Engineered protein scaffolds and novel peptide epitopes for the development of synthetic reporters

Abstract

Provided herein are synthetic reporters that can be used in multiplexed, cell-based immunoassays and are capable of reporting modulations in cell signaling pathways. Also provided herein are nucleic acids and vectors encoding the synthetic reporters under the control of a promoter. The nucleic acids and vectors may be used in methods of detecting an effect of a stimulus on a signal transduction pathway.

Description

DESCRIPTIONENGINEERED PROTEIN SCAFFOLDS AND NOVEL PEPTIDE EPITOPES FOR THE DEVELOPMENT OF SYNTHETIC REPORTERS

[0001] This application claims the benefit of United States Provisional Application No.63 / 754,244 filed February 5, 2025, the entire contents of which are hereby incorporated by reference.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on January 28, 2026, is named MESOPOOQ8WO_ST26.xml and is 53,812 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates generally to the field of cellular and molecular biology. More particularly, it concerns synthetic reporters and their use in cell-based assays for detecting modulations in cell signaling pathways.2. Description of Related Art

[0004] Assays to detect signaling pathway activation typically rely on luciferase or alkaline phosphatase reporters. These assays are based on promoterless luciferase-encoding vectors, into which a regulatory region of interest is cloned. The detection of luciferase following an external stimulus indicates that the regulatory region of interest was activated by the stimulus. However, the reliance on luciferase as a read-out prevents the detection of more than one or two pathway activation events in a single cell. As such, reporters that enable multiplex detection of signaling pathway activation events are needed.4919-2079-0411 , v. 1SUMMARY

[0005] Provided herein are three -helices reporter scaffold or synthetic reporter proteins comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1; wherein positions 35-54 are a first peptide sequence; wherein positions 89-108 are a second peptide sequence; wherein the first peptide sequence and the second peptide sequence are each independently 2 to 20 amino acids in length. The scaffold proteins may comprise an amino acid sequence having 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% identity to the amino acid sequence of SEQ ID NO: 1. The scaffold proteins may comprise the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of at least one of the first peptide and the second peptide may be identical to the amino acid sequence of SEQ ID NO: 16.

[0006] Provided herein are four-helices reporter scaffold or synthetic reporter proteins comprising the amino acid sequence of SEQ ID NO: 2; wherein positions 33-52 are a first peptide sequence; wherein positions 81-100 are a second peptide sequence; wherein positions 134-153 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length; and wherein at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is at least 6 amino acids in length. The amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence may be identical to the amino acid sequence of one of SEQ ID NO: 17-19.

[0007] Provided herein are |3-sandwich reporter scaffold or synthetic reporter proteins comprising the amino acid sequence of SEQ ID NO: 4, wherein positions 55-74 are a first peptide sequence; wherein positions 89-108 are a second peptide sequence; wherein positions 122-141 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length; and wherein at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is at least 6 amino acids in length. The amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence may be identical to the amino acid sequence of one of SEQ ID NO: 23-25.24919-2079-0411 , v 1

[0008] Provided herein are a-[ -a sandwich reporter scaffold or synthetic reporter proteins comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 3, wherein positions 36-55 are a first peptide sequence; wherein positions 67-86 are a second peptide sequence; wherein positions 129-148 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length. The scaffold proteins may comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 3. The scaffold proteins may comprise the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence may be identical to the amino acid sequence of one of SEQ ID NO: 20-22.

[0009] Provided herein are 0-sandwich reporter scaffold or synthetic reporter proteins comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 5; wherein positions 55-74 are a first peptide sequence; wherein positions 89-108 are a second peptide sequence; wherein positions 122-141 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length. The scaffold proteins may comprise an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 5. The scaffold proteins may comprise the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence may be identical to the amino acid sequence of one of SEQ ID NO: 20-22.

[0010] In any of the reporter scaffold or synthetic reporter proteins provided herein, the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence may:(a) be 8 to 12 amino acids in length;(b) contain at least 2 but no more than 4 of any combination of amino acids R, E, D, Q, and N;(c) contain at least 5 but no more than 9 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and34919-2079-0411 , v 1(e) not contain amino acids C, K, or P.

[0011] In any of the reporter scaffold or synthetic reporter proteins provided herein, the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence may:(a) be 10 amino acids in length;(b) contain 3 of any combination of amino acids R, E, D, Q, and N;(c) contain 7 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and (e) not contain amino acids C, K, or P.

[0012] In any of the reporter scaffold or synthetic reporter proteins provided herein, the amino acid sequences of the first peptide sequence, the second peptide sequence, and the third peptide sequence may each independently be identical to the amino acid sequence of one of SEQ ID NOs: 6-30.[0013| In any of the reporter scaffold or synthetic reporter proteins provided herein, the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence may be identical to the amino acid sequence of one of SEQ ID NOs: 6-14.

[0014] Any of the reporter scaffold or synthetic reporter proteins provided herein may comprise an affinity tag sequence. The affinity tag sequence may comprise a streptavidin-binding peptide sequence (SEQ ID NO: 28).

[0015] Any of the reporter scaffold or synthetic reporter proteins provided herein may comprise a signal peptide sequence. The signal peptide sequence may comprise the amino acid sequence of any one of SEQ ID NOs: 37-48.

[0016] Provided herein are polynucleotides comprising a nucleotide sequence encoding the reporter scaffold or synthetic reporter proteins described herein. Also provided herein are expression constructs comprising a promoter sequence operably linked to a nucleotide sequence encoding the reporter scaffold or synthetic reporter proteins described herein. The promoter sequence may be an inducible promoter sequence.44919-2079-0411 , v 1

[0017] Provided herein are expression vectors comprising one or more expression constructs described herein. The expression vectors may further comprise an antibiotic resistance element. The expression vectors may be viral vectors, such as, for example, adeno-associated virus viral vectors.[0018| Provided herein are recombinant cells comprising an expression construct or expression vector described herein. The expression construct may be a first expression construct, and the recombinant cell further comprises at least a second expression construct, wherein the promoter of the first expression construct differs from the promoter of the second expression construct, and further wherein the polynucleotide comprising the nucleotide sequence encoding the reporter scaffold or synthetic reporter protein of the first expression construct differs from the polynucleotide comprising the nucleotide sequence encoding the reporter scaffold or synthetic reporter protein of the second expression construct. The recombinant cells may further comprise a third expression construct and a fourth expression construct, wherein the promoter and the polynucleotide comprising the nucleotide sequence encoding the reporter scaffold or synthetic reporter protein of the third expression construct differs from the promoter and polynucleotide comprising the nucleotide sequence encoding the reporter scaffold or synthetic reporter protein of the first expression construct, the second expression construct, and the fourth expression construct.

