Fluorescent protein sensors of post-translational modifications
AssigneeAurora Biosciences Corporation
InventorAndrew B. Cubitt
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Abstract
The present invention includes a fluorescent compound that can detect an activity. such as an enzymatic activity, and exhibits quenching. The fluorescent compound can include a fluorescent protein, such as an Aequorea-related green fluorescent protein. The fluorescent compound can include a substrate site for an enzymatic activity such as a kinase activity, a phosphatase activity, a protease activity, and a glycosylase activity The fluorescent compound of the present invention can be used to detect such enzymatic activities in samples, such as biological samples, including cells. The present invention also includes nucleic acids that encode the fluorescent compounds of the present inventions, and cells that include such nucleic acids or fluorescent compounds.
Claims24
A fluorescent compound for detecting an activity, comprising:
- a fluorescent protein moiety, and
- at least one exogenous substrate recognition motif for an activity,
- wherein said fluorescent protein moiety can be converted from a first state to a second state in response to said activity,
- further wherein said fluorescent compound exhibits at least one different fluorescent property in said first state and said second state under quenching conditions.
A nucleic acid molecule coding for the expression of a fluorescent compound,
- wherein said fluorescent compound comprises
- a fluorescent protein moiety, and
- at least one exogenous substrate motif for an activity,
- further wherein said fluorescent protein moiety can be converted from a first state to a second state by an activity,
- further wherein said first state and said second state can be differentiated under quenching conditions.
A cell, comprising:
- a nucleic acid molecule coding for the expression of a fluorescent compound, wherein said fluorescent compound comprises
- a fluorescent protein moiety, and
- at least one exogenous substrate motif for an activity,
- wherein said fluorescent protein moiety can be converted from a first state to a second state by an activity,
- further wherein said first state and said second state can be differentiated under quenching conditions.
A method for determining whether a sample contains an activity, comprising:
- contacting a sample with a fluorescent compound,
- wherein said fluorescent compound comprises
- a fluorescent protein moiety, and
- at least one exogenous substrate motif for an activity,
- further wherein said fluorescent protein moiety can be converted
- from a first state to a second state by an activity,
- exciting said fluorescent compound, and
- measuring the amount of emission from said fluorescent compound.
A method for determining whether a cell exhibits an activity comprising the steps of:
- exciting a transfected host cell comprising a recombinant nucleic acid molecule,
- wherein said recombinant nucleic acid molecule comprises at least one expression control sequence operatively linked to a nucleic acid sequence coding for the expression of a fluorescent compound,
- wherein said fluorescent compound comprises
- a fluorescent protein moiety, and
- a substrate motif for an activity,
- further wherein said fluorescent compound can be converted from a first state to a second state in the presence of said activity,
- further wherein said first state and said second state and be differentiated under quenching conditions, and
- measuring the emission from said fluorescent compound.
A method for determining whether a sample contains an enzymatic activity, comprising:
- a) contacting a sample with a fluorescent compound,
- wherein said fluorescent compound comprises
- an Aequorea-related fluorescent protein moiety and
- an exogenous substrate motif for an enzymatic activity,
- further wherein said fluorescent compound can be converted from a first state to a second state by said enzymatic activity,
- further wherein said first state and said second state can be differentiated under quenching conditions,
- b) exciting said fluorescent compound, and
- c) measuring the amount of fluorescence emitted from said sample, whereby the amount of quenching that is consistent with the presence of said enzyme activity indicates the presence of said enzyme activity in said sample.
A compound identified by the method comprising the steps of:
- a) contacting a sample with a fluorescent compound,
- wherein said fluorescent compound comprises
- a fluorescent protein moiety and
- an exogenous substrate motif for an activity,
- further wherein said fluorescent compound can be converted from a first state to a second state by said activity,
- further wherein said first state and said second state can be differentiated under quenching conditions,
- b) exciting said fluorescent compound, and
- c) measuring the amount of fluorescence emitted from said sample.
Dependencies are inferred from the claim text.