Sequential reporter enzyme luminescence (SRL) methods and compositions for practicing the same
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsTom Wehrman, Georges J. Von Degenfeld, Helen M. Blau
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Abstract
Sequential reporter enzyme luminescence (SRL) methods are provided. In the subject methods, the activity of a reporter enzyme is evaluated using a secondary reporter system that employs a product of a reporter enzyme mediated reaction as a luminescent substrate, e.g., luciferase substrate. Also provided are kits and other compositions that find use in practicing the subject methods.
Claims51
A method of evaluating the activity of a reporter enzyme associated with an intact cell, said method comprising:
- (a) contacting an intact cell associated with a reporter enzyme with a prosubstrate, wherein said intact cell comprises a luminescent enzyme and wherein said prosubstrate includes:
- (i) a reporter enzyme cleavable domain that is cleavable by said reporter enzyme;
- and
- (ii) a luminescent enzyme substrate for said luminescent enzyme, wherein said reporter enzyme cleavable domain impairs the ability of said luminescent enzyme substrate from acting as a substrate for said luminescent enzyme;
- and
- (b) detecting a signal generated from conversion of said luminescent enzyme substrate to a luminescent product by said luminescent enzyme to evaluate the activity of said reporter enzyme.
A method of evaluating the activity of a reporter enzyme associated with a cell in vitro, said method comprising:
- (a) contacting a cell associated with a reporter enzyme in vitro with prosubstrate, wherein said cell comprises a luciferase and wherein said prosubstrate includes:
- (i) a reporter enzyme cleavable domain that is cleavable by said reporter enzyme;
- and
- (ii) a luciferase substrate for said luciferase, wherein said reporter enzyme cleavable domain impairs the ability of said luciferase substrate from acting as a substrate for said luciferase;
- and
- (b) detecting a signal generated from conversion of said luciferase substrate to a luminescent product by said luciferase to evaluate the activity of said reporter enzyme.
A method of evaluating the activity of a reporter enzyme in a multicellular organism, said method comprising:
- (a) administering to a multicellular organism associated with a reporter enzyme a prosubstrate, wherein said multicellular organism comprises a luciferase and wherein said prosubstrate includes:
- (i) a reporter enzyme cleavable domain that is cleavable by said reporter enzyme;
- and
- (ii) a luciferase substrate for said luciferase, wherein said reporter enzyme cleavable domain impairs the ability of said luciferase substrate from acting as a substrate for said luciferase;
- and
- (b) detecting a signal generated from conversion of said luciferase substrate to a luminescent product by said luciferase to evaluate the activity of said reporter enzyme.
Dependencies are inferred from the claim text.