WO2024229346A2 - Methods and platform related to fluorescent protein biosensors - Google Patents

Abstract

The present disclosure provides a signal protein for detecting free adenine diphosphate ribose (free ADPR) and methods of use thereof.

Description

METHODS AND PLATFORM RELATED TO FLUORESCENT PROTEIN BIOSENSORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/463,771, filed May 3, 2023, incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via Patent Center encoded as XML in UTF-8 text. The electronic document, created on May 3, 2024, is entitled “10046-512W01_ST26.xml”, and is 7,830 bytes in size.

GOVERNMENT SUPPORT CLAUSE

[0003] This invention was made with government support under Grant no. GM126897 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0004] ADPR is derived from the enzymatic cleavage of the metabolite NAD+, or oxidized nicotinamide adenine dinucleotide. Free ADPR concentrations are reported to reflect the activities of NAD+ consuming enzymes, such as pro-neurodegenerative enzyme SARM1 and human immunoenzyme CD-38. The lack of methods to monitor free ADPR concentrations in real-time and in physiological contexts represents a major barrier for screening and testing SARM1 blockers or CD-38 targeting approaches, with relevance to neurodegeneration, cancer, and cardiovascular disease. The major challenges for studying free ADPR include that its levels fluctuate and spike very locally within minutes. The sources and concentrations of free ADPR in cells are unknown; how free ADPR concentrations are regulated in cells is also unknown. Additionally, as an intermediary in signaling pathways, free ADPR is the required agonist for the opening of human calcium channel Transient Receptor Potential Melastatin family member 2 (TRPM2). As such, intracellular free ADPR concentrations are expected to play a role in controlling fever responses, synaptic activity, cell death mechanisms, as well as inflammatory and oxidative stress responses. [0005] What is needed in the art is a modified nudix-related transcriptional regulator signal protein which can detect various target molecules such as free ADPR.

SUMMARY OF THE INVENTION

[0006] The present invention provides a modified signal protein and methods detecting the presence/modulation and quantifying a target molecule using said signal protein.

[0007] In one aspect, disclosed herein is a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the signal protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and the target molecule.

[0008] In one aspect disclosed herein is a method for determining presence of a target molecule, the method comprising a) providing a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target, b) exposing the signal protein to the target molecule under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal, and c) detecting the signal, thereby determining the presence of the target molecule.

[0009] In one aspect, disclosed herein is a method for quantifying a target molecule, the method comprising a) providing a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target, b) exposing the signal protein to the target molecule under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal, and c) detecting the amount of signal produced in step b), thereby determining the amount of the target molecule present.

[0010] In one aspect disclosed herein is a method for determining modulation of a target molecule by a test compound, the method comprising a) providing a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target, b) exposing the signal protein to the target molecule and the test compound under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal, c) detecting the amount of signal produced in step b), d) comparing the amount of signal produced in step b) to a control, wherein said control was carried out without the presence of the test compound, and e) determining if a difference exists between the amount of signal produced in the presence of the test compound and the amount of signal produced in the control, wherein a significant difference indicates that the test compound modulated the target molecule.

[0011] In some embodiments, the signal protein is a modified NrtR protein. In some embodiments, the target molecule comprises free adenosine diphosphate ribose (free ADPR). In some embodiments, the signal protein is a modified L-arabinose Nudix-related transcription factor (AraR) protein. In some embodiments, the target molecule comprises L- or D-Arabinose. In some embodiments, the said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+. In some embodiments, the target molecule comprises NAD+.

[0012] In some embodiments, the output module comprises a peptide linker and a signal transducer. In some embodiments, the signal transducer is a fluorescent molecule. In some embodiments, the fluorescent molecule is cpVenus.

[0013] In some embodiments, the peptide linker transduces the conformational change to the signal transducer. In some embodiments, the nudix-related transcriptional regulator has been modified so that it can interact with a non-native target. In some embodiments, the nudix- related transcriptional regulator has been evolved to change its specificity for the target.

[0014] In some embodiments, a significant difference is more than a 5% difference. In some embodiments, modulation of the target molecule by the test compound results in an increased amount of measurable target molecule compared to the control. In some embodiments, modulation of the target molecule by the test compound results in a decreased amount of measurable target molecule compared to the control.

[0015] In some embodiments, the test compound is a small molecule such as a metal or organic compound, a polypeptide, a peptide, a natural product, a peptidomimetic, a nucleic acid, a lipid, lipopeptide, or a carbohydrate. In some embodiments, the test compound is an agonist or an antagonist of the target molecule. BRIEF DESCRIPTION OF FIGURES

[0016] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0017] FIG 1 shows the apo- (green, PDB 3GZ5) and ligand-bound (yellow, PDB 3GZ8) solved so NrtR structures are superimposed. Inset, enlarged view of the lost helical secondary structure when free ADPR is bound (yellow).

[0018] FIGS. 2A, 2B, and 2C show mutated amino acid residues and confocal images of free ADPR. FIG. 2A shows the position of mutated residues (lysine and serine, highlighted in cyan) relative to nucleic acid in PDB 3GZ6. FIGS. 2B and 2C show the confocal fluorescent images of transiently transfected free ADPR sensor in HEK293T cells before (FIG. 2B) and after (FIG. 2C) introduction of mutations K435A and S436D. Formation of puncta was alleviated with the mutations (FIG. 2C).

[0019] FIG. 3 shows the introduction of D122N (closed circles) improved the brightness of the sensor and reduced its sensitivity to non-ligand changes between pH 7.4 and pH 8.0.

[0020] FIGS. 4A and 4B shows a Coomassie image and fluorescence intensity of response of the sensor to free ADPR. FIG. 4A shows the Coomassie of affinity-isolated free ADPR sensor (arrow). The expected molecular weight for the free ADPR sensor is ~54kDa. FIG. 4B shows the free ADPR sensor’s fluorescence intensity decreases with increasing free ADPR concentrations. The control is a binding mutant (R98E). Values are mean of n=3 and error bars represent SD.

[0021] FIGS. 5A-B show that the sensor responds specifically to free ADPR and modified free ADPR (2’-deoxy ADPR, O-Acetyl ADPR and phospho ADPR) molecules. FIG. 5A shows that the sensor responds to free and modified free ADPR but does not respond to similar molecules including 2’ -cyclic ADPR, 3 ’-cyclic ADPR, cyclic ADPR, NAAD⁺, NAD⁺, NADH, NaADP⁺, Ribulose-5-Phosphate (R5P), AMP, ADP and ATP. FIG. 5B shows that the control (R98E) does not respond to any tested molecule. The bar graph represents the mean ± SD.

