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.

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.