FRET Biosensor for Detecting and Reporting NAD+/NADH Ratio Changes

Cross Reference

This application claims priority to U.S. Application Serial No. 17/812,030 filed July 12, 2022, incorporated by reference herein in its entirety.

Sequence Listing Statement:

A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on June 26, 2023 having the file name “22- 0505-WO_SeqList.xml” and is 389,395 bytes in size.

Background

Nicotinamide adenine dinucleotide NAD(H) is a key cofactor for electron transfer in metabolism. Reactions utilizing NAD(H) as a cofactor are extremely important for glycolysis and mitochondrial metabolism and thus for cellular survival and normal functioning.

NAD(H) exists in two forms: oxidized NAD+ and reduced NADH. The ratio of free concentrations of oxidized and reduced forms of NAD(H) (NAD+/NADH) is an important indicator and regulator of cellular reduction-oxidation (redox) state. NAD+/NADH ratio has been reported to regulate embryonic development, gene expression, aging, and cell death. Additionally, NAD+/NADH ratio and thus cellular redox state has been implicated in development of a number of pathological conditions, such as cancer and diabetes.

Standard methods of assessing NAD+/NADH ratio changes in cells are end point assays which require cell lysis and often involve time-consuming sample preparation and/or assay workflows.

Summary

In a first aspect, the disclosure provides fusion proteins, comprising the genus Xl-Bl- X2-B2-X3-B3-X4, wherein:

XI comprises the amino acid sequence of a first Rex protein domain (RexA),

1

SUBSTITUTE SHEET ( RULE 26) one of X2 and X4 comprises a fluorescence resonance energy transfer (FRET) acceptor polypeptide having an acceptor excitation wavelength and FRET emission wavelength, and the other of X2 and X4 comprises a FRET donor polypeptide having a donor excitation wavelength and a donor emission wavelength;

X3 comprises the amino acid sequence of a second Rex protein domain (RexB),

Bl, B2, and B3 are independently absent or comprise an amino acid linker; wherein the XI and X3 domains are capable of forming a homodimer that can bind to either NADH or NAD+ and changing conformation of the fusion protein and causing interaction of the FRET acceptor polypeptide and the FRET donor polypeptide.

In one embodiment, XI comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the ammo acid sequence of SEQ ID NO:1 (RexA), wherein residues in parentheses are optional and may be present or absent

(M) KVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPV LKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGV IEHVDLLP QRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTR LSFAILN P ( SEQ ID NO : 1 ) ; and

X3 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO:2 (RexB).

In another embodiment, the FRET acceptor polypeptide comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of one or more of SEQ ID NOS: 3-5, and identical at the CYG chromophore. In a further embodiment, the FRET donor polypeptide comprises an amino acid sequence at least 85%, 87%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6, 7, or 155, and identical at the TYG chromophore.

In one embodiment, the fusion protein comprises the genus X1-B1-X2-B2-X3-B3-X4, wherein: one of X2 and X4 comprises an ammo acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6 or 7; and

2

SUBSTITUTE SHEET ( RULE 26) the other of X2 and X4 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO:3, 4, or 5.

In one embodiment, XI comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 8 or 9 (RexA), wherein residues in parentheses are optional and may be present or absent

(M) KVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPV LKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGV IEHVDLLP QRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTR LSFAILN PT (SEQ ID NO : 8) ; or

(M) KVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPV LKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGV IEHVDLLP QRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTR LSFAILN PTW (SEQ ID NO : 9)

In another embodiment, X2 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.

In one embodiment, X3 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In another embodiment, X4 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12.

In another embodiment, the fusion protein comprises the genus X1-B1-X2-B2-X3- B3-X4-X5, wherein X5 comprises the amino acid sequence MDELYK (SEQ ID NO: 156), EASMDELYK (SEQ ID NO: 157), or- EASTSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 158) .

In various embodiments, the fusion protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO:13-93, 95-154, and 191-193.

In a second aspect, the disclosure provides control fusion protein comprising the fusion protein of any embodiment of the first aspect, with the proviso that XI and X3

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SUBSTITUTE SHEET ( RULE 26) comprises a mutation to confer non-responsiveness to changes in the ratio of NAD+/NADH. In one embodiment, the mutation comprises

(a) a G89A mutation in XI relative to SEQ ID NO: 1 residue numbering, and

(b) a G84A mutation in X3 relative to SEQ ID NO:2 residue numbering.

In another embodiment, the control fusion protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 13-93, 95-154, and 191-193, but having a G89A mutation.

In other aspects, the disclosure provides a polynucleotide encoding the fusion protein or control fusion protein, expression vectors encoding the polynucleotide operatively linked to a promoter sequence capable of directing expression of the polynucleotide, host cells comprising the fusion protein, control fusion protein, polynucleotide, and/or expression vector, and kits comprising various combinations of the fusion proteins, control fusion proteins, polynucleotides, expression vectors, and host cells of the disclosure.

In a further aspect, the disclosure provides methods for determining an NAD+/NADH ratio change in a cell of interest, comprising of expressing a FRET biosensor in a cell that undergoes a detectable change upon binding of the FRET biosensor to NAD(H) in the cell, and performing live cell imaging to determine the ratio of NAD+/NADH inside living cells.

Description of the Figures

Figure 1A Directed evolution of construct 14 produced a variant (1-F8) with substantially improved signal window. Purified protein 1-F8 was mixed with NAD+ and NADH so in one case NAD+/NADH ratio was 1 and in another 10,000. Total concentration of NAD+ was kept constant at 80 uM in both cases. Excitation spectra of 1-F8 with low and high NAD+/NADH ratio were recorded on a plate reader and then normalized and overlaid. The substantial difference between the spectra (indicated with the arrow) signifies the large signal window of the resulting biosensor.

Figure IB. Directed evolution of construct 10 produced a variant (10-1 1-F7) with improved signal window. Purified protein 10-11-F7 was mixed with NAD+ and NADH so in one case NAD+/NADH ratio was 1 and in another 10,000. Total concentration ofNAD+ was kept constant at 80 uM in both cases. Excitation spectra of 10-1 1-F7 with low and high NAD+/NADH ratio were recorded on a plate reader, and then normalized and overlaid. The noticeable difference between the spectra (indicated with the arrow) signifies now detectable signal window of the resulting biosensor.

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SUBSTITUTE SHEET ( RULE 26) Figure 2. Signal window of selected clones from libraries measured in vitro (purified proteins). Proteins were expressed, purified and treated as described in Fig.l legends, and their excitation spectra was recorded the same way as well. To calculate the signal window, the areas under the high NAD+/NADH ratio and low NAD+/NADH ratio curves were calculated for the 400-526 nm wavelength interval, which is where the FRET signal change is observed. The difference between two calculated areas, normalized to the smallest area of these two, is presented in Figure 2 on the bar graph as signal window/lowest signal. Proteins with higher signal window values were selected for further characterization in mammalian cells.

Figure 3. EC50 (sensitivity) curves of selected clones from libraries based on constructs #10 and #14.

Proteins were expressed, purified, and treated with the mixture of NAD+ and NADH similarly as in the description above. To plot FRET ratios, the proteins were excited at 460 nm and emissions at 510 nm and 560 nm were collected. The FRET ratio was calculated as emission at 560 nm divided by emission at 510 nm, and the values were normalized to the FRET ratio value at the highest NAD+/NADH ratio. Normalized FRET ratio was measured for each protein at different NAD+/NADH ratios, ranging from 1 to 10,000. For each NAD+/NADH ratio final NAD+ concentration was kept constant at 80 uM and NADH concentration was varied.

Figure 4. Determining signal window in mammalian cells.

