Real-time monitoring

Abstract

Provided herein are methods for the real-time monitoring of an intracellular event or response. In particular, the methods provided herein monitor the conversion of a pro-substrate to a substrate for a protein sensor as a result of an intracellular event or response.

1 . A method of detecting an intracellular event, condition, or response comprising:
 detecting a signal generated by the interaction of a substrate and a protein sensor;   wherein the substrate is the product of an interaction of the pro-substrate with an agent associated with the intracellular event, condition, or response;   wherein the presence of the signal indicates the occurrence of the intracellular event, condition, or response in real time;   wherein the absence of an agent associated with the intracellular event, condition, or response results in the real-time absence of the signal;   and wherein the magnitude of the signal correlates with the amount of the agent.   
     
     
         2 . The method of  claim 1 , wherein the detecting comprises one or more of: real-time monitoring, continuous monitoring of said signal, and continuous monitoring of intracellular events in live cells over extended period of time. 
     
     
         3 . The method of  claim 1 , wherein the pro-substrate is stable and continuously enters the cell. 
     
     
         4 . The method of  claim 1 , wherein the pro-substrate is converted to a substrate by an intracellular event, condition or response and continuously exits the cell. 
     
     
         5 . The method of  claim 1 , wherein the substrate generates a signal by interaction with a protein sensor placed outside the cell. 
     
     
         6 . The method of  claim 1 , wherein the substrate is unstable. 
     
     
         7 . The method of  claim 1 , wherein the substrate does not accumulate extracellularly following the interaction of the pro-substrate with the agent associated with the intracellular event, condition, or response. 
     
     
         8 . The method of  claim 1 , wherein substantially all of the substrate generated by interaction of the pro-substrate with the agent associated with the intracellular event, condition, or response is either degraded or utilized by the protein sensor. 
     
     
         9 . The method of  claim 1 , wherein the signal generated by the interaction of the substrate and the protein sensor only persists when substrate is continually produced by interaction of the pro-substrate with the agent associated with the intracellular event, condition, or response. 
     
     
         10 . The method of  claim 1 , wherein increases and decreases in the concentration of the agent associated with the intracellular event, condition, or response result in corresponding increases and decreases in the signal generated by the interaction of the substrate and the protein sensor. 
     
     
         11 . The method of  claim 1 , wherein the signal is a real-time signal. 
     
     
         12 . The method of  claim 1 , wherein the signal is a non-accumulated signal. 
     
     
         13 . The method of  claim 1 , wherein the substrate is capable of exiting the cell. 
     
     
         14 . The method of  claim 1 , wherein the protein sensor is provided extracellularly. 
     
     
         15 . The method of  claim 14 , wherein the protein sensor is incapable of entering the cell. 
     
     
         16 . The method of  claim 15 , wherein the interaction of the substrate and the protein sensor occurs extracellularly. 
     
     
         17 . The method of  claim 1 , wherein the live cells are not engineered to express the protein sensor. 
     
     
         18 . The method of  claim 1 , wherein the protein sensor retains activity over the time scale of the assay. 
     
     
         19 . The method of  claim 1 , wherein the protein sensor comprises a luciferase enzyme. 
     
     
         20 . The method of  claim 19 , wherein the luciferase enzyme comprises an  Oplophorus , beetle,  Renilla  or  Gaussia  luciferase. 
     
     
         21 . The method of  claim 20 , wherein the luciferase enzyme comprises an  Oplophorus  luciferase with enhanced protein stability, enhanced bioluminescence, and/or enhanced signal stability. 
     
     
         22 . The method of  claim 19 , wherein the pro-substrate comprises a pro-furimazine, pro-furimazine derivative, pro-coelenterazine, pro-coelenterazine derivative or analog, pro-luciferin, or pro-luciferin derivative. 
     
     
         23 . The method of  claim 1 , wherein the agent comprises a caspase, a protease, a reactive oxygen species, a cytochrome P450 (CYP450) enzyme, or monoamine oxidase (MAO), reductase, dehydrogenase, or oxidoreductase. 
     
     
         24 . A kit for detecting an intracellular event or response, the kit comprising:
 a) a protein sensor, wherein the protein sensor generates a detectable signal upon interaction with a substrate, and   b) a pro-substrate, wherein the pro-substrate is capable of entering a cell, wherein interaction of the pro-substrate with an agent associated with the intracellular event, condition, or response converts the pro-substrate into a substrate for the protein sensor.   
     
     
         25 . A method of detecting an event, condition, or response comprising:
 detecting a signal generated by the interaction of a substrate and a protein sensor;   wherein the substrate is the product of an interaction of the pro-substrate with an agent associated with the event, condition, or response;   wherein the presence of the signal indicates the occurrence of the event, condition, or response in real time;   wherein the absence of an agent associated with the event, condition, or response results in the real-time absence of the signal;   and wherein the magnitude of the signal correlates with the amount of the agent.