[0019] Provided herein are recombinant cell comprising a plurality of different expression constructs as described herein, wherein each different expression construct has a different promoter and encodes a different protein from the other different expression constructs in the plurality of different expression constructs. The recombinant cells may comprise from one to fifty different expression constructs.

[0020] Provided herein are recombinant, non-human animals, comprising a polynucleotide, expression construct, expression vector, or recombinant cell as described herein.

[0021] Provided herein are polypeptides comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 or an amino acid that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at54919-2079-0411 , v 1least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0022] Provided herein are methods of detecting an effect of a stimulus on a signal transduction pathway, the method comprising (a) contacting a recombinant cell or recombinant, non-human animal as described herein with the stimulus; and (b) detecting a change in expression of a synthetic reporter. The method may be further defined as a method for screening compounds to detect effects on cell signaling.

[0023] The synthetic reporter may comprise an affinity tag, where the synthetic reporter is detected using an antibody that binds to the affinity tag. The synthetic reporter may comprise an affinity tag, where the synthetic reporter is detected using an antibody that binds to the affinity tag and an antibody that binds to an epitope of the synthetic reporter. The synthetic reporter may be detected using an antibody that binds to an epitope of the synthetic reporter.

[0024] The recombinant cell or recombinant, non-human animal may comprise two or more expression constructs each having a different promoter and encoding a different synthetic reporter. The recombinant cell or recombinant, non-human animal may comprise ten or more expression constructs each having a different promoter and encoding a different synthetic reporter. Each synthetic reporter may be detected using an antibody that specifically binds to an epitope that is unique to the synthetic reporter. The antibody for each synthetic reporter may be present at a spatially discrete location on a microarray, where each discrete location only has one type of antibody to detect one of the synthetic reporters. Detecting a signal at a discrete location would indicate that the signaling pathway associated with that synthetic reporter was activated.

[0025] Provided herein are methods of detecting an effect of at least 3 stimuli on signal transduction pathways, the method comprising: (a) contacting cells comprising expression cassettes for at least three different synthetic reporters, wherein each synthetic reporter is under the control of a different regulatory element; and (b) detecting a change in expression for each of the synthetic reporters. Also provided are methods of detecting an effect of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, from 3 to 12, from 3 to 10, from 3 to 8, from 3 to 6, from 4 to 12, from 4 to 10, from 4 to 8, from 4 to 6, from 6 to 12, from 6 to 10, from 6 to 8, from 8 to 12, or from 8 to 10 stimuli using an equal number of64919-2079-0411 , v 1different synthetic reporters (i.e., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, from 3 to 12, from 3 to 10, from 3 to 8, from 3 to 6, from 4 to 12, from 4 to 10, from 4 to 8, from 4 to 6, from 6 to 12, from 6 to 10, from 6 to 8, from 8 to 12, or from 8 to 10 different synthetic reporters). Each synthetic reporter may comprise a first epitope site and a second epitope site, wherein each of the first epitope site and the second epitope site is independently disposed between an alpha helix and a beta sheet, a first alpha helix and a second alpha helix, or a first beta sheet and a second beta sheet, wherein each synthetic reporter has a unique first epitope and / or wherein each synthetic reporter has a unique second epitope. The cells may comprise expression cassettes for at least ten different synthetic reporters, wherein each synthetic reporter is under the control of a different regulatory element. The method may be further defined as a method for screening compounds to detect effects on cell signaling.

[0026] Each synthetic reporter may comprise a unique affinity tag, wherein each synthetic reporter is detected using an antibody that binds to its affinity tag. Each synthetic reporter may comprise a unique affinity tag, wherein each synthetic reporter is detected using an antibody that binds to the affinity tag and an antibody that binds to an epitope of the synthetic reporter. Each of the synthetic reporters can be detected using an antibody that binds to the unique first and / or second epitope of the synthetic reporter.

[0027] The antibody for each synthetic reporter may be present at a spatially discrete location on a microarray, where each discrete location only has one type of antibody to detect one of the synthetic reporters. Detecting a signal at a discrete location would indicate that the signaling pathway associated with that synthetic reporter was activated.

[0028] Provided herein are methods for detecting a microRNA in a sample, the method comprising:(a) contacting the sample with a first capture probe and a first detection probe under conditions to allow hybridization between the microRNA and the first capture probe and the first detection probe to form a binding complex if the microRNA is present in the sample, wherein the first capture probe comprises a first single stranded nucleotide segment having a sequence that is complementary to a first portion of the microRNA and a capture moiety, wherein the detection probe comprises (i) a second single stranded nucleotide segment having a sequence that is complementary to a second portion of the microRNA, (ii) a double stranded nucleotide segment74919-2079-0411 , v 1that encodes a synthetic reporter, and (iii) a promoter, wherein the promoter is operably linked to the double stranded nucleotide segment that encodes the synthetic reporter;(b) capturing the binding complex on a solid support coated with a binding partner for the capture moiety;(c) releasing the binding complex from the solid support;(d) subjecting the released sample to a cell-free protein synthesis reaction to produce the synthetic reporter encoded by the detection probe; and(e) detecting the synthetic reporter, thereby detecting the microRNA.[0029| The methods may further comprise washing the solid support between steps (b) and (c). The capture moiety may be biotin. The synthetic reporter may comprise a first epitope site and a second epitope site, wherein each of the first epitope site and the second epitope site is independently disposed between an alpha helix and a beta sheet, a first alpha helix and a second alpha helix, or a first beta sheet and a second beta sheet, wherein each synthetic reporter has a unique first epitope and / or wherein each synthetic reporter has a unique second epitope. The synthetic reporter may be any of the reporter scaffold or synthetic reporter proteins described herein.[0030| The synthetic reporter may comprise an affinity tag, wherein the synthetic reporter is detected using an antibody that binds to the affinity tag. The synthetic reporter may comprise an affinity tag, where the synthetic reporter is detected using an antibody that binds to the affinity tag and an antibody that binds to an epitope of the synthetic reporter. The synthetic reporter can be detected using an antibody that binds to an epitope of the synthetic reporter.