[0022] FIGS. 6A and 6B show fluorescence intensity relative to temperature. FIG. 6A shows that both sensor and control (R98E) have a moderate but significant change in fluorescence with temperature. The bar graph represents the mean ± SD, ANOVA < 0.05 with post-hoc Dunnett’s Test *p < 0.05 and **p < 0.01. FIG. 6B shows the change in fluorescence of the sensor can be normalized to the control.

[0023] FIGS. 7 A and 7B show that the free ADPR sensor (7 A) and TRPM2 (7B) respond to increased free cytosolic ADPR following H2O2 treatment as indicated. Fluorescence of the free ADPR sensor responds directly; TRPM2 responds with an influx of intracellular Ca²⁺, which is reported by increased fluorescence of Ca²⁺ sensor GCaMP7s. Values are mean (F/Fo) ± SD, analyzed by confocal microscopy.

[0024] FIG. 8A-B shows the Nudix-like transcription factor (TF) members as a platform for engineering sensors based on shared features. (A) shows arabinose sensor; (B) shows nudix- based NAD sensor.

DETAILED DESCRIPTION

[0025] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. [0026] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Terminology

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0028] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

[0029] The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0030] As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0031] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur.

[0032] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0033] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0034] Reference is made herein to nucleic acid and nucleic acid sequences. The terms “nucleic acid” and “nucleic acid sequence” refer to a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. It should be understood that a nucleotide, oligonucleotide, polynucleotide, or fragments thereof can be used interchangeably. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be singlestranded or double-stranded and may represent the sense or the antisense strand).

[0035] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and/or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).

[0036] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0037] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0038] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4- dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine).

[0039] Variants comprising insertions or additions relative to a reference amino acid sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues.

[0040] Fusion proteins are also contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein, or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein, or variant thereof. The heterologous protein(s) may be fused at the N-terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0041] As used herein, a “signal protein” refers to a peptide or polypeptide sequence bound or fused to a peptide linker and a signal transducing molecule used to convey an interaction between said signal protein and its target molecule. Herein, the signal protein comprises a transcription regulating protein bound to the signal transducing molecule by the peptide linker. [0042] A “peptide linker” refers to short (about 4-50 amino acids in length) peptides that serve to connect two or more biomolecules, including, but not limited to proteins, polypeptides, peptides, nucleic acids, lipids, carbohydrates, and derivatives thereof. Peptide linkers are generally used to maintain cooperative interactions between biomolecules or preserve biological activities of the biomolecules.

[0043] As used herein, a “signal transducer” or a “signal transducing molecule” refers to any biomolecule, such as a probes (e.g., fluorescent probes or fluorescent molecules), peptide, polypeptide, protein, nucleic acid, hormones, neurotransmitters, growth factors, cytokines, and chemokines that bind to a specific protein domain to initiate a series of events, including, but not limited to conformational changes, light emission, biomolecule interactions, and biomolecule synthesis or degradation.

[0044] As used herein, a “transcription factor” or a “transcription regulator” refers to a protein or family of proteins that control the rate of transcription of genetic material, or the conversion of DNA into messenger RNA (mRNA). Transcription factors or transcription regulators are able to regulate transcription by binding to specific DNA sequences to enhance or repress transcription.

[0045] An “output module” refers to a region or domain within a signal protein that receives input (e.g., an interaction between the signal protein and the target molecule) and thereafter produces a detectable signal.

[0046] A “target molecule” refers to a biomolecule, including, but not limited to carbohydrates, lipids, amino acids, nucleotides, nucleic acids, peptides, polypeptides, that is recognized by a specific signal protein. Target molecules generally comprises chemical moieties or domains that can bind or interact with the signal protein to initiate a series of events, including, but not limited to conformational changes, light emission, biomolecule interactions, and biomolecule synthesis or degradation.

[0047] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.

[0048] The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0049] Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0050] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.

[0051] A variant polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit one or more biological activities associated with binding a ligand and/or binding DNA at a specific binding site.

[0052] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0053] The term “ vitro” as used herein refers to the performance of a biochemical reaction outside a living cell, including, for example, in a microwell plate, a tube, a flask, a tank, a reactor and the like, for example a reaction to form an alkaloid compound.

[0054] The term “ vivo” as used herein refers to the performance of a biochemical reaction within a living cell, including, for example, a microbial cell, or a plant cell, for example to form an alkaloid compound.

[0055] The disclosed technology relates to “biosensors.” As disclosed herein, a “biosensor” is a molecule or a system of molecules that can be used to bind to a ligand (or target molecule) and provide a detectable response based on binding the ligand. In some cases, “biosensors” may be referred to as “molecular switches.” Biosensors and molecular switches are disclosed in the art. (See, e.g., Ostermeier, Protein Eng. Des. Sei. 2005 August; 18(8):359-64; Wright et al., Curr. Opin. Chem. Biol. 2007 June; l l(3):342-6; Roberts, Chem. Biol. 2004 November; 11(11): 1475-6; and U.S. Pat. Nos. 8,771,679; 8,679,753; and 8,338,138; the contents of which are incorporated herein by reference in their entireties). Biosensors and molecular switches have been utilized in recombinant microorganisms. (See, e.g., Rogers et al., Curr. Opin. Biotechnol. 2016 Mar. 18; 42:84-91; and U.S. Published Application Nos. 2010/0242345 and 2013/0059295; the contents of which are incorporated herein by reference in their entireties). [0056] A “substrate-promiscuous regulator” refers to any protein with the ability to bind to and report on the concentration of more than one chemical. For instance, the naturally occurring promiscuous regulators from which the biosensors disclosed herein are derived has been reported to bind to several different unrelated chemicals (Yamasaki, S., Nikaido, E., Nakashima, R. et al. Nat Commun 2013). Another common feature of substrate-promiscuous regulators is that the chemicals they bind are often structurally unrelated, but share some common general feature, such as being hydrophobic.