Proteins were expressed in HEK 293 mammalian cells following transient transfection with the plasmids encoding those proteins. Transfected cells were imaged using an Incucyte® SX5 equipped with a Metabolism Optical Module (Sartorius) and the data was processed using the built-in ATP analysis software module that allows quantification of average FRET ratio in all biosensor-expressing cells in the image. To measure signal window, cells were treated with either 10 mM lactate or 20 mM pyruvate. The former drives the NAD+/NADH ratio, and thus FRET signal down, and the latter drives the NAD+/NADH ratio, and thus FRET signal up. The difference between highest and lowest FRET ratios is the signal window in mammalian cells.

Figure 5. Restoring the brightness of mKOk in clone 1-F8 in mammalian cells. HEK293 cells were transfected with the plasmids encoding the respective constructs. Following protein expression, cells were imaged using Incucyte® SX5 using orange and near-infrared imaging channels. The former channel was used to collect the signal from mKOk while the latter was used to collect emission from a near-infrared protein which came

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SUBSTITUTE SHEET ( RULE 26) from another plasmid that was co-transfected into the same cells. This near-infrared protein’s emission was used for the biosensor protein (mKOk) expression normalization purposes. Construct 1-F8 1-2 shows restoration of the mKOk brightness to the level expected for normally functioning mKOk.

Figure 6. Signal window of 1-F8 1-2 construct remained unchanged in mammalian cells compared to signal window of 1-F8. HEK293 cells were transfected with the plasmids encoding the respective constructs. Following protein expression, cells were treated with either 10 mM lactate or 20 mM pyruvate and then imaged using an Incucyte® SX5 equipped with a Metabolism Optical Module (Sartorius). The data was processed using the built-in ATP analysis software module that allows quantification of average FRET ratio in all cells in the image. The difference between highest and lowest FRET ratios is the signal window in mammalian cells.

Detailed Description

Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; and Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004.

The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless explicitly stated otherwise or the context clearly indicates otherwise.

All embodiments disclosed herein can be combined unless the context clearly indicates otherwise.

In a first aspect, the disclosure provides fusion proteins, comprising the genus X1-B1-X2-B2-X3-B3-X4, wherein:

XI comprises the amino acid sequence of a first Rex protein domain (RexA), one of X2 and X4 comprises a fluorescence resonance energy transfer (FRET) acceptor polypeptide having an acceptor excitation wavelength and FRET emission wavelength, and the other of X2 and X4 comprises a FRET donor polypeptide having a donor excitation wavelength and a donor emission wavelength;

6

SUBSTITUTE SHEET ( RULE 26) X3 comprises the amino acid sequence of a second Rex protein domain (RexB),

Bl, B2, and B3 are independently absent or comprise an amino acid linker; wherein the XI and X3 domains are capable of forming a homodimer that can bind to either NADH or NAD+ and changing conformation of the fusion protein and causing interaction of the FRET acceptor polypeptide and the FRET donor polypeptide.

The fusion protein comprises two truncated subunits of Rex protein that are capable of forming a homodimer that can bind to either NADH or NAD+ and changing conformation of the fusion protein and causing interaction of the FRET acceptor polypeptide and the FRET donor polypeptide. The fusion proteins of this first aspect can be used, for example, to detect and measure NADH/NAD+ ratios in living cells, as detailed in the examples that follow.

In various embodiments, the first and second Rex protein domains may comprise truncated subunits of Rex proteins from Thermus aquaticus (NCBI GenBank AF061257.1), Streptomyces coelicolor (GenBank AL9391.1) or Bacillus subtilis (GenBank AL009126.1). In one embodiment, the first and second Rex protein domains may comprise truncated subunits of Rex proteins from Thermus aquaticus (T-Rex).

In some embodiments:

XI comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO:1 (RexA), wherein residues in parentheses are optional and may be present or absent (M) KVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPV LKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGV IEHVDLLP QRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTR LSFAILN P ( SEQ ID NO : 1 ) ; and

X3 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO:2 (RexB) AAISRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVP VLKRELRH ILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLL PQRVPGRI EIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILN PKWRE ( SEQ ID NO : 2 ) .

FRET is non-radiative transfer of energy from an excited donor fluorophore to a suitable acceptor fluorophore in proximity to the donor. For selection of FRET fluorophore donor/acceptor polypeptide pairs for use in the fusion proteins of the disclosure, the

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SUBSTITUTE SHEET ( RULE 26) absorption and emission wavelengths of each are considered. Based on the teachings herein, one of skill in the art can readily determine which of various fluorophores are to be used as FRET donor/ acceptor polypeptide pairs in a particular application.

Any suitable polypeptide fluorophores may be used, including but not limited to, mKOk, mKO, mK02, and truncations thereof and its derivatives; any of green fluorescent protein and derivatives such as BFP, EBFP, EBFP2, ECFP, RFP, and YFP; and other polypeptide fluorophores.

In one embodiment, X2 comprises a FRET acceptor polypeptide and X4 comprises a FRET donor polypeptide. In another embodiment, X2 comprises a FRET donor polypeptide and X4 comprises a FRET acceptor polypeptide.

In one embodiment, the FRET acceptor polypeptide has a maximal acceptor excitation wavelength in a range of 420 and 710 nm, or in a range of 500 to 560 nm and an acceptor maximal emission wavelength in a range of 460 nm and 720 nm, or a range of 530 to 580 nm.

In another embodiment, the FRET acceptor polypeptide comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of one or more of SEQ ID NOS: 3-5, and identical at the CYG chromophore. Residues in parentheses are optional throughout. The CYG chromophore is highlighted. mKOk (SEQ ID NO: 3)

IKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVS HVFCYGHRVFT KYPEEIPDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSV DWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLV RKTEGNIT EQVEDAVAHS mKO (SEQ ID NO:4)

(MSVIK) PEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAKGGPMPFAFDLVSHVFCY G HRPFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIM QNQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKKILKMPGSHYI SHRLVRKT EGNITELVEDAVA (HS ) mK02 (SEQ ID NO:5)

(MVSVI ) KPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFC Y GHRVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHISLRGNTFYHKSKFTGVNFPADGPI MQNQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQMKTTYKAAKEILEMPGDHY IGHRLVRK TEGNITEQVEDAVA (HS )

In one embodiment, all optional amino acid residues in the FRET acceptor polypeptide are present.

8

SUBSTITUTE SHEET ( RULE 26) In another embodiment, the FRET donor polypeptide has a maximal donor excitation wavelength in a range of 350 nm to 670 nm, or in a range of 450 to 500 nm and a maximal donor emission wavelength in a range of 420 nm to 700 nm, or in a range of 480 to 515 nm. In a further embodiment, the FRET donor polypeptide comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 6, 7, or 155, and identical at the TYG chromophore. mEGFP (SEQ ID NO:6)

LFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL TYGVQCFSRY PDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE YNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS TQSKLSKD PNE KRDHMVL L E FVTAAGI TL

Circularly permuted (cpm) EGFP(173/174): SEQ ID. NO: 7

( DG) SVQLADHYQQNTPIGDGPVLLPDNHYLSTQS (A/K) LSKDPNEKRDHMVLLEFVTAAGITLGMD ELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIE (EA) cpmEGFP(145/146) SEQ ID NO: 155

YNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYL STQSKLSKDPN EKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSV SGEGEGD ATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQER TI FFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

Exemplar}' FRET donor polypeptides having the requisite amino acid sequence identity to SEQ ID NO:7 and which can be used in the fusion proteins of the disclosure are listed in Table 1 below.

Table 1

9

SUBSTITUTE SHEET ( RULE 26)

10

SUBSTITUTE SHEET (RULE 26)

11

SUBSTITUTE SHEET (RULE 26)

12

SUBSTITUTE SHEET (RULE 26)

In one embodiment, the fusion protein comprises the genus X1-B1-X2-B2-X3-B3-X4, wherein: one of X2 and X4 comprises an ammo acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6 or 7; and the other of X2 and X4 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3, 4, or 5. In another embodiment, one of X2 and X4 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6; and the other of X2 and X4 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:3. In a further embodiment, X2 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6; and X4 comprises an ammo acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:3.