[0031] The sample may comprise two or more microRNAs and where step (a) comprises a detection probe and a capture probe for each microRNA, where each detection probe encodes a unique synthetic reporter. Each synthetic reporter can be detected using an antibody that specifically binds to an epitope that is unique to the synthetic reporter. The antibody for each synthetic reporter may be present at a spatially discrete location on a microarray, where each discrete location only has one type of antibody to detect one of the84919-2079-0411 , v 1synthetic reporters. Detecting a signal at a discrete location indicates that the microRNA associated with that synthetic reporter was present.

[0032] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0033] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.[0034| FIG. 1 provides an illustration of a 3D ribbon model of a three-helices scaffold protein 101 containing epitope insertion region 102 and epitope insertion region 103. SEQ ID NO: 1 is the amino acid sequence of a three -helices scaffold containing epitope insertion regions 102 and 103.

[0035] FIG. 2 provides an illustration of 3D ribbon model of a four-helices scaffold protein 201 containing epitope insertion region 202, epitope insertion region 203, and epitope insertion region 204. SEQ ID NO: 2 is the amino acid sequence of a four-helices scaffold containing epitope insertion regions 202, 203, and 204.

[0036] FIG. 3 provides an illustration of 3D ribbon model of an a-p-u sandwich scaffold protein 301 containing epitope insertion region 302, epitope insertion region 303, and epitope insertion region 304. SEQ ID NO: 3 is the amino acid sequence of an a-P-a sandwich scaffold protein containing epitope insertion regions 302, 303, and 304.

[0037] FIG. 4 provides an illustration of 3D ribbon model of a -sandwich scaffold protein 401 containing epitope insertion region 402, epitope insertion region 403, and epitope insertion region 404. SEQ ID NO: 4 is the amino acid sequence of a first P-sandwich scaffold protein containing epitope insertion regions 402, 403, and 404. SEQ ID NO: 5 is the amino94919-2079-0411 , v 1acid sequence of a second (3-sandwich scaffold protein containing epitope insertion regions 402, 403, and 404.

[0038] FIG. 5 provides the amino acid sequences of four representative scaffold proteins. SEQ ID NO: 33 is the amino acid sequence of an exemplary three-helices scaffold. SEQ ID NO: 34 is the amino acid sequence of an exemplary four-helices scaffold. SEQ ID NO: 35 is the amino acid sequence of an exemplary a-(3-a sandwich scaffold protein. SEQ ID NO: 36 is the amino acid sequence of an exemplary P-sandwich scaffold protein. The sequences underlined with a dashed line are streptavidin affinity tag sequences. The underlined sequences identify the epitope insertion regions of the scaffolds.

[0039] FIG. 6A provides an illustration providing an overview of a multiplexed cellular signaling assay.

[0040] FIGS. 6B and 6C provide an overview of a split-probe cell-free protein synthesis assay for the detection of a target microRNA sequence.[0041| FIG. 7 provides an SDS-PAGE analysis of in vitro translated candidate scaffolds. Lane 1: 5DMA; Lane 2: 5DMA-E2-T7: Lane 3: 2N8O; Lane 4: 2N8O-E2-T7: Lane 5: 5JCV-A; Lane 6: PsbP123; Lane 7: 5JCV-B; Lane 8: FimA120.

[0042] FIGS. 8A and 8B provide an SDS-PAGE analysis of scaffolds with and without heat treatment in elution buffer. Lane 1 : ladder; Lane 2: 5DMA without heat treatment; Lane 3: 2N8O without heat treatment; Lane 4: PsbP123 without heat treatment; Lane 5: FimA120 without heat treatment; Lane 6: 5DMA with heat treatment; Lane 7 : 2N8O with heat treatment; Lane 8: PsbP123 with heat treatment; Lane 9: FimA120 with heat treatment; Lane 10: 5DMA flow through; Lane 11: 2N8O flow through; Lane 12: PsbP123 flow through; Lane 13: FimA120 flow through.

[0043] FIG. 9 provides an SDS-PAGE analysis of the expression and purification of the diversified protein scaffolds.

[0044] FIGS. 10A-10C provide SDS-PAGE analyses of the expression and purification of synthetic reporters. FIG. 10A is for 3H-1 synthetic reporters, with the arrow indicating wt 3H-1 at 13.1 kDa. FIG. 10B is for FimA-3 synthetic reporters, with the arrow indicating wt104919-2079-0411 , v 1FimA-3 at 12.7 kDa. FIG. IOC is for PsbP-3 synthetic reporters, with the arrow indicating wt PsbP-3 at 18.2 kDa.

[0045] FIGS. 11A and 11B provide ELISA data demonstrating the antibodyaccessibility of the FLAG epitopes present in various synthetic reporter epitope positions. FIG.11 A is for synthetic reporters having a single FLAG epitope. For each group of bars, the left bar is “Day 1 ” and the right bar (if present) is “Day 2”). FIG. 1 IB is for synthetic reports having two reporter epitopes - one FLAG epitope plus one of cMyc, E2, HA, or T7. For each group of bars, the leftmost bar is “Harvest”, the rightmost bar (if present) is “Blank”, and the middle bar is “Elution”.DETAILED DESCRIPTION

[0046] Provided herein are synthetic reporters that can be used in various applications, including multiplexed, cell-based immunoassays and are capable of reporting modulations in cell signaling pathways. A nucleic acid encoding a synthetic reporter will be operably linked to a transcription regulatory element such that modulation of the transcription regulatory element’s activity will alter the expression of the synthetic reporter. The transcription regulatory element may be activated or repressed in response to a stimulus.

[0047] When a transcription regulatory element is activated, the expression level of the synthetic reporter operably linked thereto will increase. The transcription regulatory element may be inactive or minimally active in the absence of a stimulus such that the synthetic reporter is not detectable, or only detectable at low levels, in the absence of the stimulus. A stable synthetic reporter with a long half-life may be used to ensure that sufficient levels of the synthetic reporter accumulate.

[0048] When a transcription regulatory element is repressed, the expression level of the synthetic reporter operably linked thereto will decrease. The transcription regulatory element may be active in the absence of a stimulus such that the synthetic reporter is expressed at detectable levels in the absence of the stimulus. A synthetic reporter with a short half-life may be used to ensure that decreased levels of the synthetic reporter can be detected over time.