[0057] The systems, components, and methods disclosed herein may be utilized for sensing a ligand or a substrate or a metabolite in a cell or a reaction mixture. The disclosed systems, components, and methods typically include and/or utilize an engineered (non-naturally occurring) biosensor. The biosensors disclosed herein bind the ligand and modulate expression of an output signal, such as a reporter gene, which can be operably linked to a promoter that is engineered to include specific binding sites for the input signal. The difference in expression of the output signal in the presence of the ligand versus expression of the output signal in the absence of the ligand can be correlated to the concentration of the ligand in a reaction mixture. [0058] The term “interaction” refers to an action that occurs as two or more biomolecules have an effect on one another either with or without physical contact. In terms of biological interactions, cell, proteins, and other biomolecules can have said effects on one another to impact biological functions, such as cell signaling pathways.

[0059] As used herein, “modulating expression” may include “repressing expression” and/or “inhibiting expression,” and “modulating expression may include “de-repressing expression” and/or “activating expression.” As such, in some embodiments, when the biosensor is not bound to a ligand, the biosensor may repress expression and/or inhibit expression from a promoter that is engineered to include specific binding sites for the DNA-binding protein, and when the biosensor is bound to the ligand the biosensor may de-repress and/or activate expression from the promoter. De-repression and/or activation of the expression of the reporter gene then can be correlated with the presence of the ligand. In other embodiments, when the biosensor is bound to a ligand, the biosensor may repress expression and/or inhibit expression, and when the biosensor is not bound to the ligand the biosensor may de-repress expression and/or activate expression. A decrease in expression of the reporter gene then can be correlated with the presence of the ligand.

[0060] The disclosed biosensors, systems, and methods may be utilized and/or performed using any suitable cell. Suitable cells may include prokaryotic cells and eukaryotic cells.

[0061] “Quantify”, “quantifying”, “quantification”, and any other grammatical variations thereof refer to the process of acquiring numerical values to determine, express, or measure an amount of a substance or signal.

[0062] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0063] As used herein, the term “chemical compound” and “compound”, refers to a chemical substance consisting of two or more different types of atoms or chemical elements in a fixed stoichiometric proportion. These compounds have a unique and defined chemical structure held together in a defined spatial arrangement by chemical bonds. Chemical compounds can be held together by covalent bonds, ionic bonds, metallic ions, or coordinate covalent bonds.

[0064] As used herein, the term “lipid” or “lipid-like” refers to a macromolecule that is soluble in nonpolar solvents. These molecules are usually hydrophobic or amphiphilic molecules; the amphiphilic nature of some lipids allows formation of structures such as vesicles, liposomes, membranes, and nanoparticles. Lipids can be categorized into fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and prenol lipids.

[0065] As used herein, a “carbohydrate” refers to a large family of organic compounds including, but not limited to sugars, starch, and cellulose, containing hydrogen and oxygen in similar ratios to water (2: 1) and used as structural materials in numerous biomolecules, such as DNA or RNA, and for energy storage with living tissues. Carbohydrates can be categorized into four groups including monosaccharides, disaccharides, oligosaccharides, and polysaccharides, wherein monosaccharides and disaccharides are the smallest forms of carbohydrates, commonly referred to as sugars, whereas oligosaccharides and polysaccharides are larger, complex structures used for energy storage of the structural foundation of nucleic acids and nucleotides.

[0066] As used herein, a “lipopeptide” refers to a biomolecule comprising a lipid bound to an amino acid sequence.

[0067] As used herein, a “peptidomimetic” refers to a small biomolecule comprising amino acid-like properties and structures designed to mimic a peptide, polypeptide, or protein. Peptidomimetic s can be generated from the modification of an existing peptide, or by designing similar systems that mimic peptides, including, but not limited to peptiods and beta-peptides.

[0068] An “agonist” refers to a chemical composition or compound that activates a receptor protein to produce a biological response. Control samples (untreated with agonists) are assigned a relative activity value of 0%. Inhibition of a described target protein is achieved when the activity value relative to the control increases by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more percent.

[0069] The term “detect” or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.

[0070] ‘ ‘Inhibitors” or “antagonist” of activity are used to refer to inhibitory molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein, e.g., ligands, antagonists, and their homologs and mimetics. Inhibitors are agents that, e.g., bind to, partially or totally block stimulation or activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, e.g., antagonists. Control samples (untreated with inhibitors) are assigned a relative activity value of 100%. Inhibition of a described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5%, or 1% or less.

[0071] A “conformational change” refers to a structural event wherein the structure of a protein changes in response to receiving an input signal, such as a ligand or target molecule binding, chemical modification, or a change in environment (e.g., pH, salt, osmolarity, and temperature changes).

Compositions and Compounds

[0072] The present invention provides a modified signal protein and methods detecting the presence/modulation and quantifying a target molecule using said signal protein.

[0073] The nudix-related transcriptional regulator (NrtR) proteins are a family of proteins responsible for regulation of various aspects of nicotinamide adenine dinucleotide (NAD+) biosynthetic pathways as well as other metabolic pathways including sugar pentoses utilization and biogenesis of phosphoribosyl pyrophosphate. NrtR proteins comprises binding domains targeting numerous genes for expression. Herein, the NrtR proteins have been modified to target specific metabolic intermediates, secondary messengers, carbohydrates, and nucleotide molecules, including, but not limited to adenosine diphosphate ribose (free ADPR or ADP ribose), arabinose, and NAD+. [0074] Thus in one aspect, disclosed herein is a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the signal protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and the target molecule.

[0075] It should be noted that the signal protein possesses attributes of a biosensor, wherein the signal protein comprises a binding domain that binds to the target molecule. The interaction between the signal protein and the target molecule causes conformational change, or a physical/structural change to the signal protein. Another domain of the signal protein comprises an output module, which receives an input signal in the form of the conformational change to the signal protein. Following the conformational change, the output module comprising a peptide linker and a signal transducer, emits a detectable signal in the form of light, a change in color, a change in emission intensity, a change in fluorescence lifetime, a change in temperature, or a change in pH. The presence of said detectable signal indicates the presence of the target molecule.

[0076] In some embodiments, the signal protein is a modified NrtR protein. In some embodiments, the target molecule comprises free adenosine diphosphate ribose (free ADPR). In some embodiments, the signal protein is a modified L-arabinose Nudix-related transcription factor (AraR) protein. In some embodiments, the target molecule comprises L- or D-Arabinose. In some embodiments, the said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+. In some embodiments, the target molecule comprises NAD+.

