Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-12-28
2002-04-30
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S006120, C435S091100, C435S091200, C530S202000, C530S387100
Reexamination Certificate
active
06379884
ABSTRACT:
BACKGROUND OF THE INVENTION
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.
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.
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.
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
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.
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.
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.
REFERENCES:
patent: 4269511 (1981-05-01), Erwin
patent: 4299486 (1981-11-01), Nogami et al.
patent: 4345027 (1982-08-01), Dolbeare
patent: 4390403 (1983-06-01), Batchelder
patent: 4745285 (1988-05-01), Recktenwald et al.
patent: 5057413 (1991-10-01), Terstappen et al.
patent: 5166052 (1992-11-01), Cercek et al.
patent: 5225326 (1993-07-01), Bresser et al.
patent: 5278048 (1994-01-01), Parce et al.
patent: 5302349 (1994-04-01), Dandiliker et al.
patent: 5304487 (1994-04-01), Wilding et al.
patent: 5395503 (1995-03-01), Parce et al.
patent: 5460945 (1995-10-01), Springer et al.
patent: 5496697 (1996-03-01), Parce et al.
patent: 5534416 (1996-07-01), Millard et al.
patent: 5626134 (1997-05-01), Zuckerman
patent: 5637469 (1997-06-01), Wilding et al.
patent: 5660991 (1997-08-01), Lakowicz et al.
patent: 5726026 (1998-03-01), Wilding et al.
patent: 5744366 (1998-04-01), Kricka et al.
patent: 5804436 (1998-09-01), Okun et al.
patent: 5804448 (1998-09-01), Wang et al.
patent: 5843640 (1998-12-01), Patterson et al.
patent: 5928868 (1999-07-01), Liu et al.
patent: 5942443 (1999-08-01), Parce et al.
patent: 6025917 (2000-02-01), Toyonaga et al.
patent: 6124436 (2000-09-01), McClanahan et al.
patent: WO9615576 (1996-05-01), None
patent: WO9702357 (1997-01-01), None
patent: WO9800231 (1998-01-01), None
patent: WO9800705 (1998-01-01), None
patent: WO9805959 (1998-02-01), None
patent: WO9858074 (1998-12-01), None
patent: WO9964840 (1999-12-01), None
patent: WO-99/64840 (1999-12-01), None
Vives et al. (Journal of Biological Chemistry, vol. 272, No. 25, 1997, 16010-16017).*
Derossi et al. (Trends in Cell Biology, vol. 8, Feb. 1998, pp. 84-87).*
Crino et al. (PNAS, vol. 95, pp. 2313-2318).*
Checovich et al. (Nature, vol. 375, May 1995, pp. 254256).*
Andersson, P.E. et al., “Biological Cell Assays on an Electrokinetic Microchip” Transducers 97 1997 International Conference on Solid-State Sensors and Actuators, Chicago (Jun. 16-19, 1997) pp. 1311-1314.
Bass, D.A. et al., “Flow Cytometric Studies of Oxidative Product Formation by Neutrophils: A Graded Response to Membrane Stimulation”J. Immunol. (1983) 130(4):1910-1917.
Brunk, K.D. et al., “Quantifying Folling Adhesion with a Cell-Free Assay: E-Selection an its Carbohydrate
Farinas Javier A.
Nikiforov Theo T.
Wada H. Garrett
Caliper Technologies Corp.
Jones W. Gary
Murphy Matthew B.
Taylor Janell E.
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