[0049] Since each transcription regulatory element — synthetic reporter pair can be distinguished based on detectable features of the synthetic reporter, a single cell may be engineered with more than one transcription regulatory element — synthetic reporter pair, thus114919-2079-0411 , v 1providing for multiplex detection of signaling pathway modulation. As such, a single cell may be engineered with two, three, four, five, six, seven, eight, nine, ten, or more, transcription regulatory element — synthetic reporter pairs. The engineered cell containing one or more transcription regulatory element — synthetic reporter pair may be in vitro or in vivo. As such, the stimulus being tested may be administered to, for example, a single cell, a cell culture plate, or to an animal comprising the engineered cells.

[0050] Selection of the transcription regulatory element provides the signaling pathway specificity of a given transcription regulatory element — synthetic reporter pair. Table 1 provides exemplary transcription regulatory elements and their associated signaling pathway. However, the assay described herein can be applied to any transcription regulatory element and its associated upstream signaling pathway.Table 1. Exemplary transcription regulatory elements124919-2079-0411 , v 1134919-2079-0411 , v 1

[0051] As an example, an assay may be designed such that the synthetic reporter is under the control of an Androgen Response Element. In this assay, stimuli may be tested for their ability to activate the androgen receptor to induce reporter expression. Alternatively, the cells may be treated with an agent that stimulates the androgen receptor (e.g., testosterone or 5a-Dihydrotestosterone (5-DHT)) to induce a basal level of reporter expression. Then stimuli (e.g., Enzalutamide, Bicalutamide, Mifepristone, and ARCC-4) may be tested for their ability to antagonize androgen receptor signaling, resulting in decreased expression of the synthetic reporter.

[0052] As an example, an assay may be designed such that the synthetic reporter is under the control of an API -responsive element. In this assay, stimuli may be tested for their ability to activate the API -responsive element to induce reporter expression (e.g., activators of the JNK signaling pathway). Alternatively, the cells may be treated with an agent that stimulates the AP-1 responsive element (e.g., Phorbol 12-Myristate 13-Acetate (PMA)) to induce a basal level of reporter expression. Then stimuli (e.g., inhibitors of the JNK signaling144919-2079-0411 , v 1pathway, e.g., CC-401) may be tested for their ability to antagonize c-Jun N-terminal kinase (JNK) signaling, resulting in decreased expression of the synthetic reporter.

[0053] As an example, an assay may be designed such that the synthetic reporter is under the control of an ARE. In this assay, stimuli may be tested for their ability to activate the ARE to induce reporter expression (e.g., activators of Nrf2). Alternatively, the cells may be treated with an agent that stimulates the ARE (e.g., oxidative stress) to induce a basal level of reporter expression. Then stimuli (e.g., inhibitors of the ARE) may be tested for their ability to antagonize Nrf2 signaling, resulting in decreased expression of the synthetic reporter.

[0054] As an example, an assay may be designed such that the synthetic reporter is under control of an IL-2 promoter and the cells also constitutively express CTLA-4. Engagement of CTLA-4 by its ligand (e.g., CD80 or CD86) inhibits transcription from the IL-2 promoter. In this assay, the cells may be treated with an agent that stimulates CTLA-4 (e.g., Raji cells), resulting in low levels of reporter expression. Then stimuli (e.g., neutralizing antibodies to CTLA-4) may be tested for their ability to induce expression of the synthetic reporter.

[0055] As an example, an assay may be designed such that the synthetic reporter is under the control of a cAMP response element (CRE). In this assay, if the cells also express the Gastric Inhibitory Polypeptide Receptor, then stimuli may be tested for their ability to activate the GIPR to induce reporter expression (e.g., activators of GIPR). Alternatively, the cells may be treated with an agent that stimulates the GIPR (e.g., GIP) to induce a basal level of reporter expression. Then stimuli (e.g., inhibitors of the GIPR) may be tested for their ability to antagonize GIPR signaling, resulting in decreased expression of the synthetic reporter.

[0056] As an example, an assay may be designed such that the synthetic reporter is under the control of a cAMP response element (CRE). In this assay, if the cells also express the Glucagon-Like Peptide 1 Receptor, then stimuli may be tested for their ability to activate the GLP-1R to induce reporter expression (e.g., activators of GLP-1R, e.g., Glucagon-like peptide 1 (7-37), Glucagon-like peptide 1 (7-36) amide, Exendin-4, Lixisenatide, GLP-1 moiety from dulaglutide, Semaglutide, Tirzepatide, Retatrutide, Danuglipron). Alternatively, the cells may be treated with an agent that stimulates the GLP-1R (e.g., GLP-1) to induce a basal level of reporter expression. Then stimuli (e.g., inhibitors of the GLP-1 R) may be tested154919-2079-0411 , v 1for their ability to antagonize GLP-1R signaling, resulting in decreased expression of the synthetic reporter.

[0057] As an example, an assay may be designed such that the synthetic reporter is under the control of a TEAD responsive element. In this assay, stimuli may be tested for their ability to inhibit the TEAD responsive element to enhance reporter expression (e.g., inhibitors of the Hippo pathway). Alternatively, stimuli (e.g., activators of the Hippo pathway) may be tested for their ability to activate the TEAD responsive element signaling, resulting in decreased expression of the synthetic reporter.

[0058] As an example, an assay may be designed such that the synthetic reporter is under the control of a phosphorylated PDX-1 responsive element. Glucose induces phosphorylation of PDX-1 in pancreatic beta cells, thus leading to upregulation of insulin gene transcription. In this assay, stimuli may be tested for their ability to modulate the phosphorylation of PDX-1, resulting in changes in synthetic reporter expression.

[0059] The synthetic reporters disclosed herein may also be used in other applications, such as reporters in a cell-free system. As an example, a construct encoding a synthetic reporter can be employed in a split-probe system in which the synthetic reporter is translated only when two independent probes are linked by a target nucleic acid sequence. In one embodiment, a split probe is designed to include a capture probe and a detector probe, each having a sequence complementary to a portion of a target nucleic acid sequence. The capture probe comprises a binding reagent for immobilizing the capture probe. The detector probe comprises a synthetic reporter-encoding sequence to serve as a template for cell-free protein amplification. Multiple split-probe pairs, each pair encoding a unique synthetic reporter, may be employed in a multiplexed assay format to detect multiple different target nucleic acids. As a further example, a construct encoding a synthetic reporter can be employed in the study of the translation factors associated with the activation of mRNA for translation and / or compounds that can upregulate or downregulate translation.I. Synthetic Reporters

[0060] The synthetic reporters provided herein comprise one or more detectable epitopes within reporter scaffolds. The synthetic reporters have low homology and crossreactivity with human proteins, are small (e.g., less than 25 kDa), and remain soluble when164919-2079-0411 , v 1modified with detectable epitopes. For example, the synthetic reporters may remain soluble after treatment at 60°C for 30 minutes.

