Methods and systems for monitoring intracellular binding reactions
First Claim
1. A method of detecting intracellular binding interactions, comprising:
- providing a biological cell having at least a first component of a binding reaction disposed therein;
contacting the cell with a second component of the binding reaction whereby the second component is internalized within the biological cell, the second component comprising a non-protein molecule, at least one of the first and second components having a fluorescent label associated therewith; and
determining an amount of binding between the first and second components within the cell by measuring a level of polarized fluorescence emitted from within the biological cell.
1 Assignment
0 Petitions
Accused Products
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.
145 Citations
33 Claims
-
1. A method of detecting intracellular binding interactions, comprising:
-
providing a biological cell having at least a first component of a binding reaction disposed therein;
contacting the cell with a second component of the binding reaction whereby the second component is internalized within the biological cell, the second component comprising a non-protein molecule, at least one of the first and second components having a fluorescent label associated therewith; and
determining an amount of binding between the first and second components within the cell by measuring a level of polarized fluorescence emitted from within the biological cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
directing a polarized excitation light at the cell;
measuring a level of polarized or depolarized fluorescence emitted from within the cell; and
comparing a level of polarized or depolarized fluorescence emitted from within the cell to a control, to determine a level of the intracellular binding reaction that occurred in the cell.
-
-
22. The method of claim 1, wherein the cell is provided in a suspension of cells.
-
23. The method of claim 1, wherein the cell is disposed in a reaction vessel having a window for optically accessing the cell.
-
24. The method of claim 23, wherein the reaction vessel comprises a test tube.
-
25. The method of claim 23, wherein the reaction vessel comprises a cuvette.
-
26. The method of claim 23, wherein the reaction vessel comprises a well in a multiwell plate.
-
27. The method of claim 23, wherein the reaction vessel comprises at least a first fluidic channel.
-
28. The method of claim 27, wherein the first fluidic channel comprises at least a first microscale fluidic channel disposed within a body structure.
-
29. The method of claim 28, wherein the microscale fluidic channel comprises a first of at least two intersecting microscale channels disposed in the body structure.
-
30. The method of claim 1, further comprising:
-
exposing said cell to a test compound prior to the determining step; and
comparing an amount of binding between the first and second components within the cell 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, the comparing step occurring after the determining step.
-
-
31. The method of claim 30, wherein the exposing and comparing steps are separately repeated for a plurality of different test compounds.
-
32. A method of monitoring activation of a cell signaling pathway, comprising:
-
providing a cell which comprises a cell signaling pathway, wherein at least one step in the cell signaling pathway comprises an intracellular binding interaction between a nucleic acid and a nucleic acid binding protein;
contacting the cell with a first nucleic acid whereby the first nucleic acid is internalized within the cell, wherein the first nucleic acid comprises a fluorescent label and is capable of being bound by the nucleic acid binding protein; and
monitoring a level of binding between the nucleic acid binding protein and the first nucleic acid by monitoring a level of depolarized fluorescence emitted from within the cell, the amount of depolarized fluorescence being indicative of a level of activation of the cell signaling pathway.
-
-
33. A method of screening for effectors of cellular function, comprising:
-
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;
inserting a first nucleic acid having a fluorescent label associated therewith into the cell, wherein the first nucleic is capable of being bound by the nucleic acid binding protein;
exposing the cell to a test compound;
determining an amount of binding between the first and second components within the cell by measuring a level of polarized fluorescence emitted from within the biological cell; and
comparing an amount of binding between the first and second components within the cell 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.
-
Specification