[0077] Exemplary target molecules include, but are not limited to free ADPR, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cyclicAMP (cAMP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), cyclic GMP (cGMP), inositol triphosphate (IP3), diacylglycerol (DAG), calcium (Ca²⁺), epinephrine, norepinephrine, acetylcholine, histamine, estrogen, testosterone, progesterone, cholesterol, corticosteroids, thyroid hormone, vitamin D, retinoic acid, nitric oxide (NO), carbon monoxide (CO), glutamate, dopamine, serotonin, glycine, gamma-aminobutyric acid (GABA), insulin, glucagon, and other signaling molecules. In some embodiments, the target molecule comprises a carbohydrate or sugar molecule, or derivatives thereof. Exemplary sugar molecules include, but are not limited to ribose, arabinose, glucose, fructose, sucrose, cellulose, galactose, lactose, maltose, starch, glycogen, dextrose, fucose, inositol, maltodextrin, mannose, ribulose, trehalose, xylose, and derivatives and isomers thereof.

[0078] In some embodiments, the target molecule comprises a nucleotide, or derivatives thereof. Exemplary nucleotides include, but are not limited to adenine, thymine, cytosine, guanine, uracil, 5-bromouracil, hypoxanthine, and derivatives and analogues thereof.

[0079] In some embodiments, the target molecule comprises a metabolic intermediate. As used herein, a metabolic intermediate refers to a molecules that are precursors or metabolites of biologically active molecules. It should be noted that metabolic intermediates may have minor importance to cellular function, but they are important regulators for enzyme functions. Exemplary metabolic intermediates include, but are not limited to malate, lactate, gluconate, citrate, oxaloacetate, oxoglutarate, acetyl CoA, fumarate, aconitate, isocitrate, ketoglutarate, succinyl CoA, succinate, pyruvate, nicotinamide adenine dinucleotide (NAD+ or NADH), nicotinamide adenine dinucleotide phosphate (NADP+ or NADPH), flavin adenine dinucleotide (FAD+ or FADH), ubiquinol, ubiquinone, and coenzyme Q.

[0080] In some embodiments, the output module comprises a peptide linker and a signal transducer. In some embodiments, the peptide linker comprises one or more amino acid selected from alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, P- Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3 -Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine,

2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine,

3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4- Diaminobutyric acid, N-Methylv aline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine, in any combination. In some embodiments, the peptide linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the peptide linker is a rigid peptide linker.

[0081] In some embodiments, the signal transducer is a fluorescent molecule, or a fluorophore. As used here, a “fluorescent molecule” or a “fluorophore” refers to a fluorescent chemical compound that absorb energy from an internal or external source, and in response emit energy in the form of light.

[0082] In some embodiments, the fluorescent molecule is circular permutation Venus (cpVenus). Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5- Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6- CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA- BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GEE; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bis aminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Eactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bisBTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy 492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FE ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC- 5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca²⁺ Dye; Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CE-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16- ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer- 1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1- 43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxystilbamidine (FluoroGold); Hydroxy tryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue- White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); or a combination thereof.

[0083] In some embodiments, the signal transducer is a colorimetric reagent. As used herein, a “colorimetric reagent” refers to a molecule or probe capable of changing color (e.g., from blue to green, from red to yellow, from clear (or colorless) to blue) upon a reaction or conformation change from a signal protein. [0084] Exemplary colorimetric reagent include, but are not limited to oxazine dyes, servon blue 5G, p-dimethylaminobenzaldehyde, 1,4-benzoquinone, ninhydrin, picryl chloride, p- quinone, vanillin, nile blue, azure A, azure B, azure C, brilliant cresyl blue, anthrone, anilic acid diphenylamine, eriogreen, m-cresol-indophenol, methylene blue, meldola blue, lissamine green B, o-dianisidine, viologen, and metal ions complexes including, but not limited to thallium, cadmium, lead, gold, iron, copper, bismuth, and aluminum.

[0085] In some embodiments, the peptide linker transduces the conformational change to the signal transducer. In some embodiments, the nudix-related transcriptional regulator has been modified so that it can interact with a non-native target. As used herein, a “native target” refers to target molecule that a signal protein naturally recognizes and binds to without prior modification to the structure of the signal protein. A “non-native target” refers to an induced or forced recognition and/or binding of the signal protein, usually requiring modification to the structure of the signal protein. In general, a native target of the nudix-related transcriptional regulator protein is a sequence of nucleotides. Herein, the nudix-related transcriptional regulator has been modified to target other molecules, including, but not limited to metabolic intermediates, carbohydrates, peptide, polypeptides, proteins, lipids, hormones, neurotransmitters, secondary messengers, nucleic acids, amino acids, and other derivatives thereof.

[0086] In some embodiments, the nudix-related transcriptional regulator has been evolved to change its specificity for the target.

Methods

[0087] In one aspect disclosed herein is a method for determining presence of a target molecule, the method comprising a) providing a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target, b) exposing the signal protein to the target molecule under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal, and c) detecting the signal, thereby determining the presence of the target molecule.

[0088] In one aspect, disclosed herein is a method for quantifying a target molecule, the method comprising a) providing a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target, b) exposing the signal protein to the target molecule under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal, and c) detecting the amount of signal produced in step b), thereby determining the amount of the target molecule present.

[0089] In one aspect disclosed herein is a method for determining modulation of a target molecule by a test compound, the method comprising a) providing a signal protein, wherein the signal protein is a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target, b) exposing the signal protein to the target molecule and the test compound under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal, c) detecting the amount of signal produced in step b), d) comparing the amount of signal produced in step b) to a control, wherein said control was carried out without the presence of the test compound, and e) determining if a difference exists between the amount of signal produced in the presence of the test compound and the amount of signal produced in the control, wherein a significant difference indicates that the test compound modulated the target molecule.

[0090] In some embodiments, the signal protein is a modified NrtR protein. In some embodiments, the target molecule comprises free adenosine diphosphate ribose (free ADPR). In some embodiments, the signal protein is a modified L-arabinose Nudix-related transcription factor (AraR) protein. In some embodiments, the target molecule comprises L- or D-Arabinose. In some embodiments, the said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+. In some embodiments, the target molecule comprises NAD+.

