the benefit of
U.S. Provisional Application No. 60/174,976, filed Jan. 6, 2000,
the disclosures of which are incorporated by reference herein in
their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Many aspects of biological research rely upon the ability to
perform extremely large numbers of chemical and biochemical assays.
Increasing the throughput of screening assays has allowed
researchers to adopt a more generalized approach to the overall
screening process, as opposed to a more rational, predefined
process. For example, in the pharmaceutical discovery process,
large libraries of different compounds are generally screened
against defined target systems to determine whether any of those
compounds have a desired effect on that system. Once a compound is
identified to have the desired effect, it is then subjected to more
rigorous analysis.
[0003] Many high-throughput screening assay systems rely upon
entirely in vitro models of biological systems. This is due, at
least in part, to the ability to accurately control substantially
all of the parameters of the model system that is being assayed to
permit correlation from assay to assay, such as the quantity and
purity of reagents, the environmental conditions of the assay, the
operator performing the assay, and the like. Specifically,
variation of any of these parameters can produce widely varying
results in the performance of a given assay.
[0004] In many cases, these in vitro systems have proven to be
effective models of the biochemical system of interest, and have
led to the identification of promising pharmaceutical candidate
compounds. However, in many instances it is desirable to use a
model system that is a closer representation of what actually
occurs in more complex systems, e.g., in vivo. Cell-based systems
offer a closer model to these relevant biological systems, and have
generally been widely adopted as screening assays. In particular,
these cell based systems typically include a more complete range of
biochemical events involved in a particular biological activity,
where the overall biological activity or simply the outcome of that
biological activity is of particular pharmacological interest. By
way of example, when a cell surface receptor binds its ligand, it
may activate a cell signaling pathway or cascade, where a protein,
e.g., a DNA binding protein, is phosphorylated altering its
activity and/or specificity. The binding of this protein to a
particular nucleic acid sequence then results in an increase or a
decrease in the level of expression of a particular gene product
encoded by that nucleic acid sequence or the gene that comes under
the control of that sequence. In looking for effectors of the
activation of the gene, one could focus individually on each step
in the pathway, and hopefully obtain a promising lead effector
compound. Preferably, however, one screens the compound against the
entire pathway, to thereby increase the chances of obtaining an
effector of any one step in the pathway. This entire pathway
screening is best carried out in whole cell systems.
[0005] It would generally be desirable to provide cell-based
assays, and particularly cell based screening assays that are more
reflective of complex biological systems. Further, it would be
desirable to provide a simple assay format for monitoring the level
of intracellular interactions, which interactions figure in a
particular pathway of interest.
SUMMARY OF THE INVENTION
[0006] In a first aspect the present invention provides a method of
detecting intracellular binding interactions. The method comprises
providing a biological cell having at least a first component of a
binding reaction disposed therein. The cell is contacted with a
second component of the binding reaction whereby the second
component is internalized within the biological cell. At least one
of the first and second components has a fluorescent label. The
amount of binding between the first and second components within
the cell is determined by measuring a level of polarized and/or
depolarized fluorescence emitted from within the biological cell.
In preferred aspects, the methods measure and/or monitor the
interaction between a protein and non-protein molecule,
intracellularly.
[0007] Another aspect of the present invention is a method of
monitoring activation of a cell signaling pathway. The method
includes providing a cell which comprises a cell signaling pathway.
At least one step in the cell signaling pathway comprises an
intracellular binding interaction between a nucleic acid and a
nucleic acid binding protein. The cell is contacted with a first
nucleic acid whereby the first nucleic acid is internalized within
the cell. The first nucleic acid comprises a fluorescent label and
is capable of being bound by the nucleic acid binding protein. A
level of binding between the nucleic acid binding protein and the
first nucleic acid is monitored by monitoring a level of polarized
and/or depolarized fluorescence emitted from within the cell. The
amount of polarized and/or depolarized fluorescence is indicative
of a level of activation of the cell signaling pathway.
[0008] A further aspect of the present invention is a method of
screening for effectors of cellular function. The method comprises
providing at least a first cell capable of at least one cellular
function that is initiated by interaction of a nucleic acid
sequence and a nucleic acid binding protein. A first nucleic acid
having a fluorescent label is inserted into the cell wherein the
first nucleic is capable of being bound by the nucleic acid binding
protein. The cell is exposed to a test compound. An amount of
binding between the first and second components within the cell is
determined by measuring a level of polarized and/or depolarized
fluorescence emitted from within the biological cell. An amount of
binding between the first and second components within the cell is
compared in the presence of the test compound to an amount of
binding between the first and second components within the cell in
the absence of the test compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 schematically illustrates a system for carrying out
the assay methods of the present invention.
[0010] FIG. 2 schematically illustrates a microfluidic device for
use in carrying out the assay methods of the present invention.
[0011] FIG. 3 schematically illustrates a system incorporating a
microfluidic device for carrying out the assay methods of the
present invention.
[0012] FIG. 4 schematically illustrates a modular detector for
performing fluorescence polarization detection in accordance with
certain aspects of the present invention.
[0013] FIG. 5 illustrates detection of binding of DNA binding
proteins to a labeled nucleic acid probe by a shift in fluorescence
polarization levels.
DETAILED DESCRIPTION OF THE INVENTION
[0014] I. General Description
[0015] The present invention is generally directed to methods and
systems for monitoring intracellular biochemical interactions, in
situ, that are indicative of important cellular events. These
methods are preferably applied in screening potential
pharmaceutical compounds, also referred to as test compounds, in
order to ascertain whether any such compounds have an effect on
those interactions or upon the cellular events that lead to or
result from such interactions. As noted above, a wide variety of
specific intracellular binding or other associative biochemical
interactions are integral steps in the biological pathways that
lead to or result from important biological events, e.g.,
pharmacologically or medically relevant events. As a result, the
ability to monitor those interactions in situ, as provided by the
instant invention, is very useful.