Bl, B2, and B3 are independently absent or comprise an amino acid linker. When any of Bl, B2, and/or B3 are present, the linker may comprise any suitable amino acid linker. In one embodiment

Bl is absent, or comprises G, SG, SHG, SAHG (SEQ ID NO: 177), SAGHG (SEQ ID NO: 178), SAAGHG (SEQ ID NO: 179), or SAAGGHG (SEQ IS NO: 180);

B2 is T or is absent; and

B3 is S, GS or is absent.

The various domains may comprise additional amino acid residues. In one nonlimiting embodiment, XI comprises an ammo acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 8 or 9 (RexA), wherein residues in parentheses are optional and may be present or absent

(M) KVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPV LKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGV IEHVDLLP QRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTR LSFAILN PT (SEQ ID NO: 8) ; or

SUBSTITUTE SHEET ( RULE 26) (M) KVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPV

LKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVR GGVIEHVDLLP

QRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAG LTRLSFAILN

PTW (SEQ ID NO : 9)

In another embodiment, XI comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8, and Bl is absent.

In one embodiment, X2 comprises the formula Z1-Z2-Z3, wherein

(a) Z2 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 6;

(b) Z1 is absent or is selected from the group consisting of E, EE, GEE, KGEE

(SEQ ID NO: 181), SKGEE (SEQ ID NO: 182), VSKGEE (SEQ ID NO: 183), and

MVSKGEE (SEQ ID NO: 184); and

(c) Z3 is absent or is selected from the group consisting of G, GM, GMD, GMDE (SEQ ID NO: 185), GMDEL (SEQ ID NO: 186), GMDELY (SEQ ID NO: 187), and GMDELYK (SEQ ID NO: 188).

In one embodiment, X2 comprises an amino acid sequence at least 90%, 91%, 92%,

93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 10.

(MVSKGEE) LFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKED GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV LLPDNHYL STQSKLSKDPNEKRDHMVLLEFVTAAGITL (GMDELYK) (SEQ ID NO : 10)

In another embodiment, X3 comprises the formula Z5-Z6-Z7, wherein

Z6 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2;

Z5 is absent or is selected from the group consisting of E, PE, VPE, and KVPE; and

Z7 is absent or is selected from the group consisting of E, EM, EMM, and EMMG

(SEQ ID NO: 189).

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SUBSTITUTE SHEET ( RULE 26) In one embodiment, X3 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence of SEQ ID NO: 11.

KVPEAAISRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTD GVGYTVPVLKR ELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEH VDLLPQRV PGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSF AILNPKW REEMMG ( SEQ ID NO : 11 )

In another embodiment, X4 comprises the formula Z9-Z 10, wherein

Z9 is absent or is selected from the group consisting of V, SV, VSV, and MVSV (SEQ ID NO: 190); and

Z10 comprises the amino acid sequence of SEQ ID NO:3.

In one embodiment, X4 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12.

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHS EASMDELYK ( SEQ ID NO : 12 )

The fusion proteins may be produced by any suitable means, including but not limited to chemical synthesis and production by recombinant cells. When produced by recombinant cells, the fusion proteins may include additional residues at the N- and/or C-terminus. For example, expression in mammalian or bacterial cells may utilize vectors that add different C- terminal tails to the fusion proteins. In one embodiment, the fusion proteins comprise the genus X1-B1-X2-B2-X3-B3-X4-X5, wherein X5 comprises the amino acid sequence EASMDELYK (SEQ ID NO: 157), MDELYK (SEQ ID NO: 156), or EASTSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 158).

In specific embodiments, the fusion protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 13-93, 95-154, and 191-193; the

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SUBSTITUTE SHEET ( RULE 26) sequences are provided in Table 2 and the examples. The table also provides signal window as demonstrated in bacterial lysates and detailed in the examples that follow.

Table 2

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SUBSTITUTE SHEET ( RULE 26)

17

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18

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

20

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21

SUBSTITUTE SHEET (RULE 26)

22

SUBSTITUTE SHEET (RULE 26)

23

SUBSTITUTE SHEET (RULE 26)

24

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

26

SUBSTITUTE SHEET (RULE 26)

27

SUBSTITUTE SHEET (RULE 26)

28

SUBSTITUTE SHEET (RULE 26)

29

SUBSTITUTE SHEET (RULE 26)

30

SUBSTITUTE SHEET (RULE 26)

31

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In one embodiment, the fusion protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO: 13-43 and 191-193. In another embodiment, the fusion protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO:13-28 and 191-193. In a further embodiment, the fusion protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:13-22 and 191-193. In a still further embodiment, the fusion protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NOT3-14 and 191-193.

In one aspect, the disclosure provides fusion proteins comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 13-93, 95-154, and 191-193, SEQ ID NO: 13-43 and 191-193, SEQ ID NO: 13-28 and 191-193, SEQ ID NO: 13-22 and 191-193, or SEQ ID NOT3-14 and 191-193.

In another aspect, the disclosure provides control fusion proteins comprising the fusion protein of any embodiment or combination of embodiments disclosed above, with the proviso that XI and X3 comprises a mutation to confer non-responsiveness to NAD+/NADH ratio change. The control fusion proteins do not bind to NAD(H) and thus the control biosensor reports on non-NAD(H) related changes of the biosensor activity (e.g., fluorescent protein brightness change due to variation of intracellular pH). Any suitable mutation to confer such non-responsiveness may be employed. In one embodiment, the mutation comprises:

(a) a G89A mutation in XI relative to SEQ ID NO: 1 residue numbering, and

(b) a G84A mutation in X3 relative to SEQ ID NO:2 residue numbering.

Those of skill in the art will be able to determine, based on the teachings herein, the position of the G89A mutation in variations of the RexA sequence (G89A in SEQ ID NOS: 8 and 9) and the G84A mutation in variations of the RexB sequence (G88A in SEQ ID NO:11) . By way of non-limiting example, the position of G84 is highlighted and underlined in SEQ

SUBSTITUTE SHEET ( RULE 26) ID N0:2 below, and SEQ ID NO: 11 includes 4 additional residues at the N-terminus, so that the mutation is G88A in the control fusion protein based on X3 comprising SEQ ID NO: 11.

AAISRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGY TVPVLKRELRH ILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLL PQRVPGRI EIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILN PKWRE ( SEQ ID NO : 2 ) .

KVPEAAISRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTD GVGYTVPVLKR

ELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGV IEHVDLLPQRV

PGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTR LSFAILNPKW

REEMMG ( SEQ ID NO : 11 )

Thus, in specific embodiments, the control fusion proteins comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 13-93, 95-154, and 191-193; the sequences are provided in Table 2 and the examples, with the proviso that each include a G89A mutation. One specific example of a control is provided in SEQ ID NO: 194. By way of example, SEQ ID NO: 13 is provided below, and the position of G89 is bolded and underlined — mutating this residue to alanine renders SEQ ID NO: 13 a control fusion protein. Similarly, each of SEQ ID NO: 13-93, 95-154, and 191-193 include a G89 that becomes a control fusion protein when G89 is mutated to G89A.

SEQ ID NO: 13:

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK

RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGG VIEHVDLLPQR

VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLT RLSFAILNPT

WSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPT

LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKG

IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLL

PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDEVPEAAI SRLITYLRILEELEAQGVHRTA

SEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVG MGRLGSALADW

PGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQK AADLLVAAGIK

GILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREIKPEMKMRYYMDGSVNG HEFTIEGEGT

GRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPE GLSWERSLEFE

DGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKLE GGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHSEASMDELYK

As will be understood by those of skill in the art, the fusion proteins of the disclosure may include additional residues at the N-terminus, C-terminus, or both that are not present in

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SUBSTITUTE SHEET ( RULE 26) the described fusion proteins; these additional residues are not included in determining the percent identity of the polypeptides of the disclosure relative to the reference polypeptide. Such residues may be any residues suitable for an intended use, including but not limited to ligands suitable for purposes of purification (His tags, etc.), and additional peptide domains that add functionality to the polypeptides.