[0091] As discussed above, target molecules which can be detected with the methods disclosed herein include, but are not limited to free ADPR, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cyclicAMP (cAMP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), cyclic GMP (cGMP), inositol triphosphate (IP3), diacylglycerol (DAG), calcium (Ca²⁺), epinephrine, norepinephrine, acetylcholine, histamine, estrogen, testosterone, progesterone, cholesterol, corticosteroids, thyroid hormone, vitamin D, retinoic acid, nitric oxide (NO), carbon monoxide (CO), glutamate, dopamine, serotonin, glycine, gamma-aminobutyric acid (GABA), insulin, glucagon, and other signaling molecules.

[0092] Detection of a target molecule, or detection of quantification and/or modulation of a target molecule, can lead to the detection, and treatment, of diseases or disorders. It can also lead to a better understanding of appropriate drug dosages, the discovery of new compounds, and/or the discovery of new uses for known compounds.

[0093] Regarding the detection of free ADPR, dysregulation of enzymes involved in the regulation of ADPR signaling has been linked to a number of inherited and acquired human diseases, such as several neurological disorders and in cancer. Conversely, the therapeutic manipulation of free ADPR has been shown to ameliorate several disorders in both human and animal models. These include cardiovascular, inflammatory, autoimmune, and neurological disorders. Examples of such diseases and disorders can be found in Kulikova, V.A., Nikiforov, A.A. Role of NUDIX Hydrolases in NAD and ADP-Ribose Metabolism in Mammals. Biochemistry Moscow 85, 883-894 (2020), as well as in Guse AH, Calcium mobilizing second messengers derived from NAD. Biochim Biophys Acta. 2015 Sep;1854(9):1132-7, both of which are herein incorporated by reference in their entirety for its teaching concerning ADPR- associated disease.

[0094] Detection and/or therapies of NAD+ allows for monitoring and treatment of numerous energetic metabolic pathways, transcriptional regulation, DNA repairing systems, and reduction-oxidation status of numerous enzymes. Alterations of NAD+ homeostasis can impact disease conditions, including, but not limited to age-related disease, cardiovascular diseases, neurological or neurodegenerative disorders, cancer, microbial infections, ischemic conditions, and autoimmune diseases. Examples of diseases and treatments thereof using NAD+ can be found in Arenas- Jal et at. Therapeutic potential of nicotinamide adenine dinucleotide (NAD). Euro J of Pharm. 2020 July; 879: 173158, Wang et al. Nicotinamide adenine dinucleotide treatment alleviates the symptoms of experimental autoimmune encephalomyelitis by activating autophagy and inhibiting the NLRP3 inflammasome. Inter Immuno. 2021 January; 90: 107092, Braidy et al. Role of Nicotinamide adenine dinucleotide and related precursors as therapeutic targets for age-related degenerative diseases: rationale, biochemistry, pharmacokinetics, and outcomes. Antioxidants and Redox Signaling. 2018 November 30; 251- 294, and Hosseini et al. Nicotinamide adenine dinucleotide emerges as a therapeutic target in ageing and ischemic conditions. Biogerontology. 2019 March 5; 20: 381-395 are incorporated by reference in their entirety for teaching of detecting NAD and its use as a therapeutic target. [0095] L- or D-arabinose are simple sugars linked to glucose and lipid metabolism, and shown to effect metabolic disorders or diseases including, but not limited to diabetes (Type I or Type II), cardiovascular disease, obesity, stroke, or combinations thereof. Treatment with L- arabinose demonstrate protective effects against metabolic syndrome, which is a combination of metabolic deficiencies that increase the risk of type II diabetes, cardiovascular diseases, obesity, or stroke. Examples of using L- or D- arabinose treatment can be found in Hao et al. Protective effects of L-arabinose in high-carbohydrate, high fat diet induced metabolic syndrome in rats. Food & Nutrition Research. 2015 Dec 10; 59(1) and Wang et al. L-arabinose suppresses gluconeogenesis through AMP-activated protein kinase in metabolic disorder mice. Food & Function. 2020 Dec 22; 12: 1745-1756, herein incorporated by reference in its entirety for its teaching of administering arabinose sugars to treatment metabolic related disorders or diseases.

[0096] L- or D- arabinose has also been linked to diseases, such as microbial infections. L- or D-arabinose has been shown to be biomarkers for active microbial infections, including, but not limited to tuberculosis caused by Mycobacterium tuberculosis (Mtb). De et al. Estimation of D-arabinose by Chromatography /Mass Spectrometry as Surrogate for Mycobacterial Lipoarabinomannan in Human Urine. PLOS ONE. 2015 Dec 3; 10(12): e0144088, herein incorporated by reference in its entirety for its teaching concerning arabinose sugars detected as biomarkers of diseases or disorders.

[0097] The methods disclosed herein can be used in a variety of applications. For example, the methods can be used in a cell-based assay, such as multiplexed or high-throughput cell arrays. Thus, the methods disclosed herein can be used to assess viability, toxicity, mitochondrial or energetics activity, nuclear activity, or other cellular functions. Examples include cell viability assays, cell proliferation assays, cytotoxicity assays, cell senescence assays, cell death assays, cell membrane or mitochondrial membrane potential assays, and nuclear or mitochondrial fragmentation assays. It can also be used in a high-throughput array, such as a positional array comprising small chips, or glass surfaces, bound by the signal protein, or a suspension arrays comprising a suspension of beads bound by the signal protein in a liquid medium. Exemplary beads include, but are not limited to silica microbeads or polystyrene beads.

[0098] Small molecule test compounds can initially be members of an organic or inorganic chemical library. As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. The small molecules can be natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity.

[0099] Particular screening applications disclosed herein relate to the testing of pharmaceutical compounds in drug research (In Vitro Methods in Pharmaceutical Research”, Academic Press, 1997, and U.S. Pat. No. 5,030,015). Assessment of the activity of candidate pharmaceutical compounds generally involves administering a candidate compound, determining any change in the morphology, marker phenotype and expression, or metabolic activity of the cells and function of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.

[0100] The screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible molecular interaction effects. In some applications, compounds are screened initially for potential toxicity (Castell et al., pp. 375-410 in “In vitro Methods in Pharmaceutical Research,” Academic Press, 1997). Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain markers, receptors, or enzymes. Effects of a drug on chromosomal DNA can be especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. The reader is referred to A. Vickers (PP 375-410 in “In vitro Methods in Pharmaceutical Research,” Academic Press, 1997) for further elaboration.

