FRET biosensor for detecting and reporting NAD+/NADH ratio changes
Inventors
Filonov, Grigory • SCHRAMM, Cicely
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Publication Number
US-12338484-B2
Publication Date
2025-06-24
Expiration Date
Abstract
FRET-based fusion protein biosensors and methods for their use in measuring an NAD+/NADH ratio change in live cells are provided.
Core Innovation
The invention relates to a polynucleotide encoding a fusion protein, wherein the fusion protein comprises an amino acid sequence selected from specified residue-length portions of defined SEQ ID NOs. The disclosed embodiments include residues corresponding to SEQ ID NO:13 through SEQ ID NO:24 and additional embodiments comprising residues of SEQ ID NO:191, SEQ ID NO:192, and SEQ ID NO:193.
The invention relates to Rex-family FRET fusion protein NAD+/NADH biosensors that form a homodimer binding NADH or NAD+. Binding drives conformational changes that bring a FRET donor/acceptor pair into interaction to report a NAD+/NADH redox state. The constructs are defined using RexA and RexB domain elements and include FRET donor and acceptor polypeptide elements, producing a biosensor signal behavior tied to NADH or NAD+ binding.
The fusion protein architecture is defined as X1-B1-X2-B2-X3-B3-X4, with an optional X5 element. Truncated RexA/RexB domains are used to form the NADH/NAD+ binding homodimer, while the FRET donor/acceptor chromophores are selected from fluorescence chromophore options, including acceptors such as mKOk, mKO, and mKO2, and circularly permuted EGFP donors such as mEGFP and cpmEGFP (173/174), with the CYG and TYG chromophores referenced.
The disclosed designs also define domain identity/percent identity thresholds for the fusion components and include optional linker elements for B1/B2/B3 to connect the domain and chromophore segments. Control fusion proteins include point mutations to render non-responsive variants, including a G89A mutation in X1 and a G84A mutation in X3. The partial content also describes control non-responsiveness to NAD+/NADH ratio change, ratio correction using control sequences, and live-cell imaging using acceptor/donor excitation/emission fluorescence images to quantify FRET ratio and NAD+/NADH ratio changes.
Claims Coverage
The provided independent claim set covers polynucleotide-encoded fusion proteins limited to specified residue-length portions from defined SEQ ID NOs. Across the independent-claim family described, the main inventive features focus on limiting the encoded fusion protein sequences to particular N-terminal residue ranges, optionally adding control variants constrained by specified point mutations, and providing polynucleotide formats in expression vectors and kits.
Polynucleotide encoding a fusion protein with specified residue-length portions
A polynucleotide encoding a fusion protein, wherein the fusion protein comprises the amino acid sequence selected from the group consisting of residues 1-867 of SEQ ID NO:13; residues 1-861 of SEQ ID NO:14; residues 1-863 of SEQ ID NO:15; residues 1-862 of SEQ ID NO:16; residues 1-865 of SEQ ID NO:20; residues 1-853 of SEQ ID NO:24; residues 1-868 of SEQ ID NO:191; residues 1-867 of SEQ ID NO:192; and residues 1-866 of SEQ ID NO:193.
Expression vector with promoter-operably linked polynucleotide
An expression vector wherein the polynucleotide encodes the fusion protein and is operatively linked to a promoter sequence.
Fusion protein limited to a specified subset of SEQ ID NOs
A polynucleotide encoding a fusion protein containing an amino acid sequence selected from residues 1-868 of SEQ ID NO:191, residues 1-867 of SEQ ID NO:192, or residues 1-866 of SEQ ID NO:193.
Polynucleotide encoding the SEQ ID NO:191 residue range
A polynucleotide in which the encoded fusion protein contains the amino acid sequence corresponding to residues 1-868 of SEQ ID NO:191.
Kit including polynucleotide and control polynucleotide with G89A control variants
A kit that includes a polynucleotide and a control polynucleotide encoding a control fusion protein whose sequence is defined by specified residue ranges from SEQ ID NOs:191-194, including variants with a G89A mutation relative to SEQ ID NOs:191-193.
Overall, the claim coverage provided centers on polynucleotides encoding Rex-family FRET NAD+/NADH biosensor fusion proteins with amino acid sequences restricted to defined N-terminal residue-length portions from specified SEQ ID NOs, with additional coverage for promoter-linked expression vectors and kit formats that include control fusion protein polynucleotides constrained by specified point mutations.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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