In one embodiment, changes relative to the reference fusion proteins comprises conservative amino acid substitution. As used herein, “conservative amino acid substitution” means ammo acid or nucleic acid substitutions that do not alter or substantially alter fusion protein or domain function or other characteristics. A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in the assays described herein to confirm that a desired activity is retained.

In another aspect, the disclosure provides polynucleotides encoding the fusion protein or control fusion protein (“control polynucleotides”) of any embodiment or combination of embodiments of the disclosure. The polynucleotides may comprise RNA or DNA. Such polynucleotides may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what polynucleotides will encode the fusion proteins of the disclosure.

In specific embodiments, the nucleic acids may comprise the following nucleotide sequence, or an RNA transcript thereof: 1-F8 1-2

ATGaaaGTTCCTGAGGCAGCCATTTCCAGACTGATTACTTATCTCCGCATTCTGGAA GAGTTGGAGGC ACAAGGTGTACACCGCACCGCCTCCGAACAACTCGGAGAGCTGGCCCAGGTCACCGCCTT TCAGGTTG ATAAGGACCTGTCCTACTTTGGCAGTTACGGAACTGACGGCGTGGGATACACTGTACCAG TCCTCAAG AGAGAACTCAGACATATCCTCGGTCTCAACAGAAAATGGGGCCTGTGTATCGTGGGGATG GGACGCCT GGGATCCGCTCTTGCTGATTGGCCTGGTTTCGGCGAGAGCTTTGAGCTGAGGGGTTTCTT TGATGTGG ACCCAGGTATGGTCGGTCGGCCGGTTCGCGGTGGTGTGATCGAACACGTGGATCTGTTGC CCCAACGC GTACCTGGTAGAATCGAAATCGCTCTGCTTACGGTCCCAAGAGAGGCAGCACAGAAAGCT GCCGACCT GCTGGTTGCAGCTGGCATCAAAGGAATCCTCAATTTCGCTCCAGTTGTACTCGAGGTTCC CAAAGAGG

62

SUBSTITUTE SHEET ( RULE 26) TGGCAGTTGAGAATGTGGACATCCTTGCCGGTCTTACGCGTCTGAGCTTTGCCATTCTGA ACCCCACG

TGGagcgcagcaggtgggcatggtATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGG GTGGTGCCCAT CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGA GGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT GGCCCACC

CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATG AAGCAGCACGA

CTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA GGACGACGGCA

ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCG AGCTGAAGGGC

ATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC AGCCACAACGT

CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCA CAACATCGAGG

ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC CCGTGCTGCTG

CCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAG CGCGATCACAT

GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGGTACC AGAAGCCGCTA

TCAGCCGCTTGATCACATACTTGAGAATCTTGGAGGAACTCGAAGCTCAGGGAGTTC ATAGAACTGCA

AGCGAGCAGTTGGGCGAACTCGCACAAGTTACAGCATTCCAAGTGGACGAAGATCTC AGTTATTTCGG

TTCCTATGGCACCGATGGTGTTGGCTATACAGTCCCTGTTTTGAAACGCGAGTTGCG CCACATTTTGG

GCCTGAATCGCAAGTGGGGATTATGCATTGTTGGCATGGGCAGGTTAGGTAGTGCAC TGGCAGACTGG

CCGGGCTTTGGTGAATCTTTCGAACTCAGAGGCTTTTTCGACGTTGATCCTGGCATG GTTGGGAGACC

TGTCAGAGGAGGCGTTATTGAGCATGTTGACCTCCTGCCACAGAGAGTCCCGGGACG CATTGAGATTG

CCCTCCTGACCGTTCCTCGCGAAGCTGCCCAAAAGGCAGCTGATTTACTAGTCGCCG CAGGTATTAAG

GGCATTTTGAACTTTGCCCCTGTGGTTCTGGAAGTGCCTAAGGAAGTTGCTGTCGAA AACGTTGATTT

CCTGGCTGGCTTGACCCGCCTTTCCTTCGCAATCCTCAATCCTAAGTGGcgcgaagt gATTAAACCAG

AGATGAAGATGAGGTACTACATGGACGGCTCCGTCAATGGGCATGAGTTCACAATTG AAGGTGAAGGC

ACAGGCAGACCTTACGAGGGACATCAAGAGATGACACTACGCGTCACAATGGCCGAG GGCGGGCCAAT

GCCTTTCGCGTTTGACTTAGTGTCACACGTGTTCTGTTACGGCCACAGAGTATTTAC TAAATATCCAG

AAGAGATACCAGACTATTTCAAACAAGCATTTCCTGAAGGCCTGTCATGGGAAAGGT CGTTGGAGTTC

GAAGATGGTGGGTCCGCTTCAGTCAGTGCGCATATAAGCCTTAGAGGAAACACCTTC TACCACAAATC

CAAATTTACTGGGGTTAACTTTCCTGCCGATGGTCCTATCATGCAAAACCAAAGTGT TGATTGGGAGC

CATCAACCGAGAAAATTACTGCCAGCGACGGAGTTCTGAAGGGTGATGTTACGATGT ACCTAAAACTT

GAAGGAGGCGGCAATCACAAATGCCAATTCAAGACTACTTACAAGGCGGCAAAAGAG ATTCTTGAAAT

GCCAGGAGACCATTACATCGGCCATCGCCTCGTCAGGAAAACCGAAGGCAACATTAC TGAGCAggtag aagatgcagtagctcattccGAAGCTAGCATGGACGAGCTCTACAAG ( SEQ ID NO : 159 )

1-F8 1-2 control

ATGaaaGTTCCTGAGGCAGCCATTTCCAGACTGATTACTTATCTCCGCATTCTGGAA GAGTTGGAGGC

ACAAGGTGTACACCGCACCGCCTCCGAACAACTCGGAGAGCTGGCCCAGGTCACCGC CTTTCAGGTTG

ATAAGGACCTGTCCTACTTTGGCAGTTACGGAACTGACGGCGTGGGATACACTGTAC CAGTCCTCAAG

AGAGAACTCAGACATATCCTCGGTCTCAACAGAAAATGGGGCCTGTGTATCGTGGGG ATGGCTCGCCT

GGGATCCGCTCTTGCTGATTGGCCTGGTTTCGGCGAGAGCTTTGAGCTGAGGGGTTT CTTTGATGTGG

ACCCAGGTATGGTCGGTCGGCCGGTTCGCGGTGGTGTGATCGAACACGTGGATCTGT TGCCCCAACGC

GTACCTGGTAGAATCGAAATCGCTCTGCTTACGGTCCCAAGAGAGGCAGCACAGAAA GCTGCCGACCT

GCTGGTTGCAGCTGGCATCAAAGGAATCCTCAATTTCGCTCCAGTTGTACTCGAGGT TCCCAAAGAGG

TGGCAGTTGAGAATGTGGACATCCTTGCCGGTCTTACGCGTCTGAGCTTTGCCATTC TGAACCCCACG

TGGagcgcagcaggtgggcatggtATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGG GTGGTGCCCAT CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGA GGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT GGCCCACC

CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATG AAGCAGCACGA

CTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA GGACGACGGCA

ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCG AGCTGAAGGGC

ATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC AGCCACAACGT CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAA CATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCG TGCTGCTG

63

SUBSTITUTE SHEET ( RULE 26) CCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGC GATCACAT

GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGGTACC AGAAGCCGCTA

TCAGCCGCTTGATCACATACTTGAGAATCTTGGAGGAACTCGAAGCTCAGGGAGTTC ATAGAACTGCA

AGCGAGCAGTTGGGCGAACTCGCACAAGTTACAGCATTCCAAGTGGACGAAGATCTC AGTTATTTCGG

TTCCTATGGCACCGATGGTGTTGGCTATACAGTCCCTGTTTTGAAACGCGAGTTGCG CCACATTTTGG

GCCTGAATCGCAAGTGGGGATTATGCATTGTTGGCATGGCCAGGTTAGGTAGTGCAC TGGCAGACTGG

CCGGGCTTTGGTGAATCTTTCGAACTCAGAGGCTTTTTCGACGTTGATCCTGGCATG GTTGGGAGACC

TGTCAGAGGAGGCGTTATTGAGCATGTTGACCTCCTGCCACAGAGAGTCCCGGGACG CATTGAGATTG

CCCTCCTGACCGTTCCTCGCGAAGCTGCCCAAAAGGCAGCTGATTTACTAGTCGCCG CAGGTATTAAG

GGCATTTTGAACTTTGCCCCTGTGGTTCTGGAAGTGCCTAAGGAAGTTGCTGTCGAA AACGTTGATTT

CCTGGCTGGCTTGACCCGCCTTTCCTTCGCAATCCTCAATCCTAAGTGGcgcgaagt gATTAAACCAG

AGATGAAGATGAGGTACTACATGGACGGCTCCGTCAATGGGCATGAGTTCACAATTG AAGGTGAAGGC

ACAGGCAGACCTTACGAGGGACATCAAGAGATGACACTACGCGTCACAATGGCCGAG GGCGGGCCAAT

GCCTTTCGCGTTTGACTTAGTGTCACACGTGTTCTGTTACGGCCACAGAGTATTTAC TAAATATCCAG

AAGAGATACCAGACTATTTCAAACAAGCATTTCCTGAAGGCCTGTCATGGGAAAGGT CGTTGGAGTTC

GAAGATGGTGGGTCCGCTTCAGTCAGTGCGCATATAAGCCTTAGAGGAAACACCTTC TACCACAAATC

CAAATTTACTGGGGTTAACTTTCCTGCCGATGGTCCTATCATGCAAAACCAAAGTGT TGATTGGGAGC

CATCAACCGAGAAAATTACTGCCAGCGACGGAGTTCTGAAGGGTGATGTTACGATGT ACCTAAAACTT

GAAGGAGGCGGCAATCACAAATGCCAATTCAAGACTACTTACAAGGCGGCAAAAGAG ATTCTTGAAAT

GCCAGGAGACCATTACATCGGCCATCGCCTCGTCAGGAAAACCGAAGGCAACATTAC TGAGCAggtag aagatgcagtagctcattccGAAGCTAGCATGGACGAGCTCTACAAG ( SEQ ID NO : 160 )

In another aspect, the disclosure provides recombinant expression vectors comprising the polynucleotides or control polynucleotides (“control expression vectors”) of any embodiment or combination of embodiments of the disclosure operatively linked to a promoter sequence capable of directing expression of the polynucleotide. "Recombinant expression vector" includes vectors that operatively link the polynucleotides to any promoter sequence capable of effecting expression of the fusion proteins. “Promoter sequences” operatively linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the polynucleotides. The promoter need not be contiguous with the polynucleotide, so long as it functions to direct polynucleotide expression. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the polynucleotide and the promoter sequence can still be considered "operably linked" to the coding sequence. Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. The promoter may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF-la) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroidresponsive). In various embodiments, the expression vector may comprise a plasmid, viralbased vector, or any other suitable expression vector.

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SUBSTITUTE SHEET ( RULE 26) In a further aspect, the present disclosure provides recombinant host cells that comprise the recombinant expression vectors or control expression vectors (“control recombinant host cells”) disclosed herein, wherein the host cells can be either prokary otic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure. A method of producing a fusion protein according to the disclosure is an additional part of the disclosure. The method comprises the steps of (a) culturing a host according to this aspect of the disclosure under conditions conducive to the expression of the fusion protein, and (b) optionally, recovering the expressed fusion protein. The expressed fusion protein can be recovered from the cell free extract or the cell culture medium.

The disclosure further provides kits, comprising

(a) the fusion protein of any embodiment of the disclosure, and the control fusion protein of any embodiment of the disclosure;

(b) the polynucleotide of any embodiment of the disclosure and the control polynucleotide of any embodiment of the disclosure;

(c) the expression vector of any embodiment of the disclosure and the control expression vector of any embodiment of the disclosure; and/or

(d) the recombinant host cell of any embodiment of the disclosure and the control recombinant host cell of any embodiment of the disclosure.

The kits can be used, for example, to carry out the methods of the disclosure.

In another aspect, the disclosure provides methods for determining an NAD+/NADH ratio change in a cell of interest, comprising of expressing a FRET biosensor in a cell that undergoes a detectable change upon binding of the FRET biosensor to NAD(H) in the cell, and performing live-cell imaging to determine the ratio of NAD+/NADH inside living cells. Any suitable FRET biosensor can be used, so long as it undergoes a detectable change upon binding of the FRET biosensor to NAD(H) in the cell. Any cell imaging system may be used, including but not limited to a live cell imaging microscope and incubator system. In one non-limiting embodiment, the live cell imaging microscope and incubator system IncuCyte® SX5 (Sartorius). The IncuCyte® SX5 hardware may be used for any method of the disclosure, and is composed of 2 components: 1) gantry and 2) controller. The gantry houses the microscope, camera, and consumable trays that enable automated image acquisition of live-cell cultures and is installed inside a standard tissue culture incubator. In the NAD+/NADH ratio change application the microscope system contains a filter module that is tailored to collecting fluorescent images in the desired spectrum (or spectra). The

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SUBSTITUTE SHEET ( RULE 26) controller contains processors, memory and data storage drives that enable image storage, data handling, database storage, file systems, automated image processing, graphing and over-the-network interaction from the client computer through a graphical user interface (GUI). The software on the controller serves two purposes: 1) server interaction, and 2) instrument control.

The gantry is installed in an incubator and houses the microscope and camera. The controller controls the microscope system and functions as a server. The controller plugs into a communications port, such as, but not limited to, an ethemet port. A graphical user interface (GUI) is loaded on to a computer and interacts with the controller (i.e., server) to control the microscope system and interact with the data. All automated image processing is completed on the controller according to aspects of the present disclosure.

The Incucyte® SX5 microscope moves to user defined locations of cell culture vessels, such as, but not limited to, 96-well plates, turns on the appropriate LED and captures images at a desired exposure time using a desired microscope objective, such as, 700 ms using the lOx objective.

Data may be calculated for each object, each well, or each set of wells, stored in a database, and displayed to the user shortly following data acquisition in the client computer through the graphical user interface. Wells may be scanned as deemed appropriate, such as every 2 hours. Following each scan, metrics are calculated and stored, for instance in the database, at those time points. For example, over the course of a 3-day experiment, 36 time points may be collected for each metric, are concatenated into a time series and can be graphed over the course of the full experimental time frame, i.e. minutes, hours, days, weeks, months.

In one embodiment, the disclosure provides a method of measuring an NAD+/NADH ratio change in a cell of interest, comprising:

(a) expressing the fusion protein of any embodiment of the disclosure in one or more first cells, and generating one or more images selected from the group consisting of:

(i) a first fluorescence image generated by detecting fluorescent signals produced by light having the FRET acceptor polypeptide emission wavelength emitted from the one or more first cells upon exposing the one or more first cells to light having the FRET donor polypeptide excitation wavelength; and/or

(ii) a second fluorescence image generated by detecting fluorescent signals produced by light having the FRET acceptor polypeptide emission wavelength emitted from

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SUBSTITUTE SHEET ( RULE 26) the one or more first cells upon exposing the one or more first cells to light having the FRET acceptor polypeptide excitation wavelength; and/or

(iii) a third fluorescence image generated by detecting fluorescent signals produced by light having the FRET donor polypeptide emission wavelength emitted from the one or more first cells upon exposing the one or more first cells to light having the FRET donor polypeptide excitation wavelength; and

(b) determining a FRET ratio in the one or more first cells by comparing the output of fluorescent signals in the first fluorescent image, the second fluorescent image, and/or the third fluorescent image; wherein an NAD+/NADH ratio in the one or more first cells is proportional to the determined FRET ratio.