[0101] Examples of methods include, but are not limited to, the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997 and U.S. Pat. No. 5,030,015. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, either alone or in combination with other drugs. The investigator determines any change in the morphology, marker phenotype, or functional activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlates the effect of the compound with the observed change.

[0102] The methods disclosed herein have important implications for patient treatment and also for clinical development of new therapeutics. Physicians select therapeutic regimens for patient treatment based upon the expected net benefit to the patient. The net benefit is derived from the risk to benefit ratio. The present invention permits selection of subjects who are more likely to benefit by intervention, thereby aiding the physician in selecting a therapeutic regimen. This might include using drugs with a higher risk profile where the likelihood of expected benefit has increased. Likewise, clinical investigators desire to select for clinical trials a population with a high likelihood of obtaining a net benefit. The present invention can help clinical investigators select such subjects or for determining entry criteria for clinical trials.

[0103] In some embodiments, a significant difference is more than a 5% difference. In some embodiments, a significant difference is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or more difference.

[0104] In some embodiments, modulation of the target molecule by the test compound results in an increased amount of measurable target molecule compared to the control. In some embodiments, modulation of the target molecule by the test compound results in a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,

58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 times or more increased amount of measurable target molecule compared to the control.

[0105] In some embodiments, modulation of the target molecule by the test compound results in a decreased amount of measurable target molecule compared to the control. In some embodiments, modulation of the target molecule by the test compound results in a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,

58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 times or more decreased amount of measurable target molecule compared to the control.

[0106] In some embodiments, the test compound is a small molecule such as a metal or organic compound, a polypeptide, a peptide, a natural product, a peptidomimetic, a nucleic acid, a lipid, lipopeptide, a carbohydrate, or any variant thereof. In some embodiments, the test compound is an agonist or an antagonist of the target molecule.

[0107] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0108] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLES

[0109] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Example 1: Fluorescent Biosensors using Nudix- related Transcription Factors

[0110] Genetically encoded fluorescent biosensors can be used to monitor intracellular metabolites with high spatial and temporal resolution in cells. In addition to facilitating the discovery of new biology, they have enabled precise, cell-based screening for modulators of specific metabolic activities. Herein, a fluorescent biosensor was developed which responds to the NAD⁺ derived signaling molecule free Adenosine 5 ’Di-Phosphate Ribose (ADPR).

Intracellular concentrations of free ADPR can reflect enzymatic activities and control TRPM2 channel opening.

[0111] ADPR is derived from the enzymatic cleavage of the metabolite NAD⁺, or oxidized nicotinamide adenine dinucleotide. Free ADPR concentrations are reported to reflect the activities of NAD⁺ consuming enzymes, such as pro-neurodegenerative enzyme SARM1 and human immunoenzyme CD-38. The lack of methods to monitor free ADPR concentrations in real-time and in physiological contexts represents a major barrier for screening and testing SARM1 blockers or CD-38 targeting approaches, with relevance to neurodegeneration, cancer, and cardiovascular disease. The major challenges for studying ADPR include that its levels fluctuate and spike very locally within minutes. The sources and concentrations of free ADPR in cells are unknown; how free ADPR concentrations are regulated in cells is also unknown. Additionally, as an intermediary in signaling pathways, free ADPR is the required agonist for the opening of human calcium channel Transient Receptor Potential Melastatin family member 2 (TRPM2). As such, intracellular free ADPR concentrations play a role in controlling fever responses, synaptic activity, cell death mechanisms, as well as inflammatory and oxidative stress responses.

Design of the free ADPR sensors.

[0112] These sensors for free ADPR concentrations can be used in different parts of live cells for dynamic measurements. These sensors are genetically encoded, and so they can be localized to different subcellular compartments using targeting sequences, are further amenable to restricted expression among cell types, and can be engineered into in vivo models.

[0113] The sensor is based on a circularly-permutated single fluorescent protein design (Baird et al, 1999 PNAS and reviewed in Nasu et al, 2021 Nat Chem Biol). For the fluorescence readout, the GFP-derived fluorescent protein, Venus, was used. Venus was circularly permutated and reforms to its fluorescent beta barrel fold by introducing a cut between amino acids 144 and 145 and connecting its original N- and C- termini with a 5 amino acid GGSGG linker sequence (circularly permutated Venus now referred to as cp Venus or cpV). Circular permutation permits attachment of an analyte binding domain in close proximity to the chromophore of Venus. The analyte binding domain confers selectivity of ligand binding to the sensor. The Nudix-like bacterial transcription factor NrtR from Shewanella oneidensis was used that is an ADPR-dependent transcriptional repressor (Rodionov et al, 2008 Nucleic Acids Res; Gao et al, 2019 eLlFE). The cpV protein was integrated between amino acids 118 and 119 in the sequence of NrtR (Uniprot Q8EFJ3). The integration site for cpV was determined through a combination of in silico analyses using solved crystal structure states of NrtR (PDB files: 3GZ5, 3GZ6, and 3GZ8; Huang et al 2009 Structure) to identify regions that underwent structural changes upon ligand binding. cpVenus was then cloned into identified sites without linkers and performed an in vitro screen with 15 potential candidates to identify chimeras that retained cpV fluorescence and that would exhibit fluctuations in their fluorescence in the presence of free ADPR ligand.

[0114] Based on these analyses, cpV integrated into site 118/119 in NrtR was chosen for further analyses. This site is adjacent to the hinge region of the ligand-binding site, and it is within an alpha-helix that undergoes a disruptive loss of secondary structure when free ADPR binds (FIG. 1). The model is that this structural change is transduced to the nearby chromophore 1 of cpV to cause a change in fluorescence intensity. This may occur through disruption of the chromophore or shifting the pKa of the chromophore.

[0115] Mutations in the DNA-binding domain of the sensor were further screened; historically this domain aberrantly caused issues including mis-localization and aggregation of sensors when expressed in cells. Due to the compact nature of NrtR, attempts to use truncation to eliminate the DNA-binding domain appeared to destabilize the whole protein and thus could not be used. Instead, three point mutations were identified and used to ablate the outward facing overall positive change on this domain. These point mutations are distinct from the ligand-binding domain. The specific sites and their mutations (K435A and S436D) were identified through empirical screening and it was determined that in combination they alleviated aggregation of the sensor when it was ectopically expressed (FIG. 2).

