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US 6,280,981 B1Granted
Compositions and methods for assaying subcellular conditions and processes using energy transfer
AssigneeMitoKor
InventorsJames A. Dykens, Gönül Veliçelebi, Soumitra S. Ghosh
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Abstract
The invention is provides compositions and methods for monitoring subcellular compartments such as organelles by energy transfer techniques that do not require specific intermolecular affinity binding events between energy transfer donor and energy transfer acceptor molecules. Provided are methods for assaying cellular membrane potential, including mitochondrial membrane potential, by energy transfer methodologies including fluorescence resonance energy transfer (FRET). Diagnostic and drug screening assays are also provided.
Claims24
A method for identifying an agent that alters mitochondrial membrane potential, comprising the steps of:
- (a) contacting, in the absence and presence of a candidate agent, a sample comprising one or more mitochondria simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the mitochondria, wherein:
- (i) the first and second energy transfer molecules each localize independently of one another to a same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and
- (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule;
- (b) exciting said energy donor molecule to produce an excited energy donor molecule;
- (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential;
- and
- (d) comparing the signal generated in the absence of the candidate agent to the signal generated in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial membrane potential.
4 more elements
A method for identifying an agent that preferentially alters mitochondrial membrane potential in mitochondria from a first biological source without substantially altering mitochondrial membrane potential in mitochondria from a second biological source, comprising the steps of:
- (a) contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more mitochondria simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the mitochondria, wherein:
- (i) the first sample is derived from a first biological source and the second sample is derived from a second biological source that is distinct from the first biological source,
- (ii) the first and second energy transfer molecules each localize independently of one another to a same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and
- (iii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule;
- (b) exciting said energy donor molecule to produce an excited energy donor molecule in the presence of each of said first and second samples;
- (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule in the presence of each of said first and second samples, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential;
- and
- (d) comparing the signal generated in the presence of each of said first and second samples in the absence of the candidate agent to the signal generated in the presence of each of said first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters mitochondrial membrane potential.
5 more elements
A method for identifying an agent that preferentially alters mitochondrial membrane potential in mitochondria from a first biological sample without substantially altering mitochondrial membrane potential in mitochondria from a second biological sample, comprising the steps of:
- (a) contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more mitochondria simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the mitochondria, wherein:
- (i) the first sample is derived from a first tissue and the second sample is derived from a second tissue that is distinct from the first tissue,
- (ii) the first and second energy transfer molecules each localize independently of one another to a same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and
- (iii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule;
- (b) exciting said energy donor molecule to produce an excited energy donor molecule in the presence of each of said first and second samples;
- (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule in the presence of each of said first and second samples, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential;
- and
- (d) comparing the signal generated in the presence of each of said first and second samples in the absence of the candidate agent to the signal generated in the presence of each of said first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters mitochondrial membrane potential.
5 more elements
A method for identifying a mitochondrial membrane potential (Δψm) stabilizing agent, comprising the steps of:
- (a) contacting, in the absence and presence of a candidate Δψm stabilizing agent, (1) an agent that alters Δψm and (2) a sample comprising one or more mitochondria simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the mitochondria, wherein:
- (i) the first and second energy transfer molecules each localize independently of one another to a same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and
- (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule;
- (b) exciting said energy donor molecule to produce an excited energy donor molecule;
- (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential;
- and
- (d) comparing the signal generated in the absence of the candidate Δψm stabilizing agent, to the signal generated in the presence of the candidate Δψm stabilizing agent, and therefrom identifying Δψm stabilizing agent.
4 more elements
Dependencies are inferred from the claim text.