In this embodiment, “comparing” the output of fluorescent signals means dividing the output of fluorescent signals in one image by the output of fluorescent signals in a different image. For example:

• the output of fluorescent signals in the first fluorescent image can be divided by the output of fluorescent signals in the second fluorescent image;

• the output of fluorescent signals in the first fluorescent image can be divided by the output of fluorescent signals in the third fluorescent image;

• the output of fluorescent signals in the second fluorescent image can be divided by the output of fluorescent signals in the first fluorescent image;

• the output of fluorescent signals in the second fluorescent image can be divided by the output of fluorescent signals in the third fluorescent image;

• the output of fluorescent signals in the third fluorescent image can be divided by the output of fluorescent signals in the first fluorescent image; or

• the output of fluorescent signals in the third fluorescent image can be divided by the output of fluorescent signals in the second fluorescent image.

The “outputs” of fluorescent signals can be determined on any suitable basis, including but not limited to on a whole image basis, per cell basis, on a per pixel basis, or using any alternative intensify measurements.

In another embodiment, the methods further comprise expressing the control fusion protein of any embodiment or combination of embodiments of the disclosure in one or more first cells, and detecting a control signal produced by light having the acceptor emission wavelength emitted from the one or more first cells. Any suitable method for using the

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SUBSTITUTE SHEET ( RULE 26) control signal to correct the determined FRET ratio may be used. In one embodiment, detecting the control signal comprises

(c) expressing the control fusion protein of any embodiment of the disclosure in one or more control cells (such as the first cells, or second cells), and generating one or more images selected from the group consisting of:

(i) a fourth fluorescence image generated by detecting fluorescent signals produced by light having the FRET acceptor polypeptide emission wavelength emitted from the one or more control cells upon exposing the one or more control cells to light having the FRET donor polypeptide excitation wavelength; and/or

(ii) a fifth fluorescence image generated by detecting fluorescent signals produced by light having the FRET acceptor polypeptide emission wavelength emitted from the one or more control cells upon exposing the one or more control cells to light having the FRET acceptor polypeptide excitation wavelength; and/or

(iii) a sixth fluorescence image generated by detecting fluorescent signals produced by light having the FRET donor polypeptide emission wavelength emitted from the one or more control cells upon exposing the one or more control cells to light having the FRET donor polypeptide excitation wavelength; and

(d) determining a control fusion FRET ratio in the one or more control cells by comparing the output of fluorescent signals in the fourth fluorescent image, the fifth fluorescent image, and/or the sixth fluorescent image; wherein alterations in the control fusion FRET ratio are determined to be the result of experimental conditions unrelated to NAD+/NADH ratio, and wherein the determined FRET ratio is corrected based on the alterations in the control fusion FRET ratio.

The one or more cells may be any cell or cell population in which determining NAD+/NADH ratio is of interest. In one embodiment, the one or more first cells are in culture in an incubator. In another embodiment, all imaging steps are performed without removing the one or more first cells from the incubator. In this embodiment, the cells are cultured in a suitable cell culture medium in an incubator, and the incubator is configured such that the cells to be assayed do not have to be removed from the incubator during observation and/or recording of assays for detecting changes in NAD+/NADH ratio.

The assays can be used, for example, to test the effect of one or more test compounds on NAD+/NADH ratio in cells of interest. Thus, in one embodiment, the methods further comprise contacting the one or more first cells with one or more test substance and determining an effect of the test substance on NAD+/NADH ratio in the one or more first

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SUBSTITUTE SHEET ( RULE 26) cells. The effect of the one or more test substance on the NAD+/NADH ratio in the one or more first cells may be determined over any time period of interest, including but not limited to continuously or intermittently over a time period in the range of 1 minute to three months.

Embodiments of the compositions and methods of the disclosure are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of the claimed compositions and methods.

Examples

16 initial FRET constructs (See Table 3) were constructed using expression vector pET28c with a T7 promoter and lac operator and expressed in E. coli. Cells were collected and lysed using a commercially-available lysis buffer. Proteins of interest were isolated from the lysates using Ni-NTA magnetic beads. Eluted proteins were subjected to buffer exchange and final protein concentrating. To test performance of the initial FRET constructs, their emission spectra was recorded on a plate reader in absence or presence of 40 uM NADH. Resulting emission spectra with or without the ligand were normalized and overlaid for each construct. Constructs with very clear and robust spectra separation were labeled as having “good response”, those with noticeable but small spectra separation were labeled as having “poor response”, and finally those having no spectra separation were labeled as having “no response”.

Table 3: Initial constructs

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SUBSTITUTE SHEET ( RULE 26)

Legend:

Rex(78-189) - WT T-Rex from aa 78 to aa 189

Rex(190-211) - WT T-Rex from aa 190 to aa 211

Rex(78-211) - WT T-Rex from aa 78 to aa 211

Rex(A) - T-Rex from aa 1 to aa 205, mutations compared to WT: S30A, R46D, K58D,

Y98W, E116G, K117M, F189I, K204T

Rex(B) - T-Rex from aa 2 to aa 211, mutations compared to WT: S30A, R46D, K47E, K58D, Y98W, E116G, K117M cpmEGFP(145/146) - mEGFP permuted between amino acids N145 and Y146 cpmEGFP(173/174) - mEGFP permuted between amino acids E173 and D174

Full sequences of the initial constructs are shown below.

Initial construct #1

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVR GGVIEHVD LLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSAY NSHNVYI MADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPN EKRDHMVL LEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDA TYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGLAGLTRLSFAILNPKWREEMMGNRKWG LCIVGMGR LGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPR EAAQKAAD LLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMG ( SEQ ID NO : 161 )

Initial construct #2

MNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLL PQRVPGRIEIA LLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSAYNSHNVYIMADKQK NGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVT AAGITLGM DELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GKLPVPW PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNGLAGLTRLSFAILNPKWREEMMGNRKWGLCIVGMGRLGSAL ADYPGFGE SFELRGFFDVDPEKVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAA GIKGILNF APWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMGMVSVIKPEMKMRYYMDGSVNG HEFTIEG EGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSL EFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS ( SEQ ID NO : 162 )

Initial construct #3

70

SUBSTITUTE SHEET ( RULE 26) MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLV SHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVR GGVIEHVD LLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAG LTRLSFA ILNPKWREEMMGNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGG VIEHVDLL PQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSAYNS HNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEK RDHMVLLE FVTAAGITLGMDELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATY GKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNGLAGLTRLSFAILNPKWREEMMG ( SEQ ID NO :

163 )

Initial construct #4

MNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLL PQRVPGRIEIA LLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKW REEMMGN RKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLLPQRVP GRIEIALL TVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSAYNSHNVYIMADKQKNG IKVNFKI RHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAA GITLGMDE LYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKG IDFKEDGNILGHKLEYNGLAGLTRLSFAILNPKWREEMMGMVSVIKPEMKMRYYMDGSVN GHEFTIEG EGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSL EFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS ( SEQ ID NO :

164 )

Initial construct #5

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVR GGVIEHVD LLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSAD GSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGG SGGMVSKG EELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFS RYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEGLAGLTRLSFAILNPKWREEMMGNRKWG LCIVGMGR LGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPR EAAQKAAD LLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMG ( SEQ ID NO : 165 )

Initial construct #6

MNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLL PQRVPGRIEIA

LLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSADGSVQLADHYQ QNTPIGDGPV

LLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEE LFTGWPILVE

LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRY PDHMKQHDFFK

SAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE YNYNSHNVYIM ADKQKNGIKVNFKIRHNIEGLAGLTRLSFAILNPKWREEMMGNRKWGLCIVGMGRLGSAL ADYPGFGE

71

SUBSTITUTE SHEET ( RULE 26) SFELRGFFDVDPEKVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAA GIKGILNF APWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMGMVSVIKPEMKMRYYMDGSVNG HEFTIEG EGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSL EFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS ( SEQ ID NO : 166)

