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US 2022/0404337 A1Published application
Real-time g-protein coupled receptor (gpcr) linked bioluminescent sensing of biological targets and processes
AssigneeMassachusetts Institute of Technology
InventorsFrances Nargi, Scott Thomas Wick, Deepak Mishra
Life of this patent
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Abstract
The invention relates to compositions and methods for making and use of a real-time cellular sensor. Components of a multipart enzyme are sequestered in different cellular compartments and only come together after receptor activation; a pool of substrate is made available in the cell to ensure real-time enzymatic output.
Claims23
A recombinant cell, comprising a constitutively expressed exogenous nucleic acid encoding:
- (a) one or more proteins that produce a measurable enzymatic output, each protein comprising at least two different components, a first component anchored to a nuclear-localized protein and a second component tethered to a plasma membrane-bound receptor by a linker that comprises a protease cleavage site, wherein at least one of each of the at least two components comes together to produce enzymatic output;
- and
- (b) an enzyme complex that synthesizes a substrate for enzymatic output of the different polypeptide components,
- wherein, binding of a ligand to the plasma-membrane-bound receptor results in activation of the receptor, cleavage of the linker by a protease, and release of the second component tethered to the receptor.
1 more element
A method of detecting receptor activation, comprising:
- (a) introducing to a cell a constitutively expressed nucleic acid encoding:
- (i) one or more proteins that produce a measurable enzymatic output, each protein comprising at least two different components, a first component anchored to a nuclear-localized protein and a second component tethered to a plasma membrane-bound receptor by a linker that comprises a protease cleavage site, wherein at least one of each of the at least two components comes together to produce enzymatic output;
- and
- (ii) an enzyme complex that synthesizes a substrate for enzymatic output of the different polypeptide components,
- wherein, binding of a ligand to the plasma-membrane-bound receptor results in activation of the receptor, cleavage of the linker by a protease, and release of the second component tethered to the receptor;
- (iii) measuring levels of enzymatic output
- wherein detectable levels of enzymatic output indicate receptor activation.
4 more elements
(canceled)
A biological circuit, comprising:
- (a) a recombinant cellular sensor, comprising a membrane-bound receptor protein, translationally fused to a first portion of a multi-part enzyme by a protease cleavable linker, wherein the first portion of the multi-part enzyme further comprises a nuclear localization sequence;
- (b) a second portion of the multi-part enzyme where the second portion of the multi-part enzyme is confined to the nucleus, mitochondria, endoplasmic reticulum, or other cell membrane-integrated protein;
- and
- (c) a pool of substrate for the multi-part enzyme
- wherein, binding of a ligand to the plasma-membrane-bound receptor results in activation of the membrane-bound receptor, cleavage of the linker at the protease cleavage site, and the first portion and the second portion of the multi-part enzyme come together, processing the pool of substrate molecules to generate a measurable signal.
2 more elements
(canceled)
A recombinant cell, comprising a constitutively active exogenous nucleic acid encoding:
- (a) a recombinant sensor, comprising:
- (i) a ligand binding domain;
- (ii) a transmembrane domain;
- (iii) a protease cleavage site;
- (iv) an anchor site sequence such as a nuclear-localization sequence;
- and
- (v) a second component of a two-part enzyme,
- wherein the protease cleavage site is between the transmembrane domain and the anchor site sequence;
- (b) a nuclear receiver comprising:
- (i) a first component of the two-part enzyme;
- (ii) a nuclear-localized protein;
- and
- (c) an enzyme complex that produces a substrate, a co-factor, or both a substrate and a co-factor.
10 more elements
19-20. (canceled)
A biosensor device comprising:
- a housing comprising
- (a) a surface for accepting a ligand,
- (b) a recombinant cell within the housing having a biological circuit, wherein the biological circuit comprises a multi-part enzyme, wherein two or more parts of the multi-part enzyme have spatially distinct localizations and wherein when the recombinant cell is exposed to the ligand, the two or more parts of the multi-part enzyme are triggered to co-localize and interact with a substrate molecule to generate a measurable immediate read-out bioluminescent signal, and
- (c) a surface for observing the bioluminescent signal.
1 more element
22-31. (canceled)
A method for sensing a ligand in real-time, comprising,
- exposing a putative ligand containing material to a biosensor device,
- examining the biosensor device for the presence of a bioluminescent signal immediately upon exposure to the putative ligand containing material, wherein the presence of a bioluminescent signal in the biosensor device indicates the presence of the ligand in the material in real-time,
- wherein the biosensor device comprises a recombinant cell having a biological circuit, wherein the biological circuit comprises a multi-part enzyme, wherein two or more parts of the multi-part enzyme have spatially distinct localizations and wherein when the recombinant cell is exposed to the ligand, the two or more parts of the multi-part enzyme are triggered to co-localize and interact with a substrate molecule to generate a measurable immediate read-out bioluminescent signal indicative of the presence of the ligand.
Dependencies are inferred from the claim text.