[0016] As the present invention is directed to in situ analytical
methods and systems, it typically requires the use of whole cell
systems that comprise the elements of the particular binding or
associative reaction of interest. Typically, in accordance with the
invention, one of the components of the binding or associative
reaction or interaction is already disposed within the whole cell
system. By this is meant that at least one component of the binding
interaction of interest is typically expressed by the cell, either
natively, or as the result of genetic manipulation.
[0017] In accordance with the present invention, a second component
of the binding reaction is inserted into the cell, e.g., from an
exogenous source. The level of interaction between the two
biochemical components is then ascertained. Depending upon the
nature of the second component and the cell type used, the
insertion of the second component may occur naturally, or it may
require the addition of a translocation signal or functionality to
the second component to facilitate the internalization of that
component.
[0018] Typically, the interaction is monitored through the
incorporation of detectable labels on at least one of the
components whereby the binding event can be detected. In
particularly preferred aspects, the second or exogenously
introduced component has a relatively rotational correlation time,
e.g., is relatively small compared to the size of the first
component, and bears a fluorescent label group or moiety. In this
case, the binding of the two components (the large endogenous
component and small fluorescently labeled exogenous component)
results in a change in the fluorescence anisotropy of the labeled
component. This is readily detected by measuring the complex's
ability to emit depolarized fluorescent light in response to
polarized activation light. This change in the fluorescence
anisotropy of the label is a result of the reduced rotational
diffusion of the labeled component when bound to the other larger
component.
[0019] In certain aspects, the relative level of interaction
between the two components is monitored in the presence of one or
more pharmaceutical test compounds, or under one or more particular
environmental conditions. By monitoring the level of this
interaction in the presence of test compounds, particular
environmental conditions, or the like, one can ascertain the
effects of such compounds or conditions on that interaction, or the
biochemical events leading up to that interaction.
[0020] In particularly preferred aspects, the present invention
provides methods, systems, kits and the like for monitoring the
level of interaction between intracellular nucleic acid binding
proteins and their target nucleic acid sequences, e.g., those to
which the proteins bind. Many cellular functions are carried out
through the initiation or reduction in the level of the
transcription of certain genes, where the products of those genes
carry out a specific cellular activity which is to be increased or
decreased. A change in the level of transcription typically begins
with the receipt of a signal by the cell indicating the need for a
particular cellular function, e.g., the presence of a hormone,
growth factor, or other signaling compound. The cell then initiates
a signal cascade that results in the initiation or reduction in
transcription of the gene and expression of the gene's product.
[0021] One of the primary ways that cells regulate the level of
transcription of genes is through the use of transcriptional
regulatory proteins that bind to sites on the cell's DNA proximal
to the gene of interest to enhance or inhibit the transcription of
that gene. Many of these transcriptional regulatory proteins are
activated during the signal cascade, e.g., via phosphorylation of
one or more residues on the protein, e.g., tyrosine, serine, and/or
threonine, giving rise to their binding affinity to the regulatory
sequence on the DNA strand. Alternatively, an inhibitory subunit
can be phosphorylated, causing release of the active binding
subunit. Thus, cellular events that are marked by changes in
transcription levels are often preceded by the binding of these
transcriptional regulatory proteins to the genetic material. As a
result, monitoring the initiation of those cellular events can be
accomplished by monitoring this binding interaction. As noted
above, simply monitoring the binding reaction, in vitro, provides
the investigator with substantially limited information as to the
overall signaling process. Further, as these interactions typically
occur within the cell, and in relatively short time-frames,
observing the interactions can be quite difficult. Specifically,
binding reactions between two or more interacting elements are
generally assayed in cell extracts using a heterogeneous assay
format. The present invention, however, utilizes a first component
of the binding interaction, e.g., a DNA probe, and looks for
changes in the optical properties of that component upon
intracellular binding of the first component by another
intracellular component. In particularly preferred aspects, the
first component is fluorescently labeled and the optical property
is the level of depolarized fluorescence that is emitted from the
cell in response to a polarized excitation light source.
[0022] II. Cells
[0023] The methods and systems of the present invention are useful
in virtually any type of biological cells, including, mammalian,
bacterial, fungal, yeast, insect, and plant cells. In particularly
preferred aspects, mammalian cells, e.g., CHO, HEK-293, L-cells,
3T3 cells, COS, THP-1 cells, blood cells, i.e., B cells, T cells,
monocytes and neutrophils, etc., and bacterial cells are used. The
specific cell type used typically varies depending upon the type of
interaction that is sought to be monitored. For example, mammalian
cells and specifically, human cells or animal cells transfected to
express human proteins are typically preferred for screening
potential human therapeutics, for their effects on mammalian and
human systems, while other types of screening or experimentation
may benefit from the use of other cell types, e.g., using bacterial
or fungal cells to screen for potential antibiotic agents, etc.
[0024] Typically, well characterized cell lines that are predictive
models of human cell functions are most preferred for their
correlatability to human systems in pharmaceutical and medical
research. Examples of preferred cell lines include, e.g., COS
cells, CHO cells, HEK-293 cells, RBL-1, Jurkat, U937 and YB-1
cells.