[0116] The mutation D122N was also incorporated to reduce the number of polar and charged side-chains in proximity to the sensor’s chromophore. The rationale was that charged residues could shift the local pKa of the chromophore promoting in its protonation. Introduction of D122N improved the brightness of the sensor and stabilized its pH-sensitivity between pH 7.4 - 8.0 (FIG. 3)

[0117] The sequence of the free ADPR sensor is shown in SEQ ID NO: 1.

In vitro.

[0118] The purified sensor (FIG. 4) decreased in fluorescence intensity with addition of free ADPR. At high concentrations, it saturated with - 80% diminishment of sensor brightness that represented an ~4-fold dose-dependent response (FIG.4B, red). As a control a binding pocket mutant (R98E) was generated that did not respond to free ADPR (FIG. 4B, black). It was determined that the sensor responds to free ADPR with a Kd ~ 2 pM ± 1 p M and is thus poised to detect intracellular free ADPR concentrations (Heiner, et al, 2006 Biochem J Gasser and Guse, 2005 J Chromatogr B Analyt Technol Biomed. Life Sci; Gasser et al, 2006 J Biol Chem). [0119] The sensor had minimal responsivity to structurally related molecules such as ADP and ATP (FIG. 5). Although fluorescence of the sensor is moderately affected by temperature and large fluctuations in pH, the non-binding control is similarly affected under parallel conditions. Thus, a parallel normalization of the sensor to its non-binding control may be possible to distinguish free ADPR-dependent changes from independent effects (FIG. 6).

In cells [0120] With confocal imaging, it was determined that the free ADPR sensor responded in live cells to an accumulation of free ADPR upon oxidation stress by acute H2O2 treatment (FIG. 7). In this experimental paradigm, H2O2 induces PARP1/2 to generate poly-ADPR chains, and due to the activities of glycohydrolases, this in turn leads to increased levels of free ADPR monomers. Accordingly, treatment with olaparib, a PARP1/2 inhibitor, eliminated sensor responses (FIG. 7). The generation of free ADPR was confirmed by a GCaMP7s sensor that monitored the resulting Ca²⁺ influx from activation of co-expressed TRPM2 (FIG. 7B). Interestingly, the data from the ADPR sensor indicated that free ADPR accumulated prior to TRPM2 channel opening. The data further indicated that the sensor’s ^appKd was appropriately tuned for in-cell measurements.

Structurally similar folds to instruct sensor design.

[0121] It was found that successful designs from the free ADPR sensor could be used to create a series of prototypes for new sensors using other Nudix-like TF family members. A major hinderance for the development for these sensors is that there is no clear rule for identifying the site of cpV integration. Each sensor has historically required an empirical screen to identify an integration site that results in transduction of the desired analyte response, and neither destabilizes the overall structure nor chromophore. Common insertional positions for cpV were found to exist across Nudix-like TF family members that can be used for sensor development. This is based on their similarities in fold and allosteric mechanism. Thus a new way to bypass screening for cpV integration among this family was identified. The identification and insertion of the fluorescent protein into analogous sites without screening immediately yielded new responsive sensor prototypes. Nudix-like TFs recognize a range of molecules, including sugars and nucleotides. Additional sensor prototypes include arabinose (using AraR, Uniprot Q8AAV8) and NAD⁺ (using NdnR, Uniprot A4QD34) (FIG. 8). Additionally, with homology prediction approaches and increased annotation of diverse prokaryotic genomes this finding can lead to the rapid development of new sensors.

[0122] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. SEQUENCE LISTINGS

1. SEQ ID NO: 1 (free ADPR Sensor)

MTEAEYLANYDPKAFKAQLLTVDAVLFTYHDQQLKVLLVQRSNHPFLGLWGLPGG

FIDETCDESLEQTVLRKLAEKTAVVPPYIEQLCTVGNNSRDARGWSVTVCYTALMSY QACQIQYNSNNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLL PDNHYLSFQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEEL FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTL

GYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNIASVSDVKWWPLADVLQMPLAFDHLQLIEQAR

ERLTQKALYSLVPGFALSEPFTLPELQHVHEVLLGKPIQGADFRRRVEQADLLIDTGL KRTERGRPANLYCLKPDTASYRFLRNLEC

2. SEQ ID NO: 2 (free ADPR Sensor no binding control R98E)

MTEAEYLANYDPKAFKAQLLTVDAVLFTYHDQQLKVLLVQRSNHPFLGLWGLPGG

FIDETCDESLEQTVLRKLAEKTAVVPPYIEQLCTVGNNSRDAEGWSVTVCYTALMSY QACQIQYNSNNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLL PDNHYLSFQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEEL FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTL

GYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNIASVSDVKWWPLADVLQMPLAFDHLQLIEQAR

ERLTQKALYSLVPGFALSEPFTLPELQHVHEVLLGKPIQGADFRRRVEQADLLIDTGL KRTERGRPANLYCLKPDTASYRFLRNLEC

3. SEQ ID NO: 3 (NAD+ sensor based on NdnR)

MPASPEIQMAVSTIIFALRPGPQDLPSLWAPFVPRTREPHLNKWALPGGWLPPHEELE

DAAARTLAETTGLHPSYLEQLYTFGKVDRSPTGRVISVVYWALVRADEALKAIPGY NSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLS FQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPI LVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQC

FARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNENVQWFPADHLPELAFDHNDIVKYALERLRTKVEYSEIA HSFLGETFTIAQLRSVHEAVLGHKLDAANFRRSVATSPDLIDTGEVLAGTPHRPPKLF RFQR 4. SEQ ID NO: 4 (Arabinose sensor based on AraR)

MKNYYSSNPTFYLGIDCIIFGFNEGEISLLLLKRNFEPAMGEWSLMGGFVQKDESVD

DAAKRVLAELTGLENVYMEQVGAFGAIDRDPGERVVSIAYYALININEYDRYNSDN

VYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSFQSK

LSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVEL

DGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARY

PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK

EDGNILGHKLEYNELVQKHNAYWVNINELPALIFDHPEMVDKAREMMKQKASVEPI

GFNLLPKLFTLSQLQSLYEAIYGEPMDKRNFRKRVAEMDFIEKTDKIDKLGSKRGAA

LYKFNGKAYRKDPKFKLGSAGLC

Claims

CLAIMS What is claimed is:

1. A signal protein comprising a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the signal protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and the target molecule.