Initial construct #7

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVR GGVIEHVD LLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAG LTRLSFA ILNPKWREEMMGNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGG VIEHVDLL PQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSADGS VQLADHY QQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSG GMVSKGEE LFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYG VQCFSRY PDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE YNYNSHNVYIMADKQKNGIKVNFKIRHNIEGLAGLTRLSFAILNPKWREEMMG ( SEQ ID NO : 167 )

Initial construct #8

MNRKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLL PQRVPGRIEIA LLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKW REEMMGN RKWGLCIVGMGRLGSALADYPGFGESFELRGFFDVDPEKVGRPVRGGVIEHVDLLPQRVP GRIEIALL TVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFSADGSVQLADHYQQNTPI GDGPVLL PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGW PILVELD GDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSA MPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMAD KQKNGIKVNFKIRHNIEGLAGLTRLSFAILNPKWREEMMGMVSVIKPEMKMRYYMDGSVN GHEFTIEG EGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSL EFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS ( SEQ ID NO : 168 )

Initial construct #9

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH

RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSY GTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDP GMVGRPVR GGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAV ENVDILA

GLTRLSFAILNPTWSAAGGHGYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDG PVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEEL FTGWPIL VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYP DHMKQHDF FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY NTKVPEAA

72

SUBSTITUTE SHEET ( RULE 26) I SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLK RELRHIL

GLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDL LPQRVPGRIEI

ALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAIL NPKWREEMMG ( SEQ ID NO : 169 )

Initial construct #10

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIE HVDLLPQR VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTRLS FAILNPT WSAAGGHGYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLP DNHYLSTQ SKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGWPILVELDGD VNGHKFS VSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAM PEGYVQER TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTKVPEAAI SRLITYLRILEE LEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRK WGLCIVGM GRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTV PREAAQKA ADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMGMVSVIK PEMKMRY YMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTK YPEEIPDY FKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKI TASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITE QVEDAVAH S ( SEQ ID NO : 170 )

Initial construct #11

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSY GTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDP GMVGRPVR GGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAV ENVDILA GLTRLSFAILNPTWSAAGGHGDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDP NEKRDHMV LLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGD ATYGKLT LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAE VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNI ETKVPEAA I SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLK RELRHIL GLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQ RVPGRIEI ALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPK WREEMMG ( SEQ ID NO : 171 )

Initial construct #12

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK

RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGG VIEHVDLLPQR

VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLT RLSFAILNPT

WSAAGGHGDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLG

MDELYKGGSGGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVP

WPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIE

LKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIETKVPEAAI SRLITYLRILEE

LEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGL NRKWGLCIVGM

73

SUBSTITUTE SHEET ( RULE 26) GRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTV PREAAQKA ADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMGMVSVIK PEMKMRY YMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTK YPEEIPDY FKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKI TASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITE QVEDAVAH S ( SEQ ID NO : 172 )

Initial construct #13

MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAF DLVSHVFCYGH RVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQ NQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIG HRLVRKTE GNITEQVEDAVAHSKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSY GTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDP GMVGRPVR GGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAV ENVDILA GLTRLSFAILNPTWSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATY GKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDG SVQLADHY QQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKTKVP EAAI SRLI TYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELR HILGLNRK WGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGR IEIALLTV PREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEM MG ( SEQ ID NO : 173 )

Initial construct #14

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK

RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGG VIEHVDLLPQR

VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLT RLSFAILNPT

WSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPT

LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKG

IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLL

PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKTKVPEAAISRLITY LRILEELEAQG

VHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWG LCIVGMGRLGS

ALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPR EAAQKAADLLV

AAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEMMGMVSVIKPE MKMRYYMDGS

VNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYP EEI PDYFKQAF

PEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDG

VLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQV EDAVAHS

( SEQ ID NO : 174 )

Initial construct #15

MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW PTLVTTLTYG

VQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGN

ILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGP VLLPDNHYLST

QSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKVPEAAI SRLITYLRILEELEAQGVHRTASEQL

GELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGRL GSALADWPGFG

ESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADL LVAAGIKGILN

74

SUBSTITUTE SHEET ( RULE 26) FAPWLEVPKEVAVENVDILAGLTRLSFAILNPTWSAAGGHGMVSVIKPEMKMRYYMDGSV NGHEFTI EGEGTGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWER SLEFEDGGSASVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDG VLKGDVTM YLKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHSTKVP EAAI SRLI

TYLRILEELEAQGVHRTASEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKR ELRHILGLNRK WGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGR IEIALLTV PREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREEM MG ( SEQ ID NO : 175 )

Initial construct #16

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIE HVDLLPQR VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTRLS FAILNPT WSAAGGHGMVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTMAEGGP MPFAFDLV SHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSLEFEDGGSASVSAHI SLRGNTFYHKSKFTGVNF PADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKLEGGGNHKCQFKTTYKAAKEILE MPGDHYIG HRLVRKTEGNITEQVEDAVAHSTKVPEAAISRLITYLRILEELEAQGVHRTASEQLGELA QVTAFQVD EDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFE LRGFFDVD PGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPW LEVPKEV AVENVDFLAGLTRLSFAILNPKWREEMMGMVSKGEELFTGWPILVELDGDVNGHKFSVSG EGEGDAT YGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNY KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFK IRHNIEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMD ELYK ( SEQ ID NO : 176)

Constructs #10 and #14 in Table 3 were used to generate protein libraries. Protein #12 was not used because it showed poor performance under dual-excitation FRET signal collection mode. Protein #16 had very similar properties to protein #14 and thus also was not used. Mutagenesis of both constructs resulted in clones with substantially improved signal windows. In vitro data also shows that signal window values identified during the screening in bacterial lysates have reliable prediction power. This is evidenced by the fact that all clones chosen from the libraries to proceed with have in vitro a substantial increase in signal window, compared to the original template protein (constructs #10 or #14). These increases are also roughly similar fold-wise between the numbers obtained in bacterial lysate screening and in the process of in vitro characterization.

Library 14-1 produced the most promising clones both in terms of signal window and sensitivity, compared to library 10-1.

Protein library DNA was generated following cloning that assembled pieces of DNA that were generated using PCRs with mixtures of primers. DNA was transformed into E. coli, and the first round of protein library expression was done in bacterial colonies on agar plates. The colonies, each expressing different library members, were imaged, and then classified

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SUBSTITUTE SHEET ( RULE 26) into groups based on their FRET ratio signal. Colonies from different groups were inoculated into 96-deep-well plates for further protein expression in liquid culture. The original construct that was used to generate the library (e.g., construct #14 in Table 2) was also inoculated on the same plates, along with non-expressing bacteria. The latter served as a negative control.

Upon completion of expression, bacteria cells were lysed, lysates were cleared by centrifugation, and protein variants were tested for their response to NADH. For that, the lysates were excited with 488 nm or 550 nm light and emission was collected at 590 nm at a plate reader. The fluorescence signal upon excitation with 488 light was divided by the signal upon excitation with 550 nm light to generate FRET ratio, which was measured for all the selected protein vanants in presence or absence of 200 uM NADH. The difference between FRET ratios with and without the ligand was the signal window - the final metric used to identify the most promising mutants.

To provide more accurate data on biosensor response to NAD+/NADH ratio changes, the most promising protein variants, identified during screening in bacterial lysates, were purified. For that, the proteins were expressed in E. coli, bacteria were harvested, lysed, and proteins were isolated using two sequential rounds of affinity chromatography on a FPLC instrument. Upon buffer exchange and protein concentrating, 50 nM of protein was mixed with NAD+ and NADH so the ratio NAD+/NADH was either 1 or 10,000. The final NAD+ concentration was kept at 80 uM (close to reported physiological concentration) and final NADH concentration was either 80 uM or 8 nM. Protein excitation spectra in presence of high or low NAD+/NADH was recorded at 590 nm emission wavelength on a plate reader, and then for each protein variant two spectra were normalized to the maximal value, plotted and overlaid. In Figure 1A, purified protein 1-F8 (SEQ ID NO: 13) was mixed with NAD+ and NADH so in one case NAD+/NADH ratio was 1 and in another 10,000.. The substantial difference between the spectra (indicated with the arrow) signifies the large signal window of the resulting biosensor. Similar data is shown in Figure IB for construct 10-1 1-7 (SEQ ID NO:153).