[0025] The cells to be monitored may be provided in either
immobilized form or as a suspension culture. For ease of
processing, however, the cells are generally provided as a
suspension of cells in an appropriate medium. In the case of these
cell suspensions, cell density may vary depending upon the type of
reaction vessel that is being employed, as well as the type of
detection method that is being employed. Typically, determination
of optimal cell densities is a matter of routine for one of
ordinary skill in the art. In the case of flow-through embodiments
of the invention, e.g., microfluidic systems, cell densities
generally range from about 1 cell/nl to about 30 cells/nl in the
reaction/detection portion of the vessel or channel. In the case of
test tube or multiwell plate based reactions, cell densities
typically range from about 1,000 cells/mm.sup.2 to about 10,000
cells/mm.sup.2. Of course, these ranges can also vary depending
upon the cell types used, the relative adherence of the cells to
the vessel surfaces as well as each other, and the like.
[0026] III. Binding Reaction
[0027] As noted previously, a wide variety of binding reactions are
of particular interest to the biological research community, e.g.,
protein-protein interactions, receptor-ligand interactions, nucleic
acid interactions, protein-nucleic acid interactions, and the like.
In the intracellular environment, many of these reaction types are
involved in the multiplicity of steps of signal transduction within
cells. For example, activation of a particular cellular event is
often triggered by the interaction between a cell surface receptor
and its ligand. The signal from the receptor is often transmitted
along via the binding of enzymes to other proteins, e.g., kinases,
etc., which then pass the signal on through the cell until the
ultimate desired response is achieved. Typically, the ultimate
response is the increased or decreased expression of a particular
gene product that is involved with the cellular event. This
increase or decrease in expression is often the result of an
increased or decreased level of interaction between the regulatory
genetic sequence and newly activated gene regulating factors or
proteins.
[0028] In accordance with the present invention, the binding
interaction of interest occurs within the cell between at least
first and second components of the interaction. The first component
comprises a component of the particular interaction that is
predisposed within the cell. By "predisposed" is meant that the
first component exists within the cell prior to commencing the
assay method. Typically, this refers to a component that is part of
the cell, e.g., is expressed by the cell naturally, or as a result
of introduction of an appropriate genetic construct within the
cell. For example, where the first component comprises a receptor
protein, a nucleic acid or nucleic acid binding protein, that
component is typically expressed by the cell, either naturally or
by virtue of an appropriate genetic construct introduced into the
cell line.
[0029] In accordance with the present invention, the second
component of the interaction of interest is typically provided, at
least in part, as an exogenous probe that mimics the natural
binding partner of the first component. Thus, the nature of the
second binding component is dictated largely by the first binding
component. For example, in the case of assays for intracellular
protein-protein interactions, the second component comprises at
least a polypeptide that includes the recognition structure or
amino acid sequence that is necessary for binding by the first or
cell associated component. In preferred aspects, monitoring of
protein-protein interactions utilizes short peptide probes instead
of the full-length exogenously introduced protein. For purposes of
the instant invention, such peptide probes are considered
non-protein molecules. These peptide probes may be recognized by
virtue of their amino acid sequence, secondary structure, or
modifications, e.g., phosphorylation in the case of kinase binding.
Typically, such peptide probes will be between about 4 and about
100 residues in length, and more preferably, less than 50 residues
in length, e.g., between about 4 and about 50. Typically, such
peptides are less than about 10 kD, and more preferably, less than
about 5 kD. Although useful in measuring protein/protein
interactions, the present methods are particularly useful in
measuring interactions between intracellular proteins and
exogenously introduced non-protein molecules. Such molecules
include, e.g., nucleic acids, carbohydrates, lipids, small
molecules, i.e., binding fragments of full length proteins,
including phosphorylated fragments, signaling molecules such as
cAMP and cGMP, diacylglycerol (DAG), and the like.
[0030] Similarly, in the case of interactions between a particular
DNA binding protein and a nucleic acid sequence, the second
component typically comprises a nucleic acid probe, typically
double stranded, that includes the appropriate recognition and
binding sequence of nucleotides necessary for binding of the
particular protein to the DNA strand. Typically, nucleic acid
probes generally range from about 5 to about 100 bases in length.
Typically, such probes range from about 10 to about 50 bases in
length, so as to provide a sufficient binding sequence, without
providing excessive sequence length that might impair
measurement.
[0031] The second exogenous component typically includes one or
more labeling groups in order to facilitate detection of the
binding event. In preferred aspects, the label group on the
exogenous second component of the interaction comprises a
fluorescent group. A wide variety of fluorescent labels having a
wide variety of excitation and emission maxima are known to those
of ordinary skill in the art and are generally commercially
available (e.g., from Molecular Probes, Eugene Oreg.). As noted
previously, the fluorescent label group is used to monitor the
binding event via a change in the level of polarized fluorescence
resulting from binding of the first and second components, as is
discussed in greater detail, below. In order to maximize the
detectable shift in fluorescence polarization, it is generally
desirable to provide for the greatest difference in rotational
diffusion rates between the unbound labeled component and the
labeled component when it is bound in a complex, e.g., with the
second component. Typically, this is accomplished by providing the
labeled component as a relatively small compound as compared to the
unlabeled component. Specifically, the labeled component typically
has a molecular weight that is equal to or less than that of the
unlabeled component, and often, less than half, a fifth or even a
tenth that of the unlabeled component. Thus, the labeled component
will typically have a rotational diffusion rate that is at least
twice that of the complex, preferably, at least 5 times that of the
complex, and often, at least ten times that of the complex.
Fluorescent polarization detection is described in greater detail,
below.
[0032] While a single label group located on the exogenously
introduced second component is preferred, in some cases, it may be
desirable to provide both the first and second components with
labeling groups to facilitate monitoring of the binding event.
[0033] As noted previously, in particularly preferred aspects, the
present invention involves the monitoring of the interaction
between a nucleic acid and an intracellular nucleic acid binding
protein. In accordance with this aspect of the invention, the
intracellular nucleic acid binding protein comprises a nucleic acid
binding protein that is expressed by the cell. Typically, these
binding proteins bind to specific nucleic acid sequences to
regulate transcription of particular genes. These binding proteins
are, in turn, activated by an upstream signaling event, e.g.,
tyrosine phosphorylation which, in turn, is in response to a
particular environmental condition or other stimulus.