2. The signal protein of claim 1, wherein said signal protein is a modified NrtR protein.

3. The signal protein of claim 1, wherein the target molecule comprises free adenosine diphosphate ribose (free ADPR).

4. The signal protein of claim 1, wherein said signal protein is a modified L-arabinose Nudix-related transcription factor (AraR) protein.

5. The signal protein of claim 1, wherein the target molecule comprises L- or D- Arabinose.

6. The signal protein of claim 1 , wherein said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+.

7. The signal protein of claim 1, wherein the target molecule comprises NAD+.

8. The signal protein of any one of claims 1-7, wherein the output module comprises a peptide linker and a signal transducer.

9. The signal protein of claim 8, wherein the signal transducer is a fluorescent molecule.

10. The signal protein of claim 9, wherein the fluorescent molecule is cpVenus.

11. The signal protein of any one of claims 8-10, wherein the peptide linker transduces the conformational change to the signal transducer.

12. The signal protein of any one of claims 1-11, wherein the nudix-related transcriptional regulator has been modified so that it can interact with a non-native target.

13. The signal protein of claim 12, wherein the nudix-related transcriptional regulator has been evolved to change its specificity for the target.

14. A method for determining presence of a target molecule, the method comprising: a) providing a signal protein comprising a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target; b) exposing the signal protein to the target molecule under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal; and c) detecting the signal, thereby determining the presence of the target molecule.

15. The method of claim 14, wherein said signal protein is a modified NrtR protein.

16. The method of claim 14, wherein the target molecule comprises free adenosine diphosphate ribose (free ADPR).

17. The method of claim 14, wherein said signal protein is a modified L-arabinose Nudix- related transcription factor (AraR) protein.

18. The method of claim 14, wherein the target molecule comprises L- or D-Arabinose.

19. The method of claim 14, wherein said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+.

20. The method of claim 14, wherein the target molecule comprises NAD+.

21. The method of any one of claims 14-20, wherein the output module comprises a peptide linker and a signal transducer.

22. The method of claim 14-21, wherein the signal transducer is a fluorescent molecule.

23. The method of claim 22, wherein the fluorescent molecule is cp Venus.

24. The method of any one of claims 21-23, wherein the peptide linker transduces the conformational change to the signal transducer.

25. The method of any one of claims 14-24, wherein the nudix-related transcriptional regulator has been modified so that it can interact with a non-native target.

26. The method of claim 25, wherein the nudix-related transcriptional regulator has been evolved to change its specificity for the target.

27. A method for quantifying a target molecule, the method comprising: a) providing a signal protein comprising a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target; b) exposing the signal protein to the target molecule under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal; and c) detecting the amount of signal produced in step b), thereby determining the amount of the target molecule present.

28. The method of claim 27, wherein said signal protein is a modified NrtR protein.

29. The method of claim 27, wherein the target molecule comprises free adenosine diphosphate ribose (free ADPR).

30. The method of claim 27, wherein said signal protein is a modified L-arabinose Nudix- related transcription factor (AraR) protein.

31. The method of claim 27, wherein the target molecule comprises L- or D-Arabinose.

32. The method of claim 27, wherein said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+.

33. The method of claim 27, wherein the target molecule comprises NAD+.

34. The method of any one of claims 27-33, wherein the output module comprises a peptide linker and a signal transducer.

35. The method of claim 27-34, wherein the signal transducer is a fluorescent molecule.

36. The method of claim 35, wherein the fluorescent molecule is cpVenus.

37. The method of any one of claims 34-36, wherein the peptide linker transduces the conformational change to the signal transducer.

38. The method of any one of claims 27-37, wherein the nudix-related transcriptional regulator has been modified so that it can interact with a non-native target.

39. The method of claim 38, wherein the nudix-related transcriptional regulator has been evolved to change its specificity for the target.

40. A method for determining modulation of a target molecule by a test compound, the method comprising: a) providing a signal protein comprising a modified nudix-related transcriptional regulator (NrtR) family protein, wherein the signal protein is modified such that a conformational change occurs in the signal protein when it binds a target molecule, and further wherein the modified protein comprises an output module, wherein said output module produces a signal upon interaction of the signal protein and its target; b) exposing the signal protein to the target molecule and the test compound under conditions such that the signal protein is capable of binding the target molecule, wherein said binding produces a signal; and c) detecting the amount of signal produced in step b); d) comparing the amount of signal produced in step b) to a control, wherein said control was carried out without the presence of the test compound; and e) determining if a difference exists between the amount of signal produced in the presence of the test compound and the amount of signal produced in the control, wherein a significant difference indicates that the test compound modulated the target molecule.

41. The method of claim 40, wherein said signal protein is a modified NrtR protein.

42. The method of claim 40, wherein the target molecule comprises free adenosine diphosphate ribose (free ADPR).

43. The method of claim 40, wherein said signal protein is a modified L- arabinose Nudix- related transcription factor (AraR) protein.

44. The method of claim 40, wherein the target molecule comprises L- or D-Arabinose.

45. The method of claim 40, wherein said signal protein is a modified NAD-responsive transcriptional repressor (NdnR) of NAD+.

46. The method of claim 40, wherein the target molecule comprises NAD+.

47. The method of any one of claims 40-46, wherein the output module comprises a peptide linker and a signal transducer.

48. The method of any one of claims 40-47, wherein the signal transducer is a fluorescent molecule.

49. The method of claim 48, wherein the fluorescent molecule is cp Venus.

50. The method of any one of claims 47-49, wherein the peptide linker transduces the conformational change to the signal transducer.

51. The method of any one of claims 40-50, wherein the nudix-related transcriptional regulator has been modified so that it can interact with a non-native target.

52. The method of claim 51, wherein the nudix-related transcriptional regulator has been evolved to change its specificity for the target.

53. The method of any one of claims 40-52, wherein a significant difference is more than a 5% difference.

54. The method of any one of claims 40-53, wherein modulation of the target molecule by the test compound results in an increased amount of measurable target molecule compared to the control.

55. The method of any one of claims 40-53, wherein modulation of the target molecule by the test compound results in a decreased amount of measurable target molecule compared to the control.

56. The method of any of claims 40-55, wherein the test compound is a small molecule such as a metal or organic compound, a polypeptide, a peptide, a natural product, a peptidomimetic, a nucleic acid, a lipid, lipopeptide, or a carbohydrate.

57. The method of any one of claims 40-56, wherein the test compound is an agonist or an antagonist of the target molecule.