[0061] The synthetic reporters are highly stable in the sample preparation and assay conditions used in the detection methods (e.g., be stable upon heat treatment, pH changes, and / or chemical treatment). However, the synthetic reporters may have varying half-lives in cells. For example, a synthetic reporter may have a long half-life (e.g., 12 hours, 24 hours, 36 hours, 48 hours, or longer) such that levels of the synthetic reporter may increase over time upon modulation of the associated transcription regulatory element in response to a stimulus. On the other hand, a synthetic reporter may have a short half-life (e.g., 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or less) such that levels of the synthetic reporter may decrease over time upon modulation of the associated transcription regulatory element in response to a stimulus.[0062| The synthetic reporters may be modified to comprise a signal peptide such that the synthetic reporter is secreted from the cell upon expression. Signal peptides are usually 16-30 amino acids long and are located at the N-terminus of most newly synthesized, secreted proteins. The signal peptides are cleaved from the newly synthesized protein inside the endoplasmic reticulum. The signal peptide from any endogenous, secreted protein may be used. Exemplary signal peptides include, but are not limited to, the IgK signal peptide sequence (MDMRVPAQLLGLLLLWLRGARC; SEQ ID NO: 37), the VH signal peptide sequence (MKHLWFFLLLVAAPRWVLS; SEQ ID NO: 38), the VL signal peptide sequence (MVLQTQVFISLLLWISGAYG; SEQ ID NO: 39), the tPA signal peptide sequence (MDAMKRGLCCVLLLCGAVFVSPS; SEQ ID NO: 40), the IL-2 signal peptide sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 41), the albumin signal peptide sequence (MKWVTFISLLFSSAYS; SEQ ID NO: 42), and the insulin signal peptide sequence (MALWMRLLPLLALLALWGPDPAAA; SEQ ID NO: 43). Additional exemplary signal peptides include:174919-2079-0411 , v 1

[0063] The synthetic reporters may be modified to comprise N- and / or C-terminal affinity tags, which allow for purification. Exemplary affinity tags include, but are not limited to, a Strep-tag (WSHPQFEK; SEQ ID NO: 28), an HA-tag (YPYDVPDYA; SEQ ID NO: 27), a Myc-tag (EQKLISEEDL; SEQ ID NO: 26), a calmodulin-binding peptide (CBP; KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 31), a polyarginine tag, a polyhistidine-tag, a Flag -tag (DYKDDDDK; SEQ ID NO: 15), biotin, an Avi-tag (GLNDIFEAQKIEWHE; SEQ ID NO: 32), a V5-tag (GKPIPNPLLGLDST; SEQ ID NO: 49), an E-tag (GAPVPYPDPLEPR; SEQ ID NO: 50), an S-tag (KETAAAKFERQHMDS; SEQ ID NO: 51), an SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 52), and a polyglutamate tag.

[0064] The reporter scaffolds may be three-helices scaffolds, four-helices scaffolds, a-(3-a sandwich scaffolds, (3-sandwich scaffolds, or -barrel scaffolds. These scaffolds contain epitope insertion regions where detectable epitopes may be inserted. Moreover, the position of each epitope insertion region is such that a detectable epitope inserted in an epitope insertion region is accessible to an appropriate detection agent (e.g., an antibody having specificity for the detectable epitope). Amino acid sequences of four representative reporter scaffolds are shown in FIG. 5. The sequence underlined with a dashed line in each of the sequences is a streptavidin affinity tag sequence. SEQ ID NO: 33 is the amino acid sequence of a three-helices scaffold. The two underlined sequences are the epitope insertion regions of the three -helices scaffold, each of which may individually be replaced with a detectable epitope. SEQ ID NO: 34 is the amino acid sequence of a four-helices scaffold. The three underlined sequences are located in the epitope insertion regions of the four-helices scaffold, each of which may individually be replaced with a detectable epitope. SEQ ID NO: 35 is the amino acid sequence of an a-|3-a sandwich scaffold protein. The three underlined sequences are located in the184919-2079-0411 , v 1epitope insertion regions regions of the a- -a sandwich scaffold protein, each of which may individually be replaced with a detectable epitope. SEQ ID NO: 36 is the amino acid sequence of a -sandwich scaffold protein. The three underlined sequences are located in the epitope insertion regions regions of the P-sandwich scaffold protein, each of which may individually be replaced with a detectable epitope.[0065| The amino acid sequences of five exemplary synthetic reporters are shown in FIGS. 1-4. The scaffold, epitope insertion regions, and inserted epitopes of these synthetic reporters were selected such that epitopes are accessible to an appropriate detection agent (e.g., an antibody having specificity for the detectable epitope) and such that the scaffold structure, as defined by the interaction of the a helices and P sheets, remains largely intact when the epitope is incoiporated in the synthetic reporter. FIG. 1 is an illustration of a 3D ribbon model of a three-helices scaffold protein 101 (SEQ ID NO: 1) containing epitope insertion regions corresponding to epitope insertion region 102 and epitope insertion region 103. FIG. 2 is an illustration of a 3D ribbon model of a four-helices scaffold protein 201 (SEQ ID NO: 2) containing epitope insertion regions corresponding to epitope insertion region 202, epitope insertion region 203, and epitope insertion region 204. FIG. 3 is an illustration of a 3D ribbon model of an a-|3-a sandwich scaffold protein 301 (SEQ ID NO: 3) containing epitope insertion regions corresponding to epitope insertion region 302, epitope insertion region 303, and epitope insertion region 304. FIG. 4 is an illustration of 3D ribbon models of P-sandwich scaffold proteins 401 (SEQ ID NOs: 4 or 5) containing epitope insertion regions corresponding to epitope insertion region 402, epitope insertion region 403, and epitope insertion region 404.