[0034] As described previously, an example of particularly
interesting DNA/DNA binding protein interactions are those
resulting from the signal transduction pathways of several classes
of growth factors and cytokine receptors, that signal through
extrinsic tyrosine kinases, e.g., the Janus kinases or "JAK", to
activate genetic transcription regulation proteins, e.g., signal
transcription activators of transcription or "STAT." Briefly, the
STAT proteins, when phosphorylated at the appropriate residue(s),
through a signal transduction event, dimerize. The dimerized STAT
proteins have a greatly enhanced affinity for DNA sequences in the
genome that enhance transcription of the downstream genes. Lamb et
al., Drug Discovery Today, 3:122-130 (1998), Gouilleux et al., EMBO
Journal 13(18):4361-4369 (1994).
[0035] The erythropoeitin (EPO) and interleukin-3 (IL-3) receptors,
for example, when activated, in turn activate the JAK2 kinase which
phosphorylates the STAT5 protein, which then binds to the PIE
(Prolactin Inducible Element) and GAS (Gamma-Interferon Activated
Site), to activate gene transcription (see, Lamb et al.,
supra).
[0036] By monitoring the level of binding of the STAT proteins,
e.g., STAT5, to their target genetic regulatory sequences, e.g. PIE
and GAS, one can monitor cytokine receptor activation, and screen
for effectors of cytokine receptor activation in cells.
[0037] In operation, a fluorescently labeled, double stranded
polynucleotide probe bearing the recognition/binding site for a
particular regulatory genetic sequence, e.g., PIE or GAS, is
introduced into the cells that are being observed. In the unbound
state, these labeled probes will emit a particular level of
depolarized fluorescence when excited with a polarized light
source, due to their relatively fast rotational diffusion rate.
When the appropriate receptor is activated, e.g., EPO or IL-3, the
signaling cascade activates the appropriate binding protein, e.g.,
STAT5, which then binds to the binding and/or recognition sequence
on the fluorescent probe. Once bound, the rotational diffusion rate
of the fluorescent label, which is now part of the bound complex,
is significantly slower, due to the larger molar volume of the
complex. The slower rotational diffusion results in emission of
more polarized (or less depolarized) fluorescence from the bound
probe as compared to the free probe. This shift in the level of
fluorescence polarization is then detected, providing a detectable
event that corresponds with receptor activation. The detection of
this binding interaction provides a direct reporter signal for
activation of a the signal transduction pathway, e.g., receptor
activation.
[0038] In many cases, e.g., where the second component comprises a
nucleic acid compound, it may be necessary to incorporate a
translocation functionality on the second component in order to
facilitate the translocation or internalization of that component
from the outside to inside the cell. As used herein, the term
"translocation functionality" refers to a chemical compound, group
or moiety that increases the cell's ability to internalize another
compound or material. Examples of such translocation
functionalities include peptide recognition/transport sequences,
liposomal compositions, or the like. Alternative translocation
methods and compositions are also utilized in accordance with the
present invention to induce uptake of the second component,
including, e.g., electroporation, cell permeating compositions
containing, e.g. PEG, porins, saponins, streptolysin or the
like.
[0039] In particularly preferred aspects, and particularly those
aspects where the second component is a nucleic acid material, the
translocation functionality comprises a polypeptide sequence that
increases the internalization of the second component. Examples of
particularly preferred peptide sequences include trojan peptides.
See Derossi et al., Trends in Cell Biol. 8(2):84-87 (1998),
incorporated herein by reference in its entirety for all purposes.
In particular, a labeled nucleic acid probe is coupled to a
relatively short polypeptide that facilitates the translocation of
the nucleic acid probe into the cell where the assayed binding
reaction occurs. Examples of preferred polypeptides include those
derived from homeodomain proteins, which generally belong to a
class of transcription factors involved in multiple morphological
processes, e.g., derived from whole or fragments of ANTENNAPEDIA
homeodomain (Antp-HD), a Drosophila transcription factor, i.e.,
amino acids 43-58 (Allinquant et al., J. Cell Biol. 128:919-927
(1995). Similarly, polypeptides derived from residues 48-60 of the
HIV-1 tat protein, e.g., having sequences homologous to this
sequence C(Acm)GRKKRRQRRRPPQC (SEQ ID NO:1)
(C(Acm)=cysacetamidomethyl), are also preferred for use as
translocation functionalities (Vives et al., J. Biol. Chem.
272:16010-16017 (1997)).
[0040] IV. Fluorescence Polarization Detection
[0041] In preferred aspects, as noted above, monitoring of the
interaction event of interest is accomplished through fluorescence
polarization detection. In particular, fluorescence polarization
detection provides a relatively simple, homogeneous detection
method for binding interactions. Measurement of differential
polarization of free and bound ligands has long been utilized to
determine relative ligand binding levels, and even to screen for
compounds or conditions that might affect that binding. To date,
such assays have been carried out in a contained fluid system,
e.g., a cuvette or multiwell plate, where fluid components of the
binding reaction, e.g., a labeled ligand and its receptor, are
mixed in the presence or absence of a compound to be tested.
Surprisingly, in accordance with the present invention,
fluorescence polarization also provides a useful measurement tool
for detecting intracellular binding reactions.