We next calculated signal window for the constructs. To calculate the signal window, the areas under the high NAD+/NADH ratio and low NAD+/NADH ratio excitation spectra curves were calculated for the 400-526 nm wavelength interval, which is where the FRET signal change is observed. The difference between two calculated areas, normalized to the lowest area of these two, is presented in Figure 2 on the bar graph as signal wmdow/lowest

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SUBSTITUTE SHEET ( RULE 26) signal. Proteins with higher signal window values were selected for further characterization in mammalian cells.

To plot the sensitivity curves, purified proteins were mixed with NAD+ and NADH so the resulting NAD+/NADH ratios varied, and then excited at 460 nm and emissions at 510 nm and 560 nm were collected. The FRET ratio was calculated as emission at 560 nm divided by emission at 510 nm, and the values were normalized to the FRET ratio value at the highest NAD+/NADH ratio. Normalized FRET ratio was measured for each protein at different NAD+/NADH ratios, ranging from 1 to 10,000. For each NAD+/NADH ratio, the final NAD+ concentration was kept constant at 80 uM and NADH concentration was varied.

Table 4 summarizes data for selected protein. EC50 values are approximate and calculated by Figure 3 curve fitting in GraphPad™ Prism (R ² values -0.99).

Signal window data presented in the table clearly shows that directed evolution substantially increased biosensor performance, with the best clones achieving 3 to 8 fold improvement compared to parental constructs #10 or #14.

Additionally, directed evolution resulted in clones with more than 10-fold variation in the value of EC50. The latter determines the sensitivity of the sensor, i.e., what range of NAD+/NADH ratio change the sensor is best applicable for. Large EC50 variation provides greater biosensor choice flexibility since some cell types/lines may have different, compared to the average, NAD+/NADH ratio change ranges. See Figure 3.

In a further study, proteins were expressed in HEK 293 mammalian cells following transient transfection with the plasmids encoding those proteins. Transfected cells were imaged using Incucyte® SX5 equipped with a Metabolism Optical Module (Sartorius) and the

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SUBSTITUTE SHEET ( RULE 26) data was processed using the built-in ATP analysis software module that allows quantification of average FRET ratio in all cells in the image. To measure signal window, cells were treated with either 10 mM lactate or 20 mM pyruvate. The former drives the NAD+/NADH ratio, and thus FRET signal down, and the latter drives the NAD+/NADH ratio, and thus FRET signal up. The difference between highest and lowest FRET ratios is the signal window in mammalian cells. Data is shown in Figure 4. Mammalian cell data demonstrates that most of generated biosensor clones have large signal windows. Signal window values above 0.2 generally allow the most robust FRET ratio change measurements. Also, most clones identified in bacterial lysates and then in vitro as promising showed large signal windows in mammalian cells as well. This indicates that biosensor screening in bacterial lysates has reliable predictive power.

Clone 1-F8 showed the largest signal window in mammalian cells and also had sensitivity suitable for reporting physiological changes of NAD+/NADH ratio. However, imaging in mammalian cells revealed that clone 1-F8 had lower, compared to other promising clones, brightness of mKOk protein. Sequence analysis of clone 1-F8 showed that Z9 was completely absent, which could affect the brightness of mKOk which is immediately adjacent to it. To attempt restoring the brightness of 1-F8 we reintroduced V into Z9 while (1) keeping the rest of 1-F8 the same (generating variant 1), (2) removing E from the N terminus of Z6 (SEQ ID NO:2) (generating variant 2), and (3) removing RE from the N terminus of Z6 (SEQ ID NO:2) (generating variant 3).

Cloning these constructs generated 1-F8 1-2 (variant 1, clone 2) (SEQ ID NO:191), 1- F8 2-3 (variant 2, clone 3) (SEQ ID NO: 192) and 1-F8 3-3 (variant 3, clone 3) (SEQ ID NO: 193). These constructs were tested in mammalian cells by comparing their brightness with the brightness of the original 1-F8 construct in the mKOk imaging channel. We also assessed signal window of the resulting constructs in comparison with the signal window of 1-F8 to confirm that the signal window was not affected substantially by the mutagenesis.

Construct 1-F8 1-2 (SEQ ID NO: 191) showed the most restoration of the brightness of mKOk, compared to 1-F8, while retaining nearly identical to 1-F8 signal window (Figure 5 and Figure 6). Thus, construct 1-F 1-2 (SEQ ID NO: 191) and its control protein (SEQ ID NO: 194) were selected to be used as the main biosensor protein sequences for imaging NAD+/NADH changes in living cells.

1-F8 1-2

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SUBSTITUTE SHEET ( RULE 26) MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIE HVDLLPQR VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTRLS FAILNPT WSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKG IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTP IGDGPVLL PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDEVPEAAI SRLITYLRILEELEAQGVHRTA SEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGR LGSALADW PGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAAD LLVAAGIK GILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREVIKPEMKMRYYMDGSVNGHE FTIEGEG TGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSLEF EDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKL EGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHSEASMDELY K ( SEQ ID N0 : 191 )

1-F8 2-3

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIE HVDLLPQR VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTRLS FAILNPT WSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPT

LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKG IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTP IGDGPVLL PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDEVPEAAI SRLITYLRILEELEAQGVHRTA SEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGR LGSALADW PGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAAD LLVAAGIK GILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWRVIKPEMKMRYYMDGSVNGHEF TIEGEGT GRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLS WERSLEFE DGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKLE GGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHSEASMDELYK ( SEQ ID NO : 192 )

1 -F8 3-3

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK RELRHILGLNRKWGLCIVGMGRLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGGVIE HVDLLPQR VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLTRLS FAILNPT WSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKG IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTP IGDGPVLL PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDEVPEAAI SRLITYLRILEELEAQGVHRTA SEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVGMGR LGSALADW PGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAAD LLVAAGIK GILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWVIKPEMKMRYYMDGSVNGHEFT IEGEGTG RPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSLEFED GGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKLEG GGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHSEASMDELYK ( SEQ ID NO : 193 )

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SUBSTITUTE SHEET ( RULE 26) 1-F8 1-2 control

MKVPEAAI SRLITYLRILEELEAQGVHRTASEQLGELAQVTAFQVDKDLSYFGSYGTDGVGYTVPVLK

RELRHILGLNRKWGLCIVGMARLGSALADWPGFGESFELRGFFDVDPGMVGRPVRGG VIEHVDLLPQR

VPGRIEIALLTVPREAAQKAADLLVAAGIKGILNFAPWLEVPKEVAVENVDILAGLT RLSFAILNPT WSAAGGHGMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPT

LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKG

IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQ NTPIGDGPVLL

PDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDEVPEAAI SRLITYLRILEELEAQGVHRTA

SEQLGELAQVTAFQVDEDLSYFGSYGTDGVGYTVPVLKRELRHILGLNRKWGLCIVG MARLGSALADW PGFGESFELRGFFDVDPGMVGRPVRGGVIEHVDLLPQRVPGRIEIALLTVPREAAQKAAD LLVAAGIK

GILNFAPWLEVPKEVAVENVDFLAGLTRLSFAILNPKWREVIKPEMKMRYYMDGSVN GHEFTIEGEG

TGRPYEGHQEMTLRVTMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEI PDYFKQAFPEGLSWERSLEF

EDGGSASVSAHI SLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLKL

EGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHSEASMD ELYK ( SEQ ID NO : 194 )

SUBSTITUTE SHEET ( RULE 26)