[0066] The detectable epitopes may be synthetic epitopes, which are orthogonal to each other, orthogonal to natural peptides, and compatible with the scaffold structure. Table 2 provides information on various exemplary epitopes that may be incorporated into one or more epitope insertion regions of the scaffold proteins disclosed herein.Table 2. Epitopes that may be incorporated into one or more epitope insertion regions of the scaffold proteins disclosed herein194919-2079-0411 , v 1Epitopes 1-9 are synthetic epitope sequences; epitopes 11-20 are epitope insertion regions (i.e., base epitopes) in one of the reporter scaffolds; epitopes 21-25 are other known reporter epitope sequences.II. Nucleic Acids

[0067] Also provided are nucleic acid molecules that encode a synthetic reporter described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules that encode only the ectodomain of the protein are also contemplated. The nucleic acid molecule can be incorporated into a vector, such as an expression vector. The nucleic acid may be a self-replicating RNA molecule. The nucleic acid may include a modified RNA molecule. Also provided are compositions comprising a nucleic acid described herein.204919-2079-0411 , v 1

[0068] Expression requires that appropriate signals be provided in the vectors and include various transcription regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of a protein described herein. Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0069] The term promoter is used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0070] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the S V40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0071] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to214919-2079-0411 , v 150 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0072] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

[0073] The transcription regulatory elements used herein may be selected based on their known relationship to an upstream signaling cascade, such that the transcription regulatory element’s activity is modulated by the activation or inhibition of the signaling cascade. As such, the expression of the synthetic reporter will be dependent on the stimulation or repression of the signaling cascade. Exemplary transcription regulatory elements and their associated signaling pathway are provided in Table 1.

[0074] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0075] An exemplary expression vector for use in mammalian cell is the pcDNA™ 3.4 vector. Exemplary expression vectors for use in bacterial cells (e.g., E. coll) include pET28a and pJLl vectors. Exemplary expression vectors for us in yeast cells include pPICZa A, B, & C.

[0076] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered224919-2079-0411 , v 1construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. They can be readily introduced in a variety of cell lines and laboratory animals.[0077| One method for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient (a) to support packaging of the construct and (b) to express a synthetic reporter that has been cloned therein.

[0078] The expression vector may comprise a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, doublestranded DNA vims, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retro vims, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenovimses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovims can infect virtually all epithelial cells regardless of their cell cycle stage.

[0079] Adenovims is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. Generation and propagation of the current adenovims vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovims genome, the current adenovims vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions.

[0080] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovims. Such cells include, e.g., Vero234919-2079-0411 , v 1cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0081] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is one exemplary starting material that may be used to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.

[0082] Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

[0083] In some embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus. AAV infects non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITR) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA.

[0084] The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

[0085] The ITR sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV244919-2079-0411 , v 1genome. The feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.III. Assay Methods

[0086] Various assay methods may be used to detect whether a given synthetic reporter was expressed by a cell or expressed in a cell-free system.

[0087] FIG. 6A provides an overview of a multiplexed cell-based assay according to one embodiment. The portion of the cell illustrated in FIG. 6A includes a cell membrane 809, a nuclear membrane 814, cell surface receptors 805, 806, 807, and 808, and signaling pathways 810, 811, 812, and 813. The cell is exposed to one or more analytes of interest 801, 802, 803, and 804. Analytes of interest 801, 802, 803, and 804, may be the same analyte or a combination of different analytes. As shown in FIG. 6A, analytes of interest 801, 802, 803, and 804, interact with cell surface receptors 805, 806, 807, and 808, which interaction activates signaling pathways 810, 811, 812, and 813. The activation of each of signaling pathways 810, 811, 812, and 813 results in transcription factors 815, 816, 817, and 818 binding their respective reporter elements in synthetic reporter expression constructs 819, 820, 821, and 822. The four unique synthetic reporters are then transcribed and translated. The synthetic reporter proteins are detected using sandwich immunoassay 823. Because each synthetic reporter expression construct 819, 820, 821, and 822 encodes a unique synthetic reporter, with each being under the control of a different promoter, the synthetic reporters can be associated with a particular transcription factor and signaling pathway.

[0088] FIGS. 6B and 6C provide an overview of a split-probe cell-free protein synthesis assay for the detection of a target microRNA sequence. Capture probe 825, comprising biotin 826, linker 826, and a first single-stranded DNA sequence 828, is combined with detection probe 830, comprising a second single-stranded DNA sequence 831, linker 832, a doublestranded DNA sequence 833 encoding a synthetic reporter as described herein, and promoter 834 operably linked to the double-stranded DNA sequence 833, and a sample containing target microRNA 829. First single-stranded DNA sequence 828 is complementary to a first portion254919-2079-0411 , v 1of target microRNA 829, and the second single-stranded DNA sequence 831 a second portion of target microRNA 829.

[0089] If microRNA 829 is present in the sample, binding complex 835 is formed. Binding complex 835 may be captured by binding biotin 834 to streptavidin coated surface 836. Binding complex 835 can then be washed to remove any unbound sample, capture probes, and detection probes. Following washing, detection probe 830 can be released from streptavidin coated surface 836 and subjected to transcription and translation in a cell-free protein synthesis system such as described in Min et al., ACS Applied Bio Materials (2024) (https: / / doi.org / 10.1021 / acsabm.4c01187). The translated synthetic reporter protein can then be detected according to the methods described herein. Multiple different split-probe pairs, each specific for a different target nucleic acid sequence and each encoding a unique synthetic reporter may be employed in a multiplexed format to detect multiple target nucleic acid sequences in the same sample.

[0090] In some embodiment, the synthetic reporter may be captured onto a solid support by a first antibody to a first epitope (e.g., an affinity tag) on the synthetic reporter. Then, a second antibody to a second epitope on the synthetic reporter may be bound to the captured synthetic reporter. In some aspects, the second antibody comprises a detectable label. In some aspects, a third antibody that binds to the second antibody (e.g., an anti-constant region antibody) may be bound to the complex. The third antibody may comprise a detectable label.