[0042] The principles behind the use of fluorescence polarization
measurements as a method of measuring binding among different
molecules are relatively straight-forward. Briefly, when a
fluorescent molecule is excited with a polarized light source, the
molecule will emit fluorescent light in a fixed plane, e.g., the
emitted light is also polarized, provided that the molecule is
fixed in space. However, because the molecule is typically rotating
and tumbling in space, the plane in which the fluoresced light is
emitted varies with the rotation of the molecule. Restated, the
emitted fluorescence is generally depolarized. The faster the
molecule rotates in solution, the more depolarized it is.
Conversely, the slower the molecule rotates in solution, the less
depolarized, or the more polarized it is. The relationship between
the polarization value (P) for a given molecule and the molecule's
"rotational diffusion rate" or "rotational correlation time,"
(sometimes termed the "rotational relaxation time") or the amount
of time it takes the molecule to rotate through an angle of
57.3.degree. (1 radian), is given by the Perrin Equation: 1 1 P - 1
3 = ( 1 P 0 - 1 3 ) ( 1 + T F T C )
[0043] where P.sub.0 is the limiting polarization value, T.sub.F is
the fluorescent lifetime of the fluorescent label and T.sub.C is
the rotational correlation time. The smaller the rotational
correlation time, the faster the molecule rotates, and the less
polarization will be observed, e.g., the more depolarized the
fluorescent emissions. The larger the rotational correlation time,
the slower the molecule rotates, and the more polarization will be
observed, e.g., the less depolarized the emitted fluorescence will
be. Rotational correlation time is related to viscosity (.eta.),
absolute temperature (T), molar volume (V), and the gas constant
(R). The rotational correlation time is generally calculated
according to the following formula:
Rotational Correlation Time=3 .eta. V/RT
[0044] As can be seen from the above equation, if temperature and
viscosity are maintained constant, then the rotational correlation
time, and therefore, the polarization value, is directly related to
the molecular volume. Accordingly, the larger the molecule, the
higher its fluorescent polarization value, and conversely, the
smaller the molecule, the smaller its fluorescent polarization
value. Typically, the binding partners for a given assay are chosen
such that T.sub.F>T.sub.C when the binding partners are not
bound and T.sub.F<T.sub.C when they are bound.
[0045] In the performance of fluorescent binding assays, a
typically small, fluorescently labeled molecule, e.g., a ligand,
antigen, oligonucleotide or nucleic acid probe, etc., having a
relatively fast rotational correlation time, is used to bind to a
much larger molecule, e.g., a receptor protein, antibody,
complementary nucleic acid target sequence etc., which has a much
slower rotational correlation time either on its own or as a part
of the complex. The binding of the small labeled molecule to the
larger molecule or in the larger complex significantly increases
the rotational correlation time (decreases the amount of rotation)
of the labeled species, namely the labeled complex over that of the
free unbound labeled molecule. This has a corresponding effect on
the level of polarization that is detectable. Specifically, the
labeled complex presents much higher fluorescence polarization than
the unbound, labeled molecule. The polarization value can then be
used to determine the level of bound and free fluorescent
compound.
[0046] Generally, the fluorescence polarization level is calculated
using the following formula:
P=[I(.parallel.)-I(.perp.)]/[I(.parallel.)+I(.perp.)]
[0047] Where I(.parallel.) is the fluorescence detected in the
plane parallel to the excitation light (also termed "polarized
fluorescence"), and I (.perp.) is the fluorescence detected in the
plane perpendicular to the excitation light (also termed
"depolarized fluorescence"). Thus, as can be seen from this
equation, the polarization value P is related to the ratio of
polarized to depolarized fluorescence.
[0048] In performing screening assays, e.g., for potential
inhibitors, enhancers, agonists or antagonists of the binding
function in question, the change in fluorescence polarization of
bound versus free labeled ligand is compared in the presence and
absence of different compounds, to determine whether these
different compounds have any effect on the binding function of
interest. In particular, in the presence of inhibitors of the
binding function, the fluorescence polarization will decrease, as
more free, labeled ligand is present in the assay. Conversely,
enhancers of the binding function will result in an increase in the
fluorescent polarization, as more complex and less free labeled
ligand are present in the assay.
[0049] V. Applications
[0050] The methods and systems of the present invention generally
have a variety of uses in the research and diagnostic fields.
[0051] A. Expression Monitoring
[0052] The most notable uses for monitoring intracellular binding
reactions in accordance with the methods and systems of the present
invention is in monitoring gene expression in response to a
particular stimulation or activation event, e.g., receptor
activation via ligand binding, or the like.
[0053] In general, a cell's response to a given stimulus can be
coupled to the increased or decreased expression of certain gene
products. This may take the form of natural expression of gene
products or engineered expression. In the case of engineered
expression, this can be done by engineering indicator cell lines
that have stimulus responsive regulatory elements controlling gene
products, such as reporter genes. Expression is then monitored by
detecting expression of the reporter gene product, typically an
enzyme (e.g., .beta.-galactosidase, luciferase, alkaline
phosphatase, etc.) that produces an easily detectable product, such
as a colored, chemiluminescent or fluorescent product. In the past,
this has been accomplished by attempting to stimulate the cells,
lyse the cells and look for increased levels of RNA resulting from
increased expression, which requires several hours for gene
transcription and a large number of complex steps, e.g., cell lysis
and purification, amplification of nucleic acids, labeling, and the
like. Both of these methods are performed after the fact as opposed
to in real time.
[0054] As noted above, changes in expression levels of genes are
typically preceded by increased or decreased interaction of that
gene's regulatory region with DNA binding, gene regulation
proteins. These changes in binding interactions are rapid,
typically occurring within 60 seconds of stimulation, and are
directly monitored using the above-described methods.
