Methods and compositions for live cell analysis of intracellular ATP
Inventors
APPLEDORN, Daniel • SCHRAMM, Cicely • Filonov, Grigory • Schroeder, Kirk
Assignees
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Publication Number
US-12158470-B2
Publication Date
2024-12-03
Expiration Date
Abstract
Disclosed herein are ATP biosensor fusion proteins and their use for assaying ATP levels in cells.
Core Innovation
The invention provides genetically encoded ATP biosensor fusion proteins that comprise a FRET donor polypeptide and a FRET acceptor polypeptide linked by defined segment options to an ATP binding protein. In particular, the FRET acceptor polypeptide is defined by the amino acid sequence of SEQ ID NO: 1 and the FRET donor polypeptide is defined by the amino acid sequence of SEQ ID NO: 4, while an ATP binding protein is defined by the amino acid sequence of SEQ ID NO: 5. The fusion protein genus is described as X1-X2-X3-X4-X5, with ATP binding protein positioned to mediate regulation of the FRET interaction.
ATP binding by the ATP binding protein causes interaction of the FRET acceptor polypeptide and the FRET donor polypeptide, so that ATP level can be quantified in live-cell via fluorescence resonance energy transfer. The described implementation uses dual-excitation ratiometric FRET imaging in which the FRET ratio is proportional to the ATP level. Control fusion proteins are also provided to enable background correction, including constructs with an ATP-nonbinding ATP binding protein mutant using the specified SEQ ID NO: 12 or 13 control forms.
The disclosure further includes polynucleotides encoding the ATP-regulated FRET fusion proteins, expression vectors, recombinant host cells, and kits that provide the defined sensor and control components. Documented use includes live-cell and automated imaging captured in an incubator without removing cells, with example analyses showing differences in the signal window for linker options and ATP depletion kinetics in drug-treated cells. The described imaging readout is used for metabolic vulnerability and toxicity categorization, including comparisons across cell lines and co-culture effects, under perturbations involving glucose/galactose switch and therapeutic agents.
Claims Coverage
The independent claim is clm-00001. It covers a polynucleotide encoding an ATP-regulated FRET fusion protein in which an ATP binding protein mediates interaction between fixed FRET acceptor and FRET donor polypeptides, with defined link segment options; dependent claims further specify expression vectors, recombinant host cells, kit formats with control polynucleotides, and additional selectable fusion protein sequences from enumerated SEQ ID NO sets.
ATP-regulated FRET fusion polynucleotide with fixed donor/acceptor and defined ATP binding protein
A polynucleotide comprising a nucleic acid encoding a fusion protein of genus X1-X2-X3-X4-X5, where X1 is a FRET acceptor polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and X5 is a FRET donor polypeptide consisting of the amino acid sequence of SEQ ID NO: 4; X3 is an ATP binding protein consisting of the amino acid sequence of SEQ ID NO: 5; X2 and X4 are selected from the enumerated options; and binding of ATP by the ATP binding protein causes interaction of the FRET acceptor polypeptide and the FRET donor polypeptide.
Operatively promoter-linked expression vector encoding the ATP-regulated FRET fusion polypeptide
An expression vector in which the polynucleotide is operatively linked to a promoter sequence that drives expression of a fusion protein.
Recombinant host cell containing the expression vector
A recombinant host cell that includes the expression vector recited in the promoter-linked expression vector claim.
Kit including the ATP-regulated fusion polynucleotide and a control polynucleotide encoding a defined control fusion protein
A kit containing the polynucleotide together with a control polynucleotide encoding a control fusion protein defined by specified SEQ ID NO sequences and allowed amino-acid segment options for X2 and X4, with X3 fixed to SEQ ID NO: 12 and X1 and X5 fixed to SEQ ID NO: 1 and SEQ ID NO: 4, respectively.
Selectable fusion protein amino acid sequences defined by enumerated SEQ ID NO options
A polynucleotide that encodes a fusion protein containing an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
Overall, the claim set centers on a polynucleotide encoding an ATP-regulated FRET fusion protein in which ATP binding by an ATP binding protein (SEQ ID NO: 5) drives interaction between fixed FRET acceptor (SEQ ID NO: 1) and fixed FRET donor (SEQ ID NO: 4) polypeptides, with enumerated X2 and X4 segment options. Dependent claims extend coverage to promoter-linked expression vectors, recombinant host cells, and kit formats that include control polynucleotides encoding defined control fusion proteins, plus additional selectable fusion protein sequences from enumerated SEQ ID NO options.
Stated Advantages
Enables quantification of ATP in live-cell via FRET where a FRET ratio is proportional to ATP level.
Provides control fusion proteins for background correction using ATP-nonbinding ATP-binding protein variants.
Supports live-cell and automated imaging captured without removing cells.
Documented Applications
Metabolic vulnerability and toxicity categorization using glucose/galactose switch and therapeutic agents.
Assessment of ATP depletion kinetics in drug-treated cells.
Comparison between cell lines and evaluation of co-culture effects in the described metabolic vulnerability/toxicity categorization context.
Use with stable cell lines and recombinant systems (including described imaging workflows) for intracellular ATP measurement.
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