[0091] The assay methods may be used in a multiplexed format by binding a plurality of different synthetic reporters to a plurality of first antibodies, where each unique synthetic reporter comprises a unique first epitope (e.g., a unique epitope introduced into the reporter). In this method, the number of unique first epitopes equals the number of unique first antibodies, such that each unique first epitope pairs with one of the unique first antibodies. In other words, each unique first antibody binds to only one of the unique first epitopes. The first antibodies may be bound, directly or indirectly, to different discrete binding domains on one or more solid supports, e.g., as in a binding array where the binding domains are individual array elements, or in a set of beads where the binding domains are the individual beads, such that discrete assay signals are generated on and measured from each binding domain. If the first antibodies for each of the synthetic reporters are immobilized in different binding domains, the different synthetic reporters bound to those domains can be measured independently. For example, the binding domains may be prepared by immobilizing, on one or more surfaces, discrete domains264919-2079-0411 , v 1of capture reagents that bind synthetic reporters. Optionally, the surface(s) may define, in part, one or more boundaries of a container (e.g., a flow cell, well, cuvette, etc.) which holds the sample or through which the sample is passed. In one embodiment, individual binding domains are formed on electrodes for use in electrochemical or electrochemiluminescence assays. Multiplexed measurement of analytes on a surface comprising a plurality of binding domains using electrochemiluminescence has been used in the Meso Scale Diagnostics, LLC, MULTIARRAY® and SECTOR® Imager line of products (see, e.g., U.S. Pat. Nos. 7,842,246 and 6,977,722, the disclosures of which are incorporated herein by reference in their entireties).

[0092] In the multiplexed format, a second antibody to a second epitope (e.g., common affinity tag, common epitope in the scaffold) that is shared among the synthetic reporters may be bound to the captured synthetic reporters. The second antibodies each comprises the same detectable label, for example, an ECL label or a fluorophore. Alternatively, a plurality of second antibodies to unique second epitopes (e.g., a unique epitopes introduced into the reporters) on the synthetic reporters may be bound to the captured synthetic reporters, where each unique synthetic reporter has a unique second epitope (e.g., a unique epitope introduced into the reporter). In this case, the second antibodies may each comprise a unique detectable label or share the same detectable label.

[0093] Still further, the detection complex and / or capture reagents (i.e., the plurality of first antibodies) can be bound, directly or indirectly, to an electrode surface, which optionally includes different discrete binding domains, as described above. The electrode surface can be a component of a multi-well plate and / or a flow cell. Electrodes can comprise a conductive material, e.g., a metal such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, a conductive allow, or the like. They may also include oxide coated metals, e.g., aluminum oxide coated aluminum. The electrode can include a working and counter electrodes which can be made of the same or different materials, e.g., a metal counter electrode and carbon working electrode. In one embodiment, electrodes comprise carbon-based materials such as carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, graphene, carbon fibers and mixtures thereof. In one embodiment, the electrodes comprise elemental carbon, e.g., graphitic, carbon black, carbon nanotubes, etc. They may include conducting carbonpolymer composites, conducting particles dispersed in a matrix (e.g. carbon inks, carbon pastes, metal inks, graphene inks), and / or conducting polymers. One embodiment is an assay module,274919-2079-0411 , v 1preferably a multi-well plate, having electrodes (e.g., working and / or counter electrodes) that comprise carbon, e.g., carbon layers, and / or screen-printed layers of carbon inks.

[0094] Provided are methods for detecting and counting individual synthetic reporter detection complexes. In one embodiment, the surface can comprise a plurality of capture reagents (e.g., antibodies) for one or more synthetic reporter that are present in a sample and the plurality of capture reagents are distributed across a plurality of resolvable binding regions positioned on the surface. Under the conditions used to carry out and analyze a measurement, a “resolvable binding region’’ is the minimal surface area associated with an individual binding event that can be resolved and differentiated from another area in which an additional individual binding event is occurring. Therefore, the method consists of binding the one or more synthetic reporter to one or more capture reagents (e.g., antibodies) on the surface, determining the presence or absence of an analyte molecule (e.g., synthetic reporter) in a plurality of resolvable binding regions on the surface, and identifying the number of resolvable binding regions that contain an analyte molecule (e.g., synthetic reporter) and / or the number of analyte domains that do not contain an analyte molecule (e.g., synthetic reporter).

[0095] The resolvable binding regions can be optically interrogated, in whole or in part, i.e., each individual resolvable binding region can be individually optically interrogated and / or the entire surface comprising a plurality of resolvable binding regions can be imaged and one or more pixels or groupings of pixels within that image can be mapped to an individual resolvable binding region. A resolvable binding region may also be a microparticle within a plurality of microparticles. The resolvable binding regions exhibiting changes in their optical signature can be identified by a conventional optical detection system. Depending on the detected species (e.g., type of fluorescence entity, etc.) and the operative wavelengths, optical filters designed for a particular wavelength can be employed for optical interrogation of the resolvable binding regions. In embodiments where optical interrogation is used, the system can comprise more than one light source and / or a plurality of filters to adjust the wavelength and / or intensity of the light source. In some embodiments, the optical signal from a plurality of resolvable binding regions is captured using a CCD camera. Other non-limiting examples of camera imaging systems that can be used to capture images include charge injection devices (CIDs), complementary metal oxide semiconductors (CMOSs) devices, scientific CMOS (sCMOS) devices, and time delay integration (TDI) devices, as will be known to those of284919-2079-0411 , v 1ordinary skill in the art. In some embodiments, a scanning mirror system coupled with a photodiode or photomultiplier tube (PMT) can be used for imaging.

[0096] The measuring step of the method can comprise imaging an optical signal from the surface (or a portion thereof) to generate an image that consists of a plurality of pixels, wherein each resolvable binding region maps to one or more pixels or groups of pixels in the image. Image analysis to identify pixels or sets of pixels having a signal indicative of a binding event (detection complex) can be accomplished using art recognized methods, for example, the wealth of image analysis algorithms and software available to identify and count labeled biological structures in fluorescence microscopy images. In one embodiment, after filtering the image to remove large-scale signal gradients, the image is converted to a binary image using a segmentation threshold. Resolvable binding regions are found by identifying contiguous regions of above-threshold intensity. Binding domains are categorized as binding events if they meet size and intensity requirements.