[0055] B. Screening Applications
[0056] While the methods and systems of the present invention have
a variety of uses in research and diagnostic applications, as noted
previously, they are most preferably applied in screening
applications, and particularly pharmaceutical screening
applications. In particular, when determining whether a particular
compound has useful pharmacological activity, it is generally
desired to screen the compound against a biochemical system model,
in vitro, where interaction of the components of the biochemical
system is mimetic of the interaction of components of an actual in
vivo system in which a particular pharmacological activity would be
desirable, e.g., inhibition of undesired reactions or occurrences
or enhancement of desired reactions. The components of the
biochemical system are exposed to the particular compound and their
level of interaction is ascertained and compared to a negative or
positive control, e.g., where no compound or a compound having a
known effect is present, respectively.
[0057] The assay methods and systems are generally parallelized,
e.g., carried out in parallel, in order to increase the number of
different test compounds that may be screened at a given time. In
conventional laboratories, this is carried out by depositing all of
the assay reagents into separate wells in multi-well microplates,
e.g., 96, 384 or 1536 well plates. A different test compound or
control is then introduced into each of the various different
wells, and the effects of each of the test compounds on the
biochemical system of interest are then determined, e.g., using a
multi-well plate reader to detect the optical signals associated
with the screening assay, e.g., fluorescence polarization
shifts.
[0058] Using these methods, one can rapidly screen large numbers or
libraries of different test compounds for potential pharmacological
activity. Typically, libraries of test compounds are derived from
any of a variety of known origins, including isolation from natural
sources, combinatorial chemistry methods, or the like. As such,
these test compounds may include isolated small molecule organic or
inorganic compounds, as well as isolates or mixtures of materials
from plant, animal, fungal or bacterial extracts, or the like.
While traditional laboratory vessels are easily employed in the
methods and systems of the invention, as described below,
continuous flow methods and systems, and particularly those
employing microfluidic channel networks and systems, as described
in greater detail, below.
[0059] The methods and systems of the invention are particularly
useful in performing such screening assays. In particular, these
assay methods and systems are used to screen libraries of potential
pharmaceutical compounds or the like for an effect on the
intracellular binding reaction of interest, or on events that
precede and lead to those intracellular binding reactions, e.g.,
receptor activation.
[0060] In performing these screening assays, a cell is initially
provided that includes at least a first component of the binding
interaction of interest, e.g., a particular DNA binding protein.
The second component of the binding interaction, e.g., a labeled
nucleic acid probe, is then introduced into the cell to permit it
to be bound by the first component. Either during or subsequent to
the introduction of the second component into the cell, the cell is
contacted with a test compound that is being screened. The cell is
also exposed to any compounds or conditions required to activate
the signaling cascade reaction that results in the binding reaction
of interest, e.g., a ligand for a particular receptor, e.g.,
cytokines, etc.
[0061] The relative level of binding of the first and second
components, e.g., as determined by comparing fluorescence
polarization of activated cells to that of non-activated cells, is
then ascertained. An increase or decrease in binding in the
presence of the test compound is indicative of a change in the
level of signal transduction events leading up to and including the
binding reaction. The precise effect of the test compound on the
overall signal transduction, e.g., whether it enhances or inhibits
the pathway will depend upon the nature of the signal pathway,
e.g., whether activation of the pathway is a negative or positive
regulatory event. For example, in many cases, pathway activation
results in increased binding of the first and second components,
e.g., DNA binding proteins and regulatory gene sequences. In those
cases, exposure of the cell to enhancers of pathway activation will
show a further relative increase in the level of binding.
Conversely, where a pathway results in reduced binding of two
components, an enhancer of the signal pathway would lead to a
decrease in the binding level.
[0062] VI. Assay Systems
[0063] A. Generally
[0064] A variety of systems may be used in carrying out the assay
methods described above. These include conventional assay systems
that rely upon the use of multi-well plates and plate reader
detection systems available from, e.g., Life Technologies,
Molecular Devices Inc., and LJL Biosystems, as well as more
advanced systems, such as microfluidic or array based
technologies.
[0065] A general schematic of a system for carrying out the methods
of the invention is illustrated in FIG. 1. As shown, the system 100
includes a reaction vessel 102, in which the assay reaction is
carried out. The reaction vessel includes a cell suspension 104
disposed therein. The reaction vessel 102 optionally includes a
conventional assay vessel, such as a test tube, or a well of a
multiwell plate, e.g., a 96, 384 or 1536 well plate. Alternatively,
the reaction vessel 102 comprises a flow through conduit, such as a
capillary channel or a channel in a microfluidic channel network,
as described below.
[0066] A detector or detection system 106 is typically disposed
within sensory communication of the contents of the reaction
vessel. The phrase "within sensory communication" generally refers
to the detector that is positioned relative to the reaction vessel
102 so as to be able to receive a particular signal from that
receptacle. In the case of optical detectors, e.g., fluorescence or
fluorescence polarization detectors, sensory communication
typically means that the detector is disposed sufficiently proximal
to the receptacle that optical, e.g., fluorescent signals are
transmitted to the detector for adequate detection of those
signals. Typically this employs a lens, optical train and/or other
detection elements, e.g., a CCD, that is focused upon a relevant
transparent or open portion of the receptacle to efficiently gather
and record these optical signals. In the case of thermal or
electrochemical signals, sensory communication requires actual
contact between portions of the detector and the contents of the
vessel, e.g., through thermal probes, electrodes, or the like.
[0067] The detector 106 is typically operably coupled to a computer
108 for receipt and processing of the signals received by the
detector. Optionally, in the case of microfluidic systems, the
computer also functions to control movement of material through the
channels of the device, e.g., by instructing a fluid controller
(not shown), as well as sampling activities for bringing test
compounds or other reagents into the channels of the device. As
described with reference to FIG. 3, below.
[0068] B. Microfluidics
[0069] As noted above, in preferred aspects, the assay systems of
the invention utilize microfluidic channels and/or channel networks
as the reaction vessels. These microfluidic systems provide for
increased throughput, reduced reagent consumption, improved
integration and automation over conventional systems, e.g., tube or
well based systems. Microfluidic screening assay methods and
systems have been described previously. See, e.g., U.S. Pat. No.
5,942,443. Similarly, use of fluorescence polarization detection in
flowing microfluidic systems has been described in, e.g., Published
International Patent Application No. PCT US99/12671, as have cell
based assay methods and systems, see, e.g., Published International
Patent Application No. PCT US99/13918. Each of the above references
is incorporated herein by reference in its entirety for all
purposes.
[0070] As used herein, the term "microfluidic" generally refers to
one or more fluid passages, chambers or conduits which have at
least one internal cross-sectional dimension, e.g., depth, width,
length, diameter, etc., that is less than 500 .mu.m, and typically
between about 0.1 .mu.m and about 500 .mu.m. In the devices of the
present invention, the microscale channels or chambers preferably
have at least one cross-sectional dimension between about 0.1 .mu.m
and 200 .mu.m, more preferably between about 0.1 .mu.m and 100
.mu.m, and often between about 0.1 .mu.m and 20 .mu.m. Accordingly,
the microfluidic devices or systems prepared in accordance with the
present invention typically include at least one microscale
channel, usually at least two intersecting microscale channels, and
often, three or more intersecting channels disposed within a single
body structure. Channel intersections may exist in a number of
formats, including cross intersections, "T" intersections, or any
number of other structures whereby two channels are in fluid
communication.
[0071] The body structure of the microfluidic devices described
herein typically comprises an aggregation of two or more separate
layers which when appropriately mated or joined together, form the
microfluidic device of the invention, e.g., containing the channels
and/or chambers described herein. Typically, the microfluidic
devices described herein will comprise a top portion, a bottom
portion, and an interior portion, wherein the interior portion
substantially defines the channels and chambers of the device. In
particular, these microfluidic devices are typically fabricated
from two or more planar solid substrates. A series of
interconnected grooves is generally fabricated into the surface of
the first of the two substrates. The second substrate is then
overlaid and bonded to the surface of the first substrate to seal
the grooves and define the integrated fluidic channels of the
device. Typically one of the substrates includes one or more ports,
e.g., holes, disposed through the substrate, that are positioned
such that the holes are in fluid communication with the integrated
channels in the complete assembled device. These holes then
function as reservoirs for fluid introduction into the channels of
the device, as well as providing electrical access points for the
various channels for use in, e.g., controlled electrokinetic
material transport systems.
[0072] One example of a microfluidic device useful in carrying out
the assays described herein is illustrated in FIG. 2. As shown, the
device 200 includes a body 202 having a main analysis channel 204
disposed within its interior. At one terminus, main channel 204 is
in fluid communication with waste reservoir 214. At the other
terminus, main channel 204 is in fluid communication with an
external sampling capillary or pipettor 220, via channel junction
216. Specifically, external capillary 220 is attached to the body
202 of the device 200, such that the channel within the capillary
is fluidly connected to the main channel 204. Additional channels
226, 228, 230 and 232 intersect the main channel 204 and connect
the main channel to reservoirs 206, 208, 210 and 212, respectively.
These reservoirs 206-212 are used to introduce reagents into the
main channel, including cell suspensions, assay reagents, buffers,
and the like. In operation, the cell suspension and second
component of the binding reaction, e.g., a nucleic acid probe, are
flowed into the main channel 204 from, e.g., reservoirs 208 and
210. Appropriate diluents are also optionally added from reservoirs
206 and 212. As shown, fluid transport is driven by application of
a vacuum source, e.g., to reservoir 214. Periodically, plugs of
fluid containing test compounds are introduced into main channel
204 from capillary 220, e.g., by placing the open end of capillary
220 into a source of the test compound and drawing a volume of the
test compound into the capillary channel and then up into the main
channel 204. The results of the assay are then detected at a point
in the main channel, e.g., detection window 234, e.g., by a
fluorescence polarization detector.
[0073] FIG. 3 schematically illustrates a microfluidic device 200
incorporated in an overall system. As shown, and with reference to
FIGS. 1 and 2, the system includes device 200. A detector 106 is
positioned to be within sensory communication with main channel
104. The device 200 is also operably connected to a flow controller
310 which directs fluid movement through the channels of the device
200. As shown, the flow controller 310 comprises a vacuum
controller that applies a negative pressure to at least one of the
wells of the device 200 (as indicated by the black arrow). Other
methods of flow control are also optionally used in the systems
described herein, including, e.g., pressure control at multiple
reservoirs, and electrokinetic control via electrodes in contact
with fluid in the various reservoirs of the device. Controlled
electrokinetic transport of materials in microfluidic channel
systems and use of such microfluidic systems in performing
screening assays is described in detail in U.S. Pat. Nos.
5,779,868, 5,858,195, 5,976,336, and 5,942,443.
[0074] The system also includes a computer 108 that is operably
coupled to the detector 106, for recording, storing and optionally
analyzing the data from the detector 106. The computer is
optionally operably coupled to the flow controller 310, as well. As
such, the computer can also instruct the operation of the flow
controller in accordance with user input and/or preprogrammed
instructions, in order to provide desired fluid flow
characteristics, e.g., rate, direction etc., through the channels
of the device. As used herein, the term "operably coupled" refers
to a connection between two components wherein one component is
capable of communicating with the other. Operable coupling includes
but is not limited to electrical, hydraulic, pneumatic, mechanical,
optical and radio communication.
[0075] C. Detection Instrumentation
[0076] As noted previously, in preferred aspects, fluorescence
polarization is used to detect intracellular binding events in the
methods of the present invention. As such, specialized detection
systems are typically incorporated into the systems of the
invention. Specifically, such systems typically include a source of
polarized excitation light that is directed at a given sample
material, e.g., through an appropriate optical train. Fluorescence
emitted from the sample is then collected, e.g., by the same
optical train, and split into fluorescence that is in the same
plane as the excitation light and fluorescence that is in the
perpendicular plane. The amount of fluorescence in each plane is
then detected.
[0077] An example of an optical detection system for use in
fluorescence polarization detection is illustrated in FIG. 4. As
shown, the fluorescence polarization detector 400 includes a light
source 402, which generates light at an appropriate excitation
wavelength for the fluorescent compounds that are present in the
assay system. Typically, coherent light sources, such as lasers,
laser diodes, and the like are preferred because of the highly
polarized nature of the light produced therefrom. The excitation
light is optionally directed through a polarizing filter 404, which
passes only light in one plane, e.g., polarized light. The
polarized excitation light is then directed through an optical
train, e.g., including microscope objective 406, which focuses the
polarized light onto the channel in the microfluidic device 200,
through which the sample to be assayed is flowing, to excite the
fluorescent label present in the sample.
[0078] Fluorescence emitted from the sample is then collected,
e.g., through objective 406, and passed through the optical train
including e.g., a dichroic beamsplitter 408 for separating
fluorescence from reflected excitation light, polarizing beam
splitter 410 for separating the emitted fluorescence into a
component that is in the plane parallel to the excitation light,
and the plane perpendicular to the excitation light. The separated
polarized light components are then separately directed to separate
detectors 412 and 414, respectively (as indicated by the symbols
.parallel. for parallel fluorescence and .perp. for perpendicular
fluorescence), where each component is quantified. Photomultiplier
tubes (PMTs) are generally preferred as light detectors for the
quantification of the light levels, but other light detectors are
optionally used, such as photodiodes, CCDs, or the like. Additional
optical elements are also optionally included, i.e., detector
focusing lenses 416 and 418, emission filter 420, laser line filter
422, laser collimation lens 424, as well as a reference detector
426 and accompanying beamsplitter 428.
[0079] As described above, the detector is typically coupled to a
computer or other processor, which receives the data from the light
detectors, and includes appropriate programming to compare the
values from each detector to determine the amount of polarization
from the sample, e.g., in accordance with the processes and
equations set forth herein. The computer also typically displays
the polarization data in an appropriate graphical format that is
convenient for the user
[0080] VII. Kits
[0081] The present invention also provides kits for the use in
practicing the above-described methods. Typically, such kits
optionally include cells or cell suspensions that include at least
a first component of a binding reaction disposed within their
interior portions, e.g., endogenous DNA binding proteins, etc.
Alternatively, the investigator may use cells of their own
selection as needed for the desired assay. The kits also typically
include at least a second component of the binding reaction as a
separate reagent, e.g., a fluorescently labeled nucleic acid probe,
that is capable of being taken into the cells and bound by the
first component. The kits optionally include such reagents as well
as additional reagents in liquid or lyophilized forms, such as
buffers, detergents, salts or other elements useful in carrying out
the assay, where all components of the kit are disposed in a common
package. In certain aspects, the kits also include the assay or
reaction receptacle, which is optionally a test tube, multiwell
plate or microfluidic device. In addition, such kits typically
include appropriate instructions for carrying out the methods
described above.
EXAMPLES
[0082] A 17 bp, fluorescein labeled double stranded DNA probe was
mixed with serial dilutions of recombinant p50 fragment of NF-Kb
DNA binding protein in binding buffer containing 12 mM HEPES, 4 mM
Tris, 60 mM KCl, 0.4 mM EDTA, 10% glycerol, 2 mM DTT and poly-dAdT,
and each was incubated for 45 minutes at room temperature. The each
of the different dilutions were then flowed through a microscale
capillary channel and monitored using a fluorescence polarization
detector as shown in FIG. 4. As can be seen, increased
concentration of binding protein, which leads to increased levels
of binding, also leads to an increase in the level of fluorescence
polarization, showing that fluorescence polarization is a useful
method of detecting these interactions.
[0083] When the Antp-HD peptide is coupled to the 17 bp fluorescein
labeled ds-DNA probe, cells expressing NF-Kb will take up the probe
from the culture medium. In the intracellular reducing environment,
the disulfide linkage should be cleaved, releasing the probe. After
the cells are loaded with the labeled probe, excess extracellular
probe can be removed by pelleting the cells, e.g., in a centrifuge,
and re-suspending the pellet in a buffered cell medium, i.e.,
Hank's Balanced Salt Solution, containing a density adjusting
material, e.g., Optiprep (Nicomet) at 11% w/v. The cell medium
should prevent the cells from settling in the reaction vessel,
e.g., microfluidic channels. The cells would then be placed in a
well of a microfluidic device designed to transport the cells along
an analysis channel, and mix the cells with test compounds. The
cells/test compound mixture would then be incubated for
approximately 60 seconds as they move along the channel past a
fluorescence polarization detector. The signals that would be
obtained from the cells are then averaged over a selected time
period for each test compound injection, e.g., 20-60 seconds. In
operation, it would be expected that signal could be detected that
would be indicative of the binding interaction in the cells, and
those signals could be compared to appropriate positive and/or
negative controls.
[0084] All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. Although the present
invention has been described in some detail by way of illustration
and example for purposes of clarity and understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the appended claims.
Sequence CWU 1
1
1 1 15 PRT Homo sapiens MISC_FEATURE (1)..(1) cysacetamidomethyl 1
Xaa Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys 1 5 10
15
* * * * *