[0097] Methods for measuring the amount of an analyte (e.g., synthetic reporter) also include techniques that measure analytes through the detection of labels which may be attached directly or indirectly (e.g., through the use of labeled binding partners of an analyte) to an analyte. Suitable labels include labels that can be directly visualized (e.g., particles that may be seen visually and labels that generate a measurable signal such as light scattering, optical absorbance, fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, magnetic fields, etc.). Labels that may be used also include enzymes or other chemically reactive species that have a chemical activity that leads to a measurable signal such as light scattering, absorbance, fluorescence, etc. The use of enzymes as labels has been well established in Enzyme-Linked ImmunoSorbent Assays, also called ELISAs, Enzyme ImmunoAssays or EIAs. In the ELISA format, an unknown amount of antigen is affixed to a surface and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme converts to a product that provides a change in a detectable signal. The formation of product may be detectable, e.g., due a difference, relative to the substrate, in a measurable property such as absorbance, fluorescence, chemiluminescence, light scattering, etc. Certain (but not all) measurement methods that may be used with solid phase binding methods according to the invention may benefit from or require a wash step to remove unbound components (e.g., labels)294919-2079-0411 , v 1from the solid phase. Accordingly, the methods of the invention may comprise such a wash step.

[0098] Labels used to detect analyte molecules can be fluorescent species that can be used in single molecule fluorescence detection, e.g., fluorescence correlation spectroscopy, and / or fluorescence cross-correlation spectroscopy. Single molecule fluorescence detection comprises flowing an eluent that includes a detectable species through a capillary, focusing a light source on a volume within the capillary to create an interrogation zone and observing the interrogation zone with a light detector to detect the passage of fluorescent molecules through the interrogation zone.

[0099] In one embodiment, an analyte(s) of interest in the sample may be measured using electrochemiluminescence-based assay formats, e.g. electrochemiluminescence (ECL) based immunoassays. The high sensitivity, broad dynamic range and selectivity of ECL are important factors for medical diagnostics. Commercially available ECL instruments have demonstrated exceptional performance and they have become widely used for reasons including their excellent sensitivity, dynamic range, precision, and tolerance of complex sample matrices. Species that can be induced to emit ECL (ECL- active species) have been used as ECL labels, e.g., i) organometallic compounds where the metal is from, for example, the noble metals of group VIII, including Ru-containing and Os-containing organometallic compounds such as the tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and related compounds. Species that participate with the ECL label in the ECL process are referred to herein as ECL coreactants. Commonly used coreactants include tertiary amines (e.g., see U.S. Patent No. 5,846,485), oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Patent No. 5,240,863). The light generated by ECL labels can be used as a reporter signal in diagnostic procedures (Bard et al., U.S. Patent No. 5,238,808, herein incorporated by reference). For instance, an ECL label can be covalently coupled to a binding agent such as an antibody, nucleic acid probe, receptor or ligand; the participation of the binding reagent in a binding interaction can be monitored by measuring ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound may be indicative of the chemical environment (see, e.g., U.S. Pat. No. 5,641,623 which describes ECL assays that monitor the formation or destruction of ECL coreactants). For more background on ECL, ECL labels, ECL assays and instrumentation for conducting ECL assays see U.S. Pat. Nos.5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519;304919-2079-0411 , v 15,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369; 6,214,552 and 5,589,136 and Published PCT Nos. WO99 / 63347; WO00 / 03233; WO99 / 58962; WO99 / 32662; WO99 / 14599; WO98 / 12539; WO97 / 36931 and WO98 / 57154, all of which are incorporated herein by reference.[01001 The methods of the invention may be applied to singleplex or multiplex formats where multiple assay measurements are performed on a single sample. Multiplex measurements that can be used with the invention include, but are not limited to, multiplex measurements i) that involve the use of multiple sensors; ii) that use discrete assay domains on a surface (e.g., an array) that are distinguishable based on location on the surface; iii) that involve the use of reagents coated on particles that are distinguishable based on a particle property such as size, shape, color, etc.; iv) that produce assay signals that are distinguishable based on optical properties (e.g., absorbance or emission spectrum) or v) that are based on temporal properties of assay signal (e.g., time, frequency or phase of a signal).[0101| In some embodiments, a measure of the concentration of analyte molecules in the sample may be determined at least in part by comparison of a measured parameter to a calibration standard. For example, the fraction of binding surfaces that comprise an analyte molecule may be compared against a calibration curve to detennine a measure of the concentration of the analyte molecule in the sample. The calibration curve may be produced by completing the assay with a plurality of standardized samples of known concentration under the conditions used to analyze the test samples. A reading may be taken for the signal related to the detection / quantification of the analyte molecules for each standardized sample, therefore allowing for the formation of a calibration curve relating the detection of the analyte molecules with a known concentration of the analyte molecule. The assay may then be completed on a sample comprising the analyte molecule in an unknown concentration, and the detection of the analyte molecules from this assay may be plotted on the calibration curve, therefore determining a measure of the concentration of the analyte molecule in the sample.

[0102] In the specific case of using an imaging technique to measure an optical signal (such as fluorescence, chemiluminescence or electrochemiluminescence) a binding event can be detected as a bright point source of light. When the surface density of point sources is low (e.g., when the probability of finding a point source in an RxR area — where R is the spatial resolution of the detection system- is less than 10%), it is likely that any observed point source is due to a single binding event. Under these conditions, counting events can provide the most314919-2079-0411 , v 1sensitive measurement. As the surface density increases, it becomes increasingly difficult to resolve and count individual binding events. Under these conditions, integrating the optical signal over the binding surface provides a more accurate measurement.

[0103] It will be evident to the skilled artisan that the methods described herein can be applied to numerous immunoassay platforms known to those skilled in the art. Various features of the immunoassay platforms may be adjusted to suit the particular platform, but those adjustments are well within the skill of the ordinary artisan. For example, the methods described herein can be applied to a bead-based format that uses coded particles. In such a system, the bead used can be magnetic or non-magnetic and the surface of the beads is modified to include one or more copies of a capture reagent. The detection reagents employed in this system are a pair of detection reagents. In one embodiment, the two detection reagents include distinguishable fluorescent labels. Alternatively, the two detection reagents are modified with nucleic acid probes, as described W02013070990, in which case, the immunoassay method includes an extension process, e.g., RCA-PLA to generate an amplified product indicative of the presence of each detection reagent that can be detected. If the detection reagents include two distinguishable fluorescent labels, the measurement step includes introducing the beads into a flow cell, and if the beads are magnetic, capturing the beads in the flow cell. If the detection reagents are modified with nucleic acid probes, the measurement step includes forming a sandwich complex on the beads, performing RCA-PLA and labeling the amplicon with fluorescently labeled detection probes. The labeled beads are then introduced into the flow cell and if the beads are magnetic, the beads are captured in the flow cell. In each embodiment, the assay can be multiplexed spectrally based on the identification of fluorescently labeled encoded beads. An excitation light source and emission light detector for multi-color detection can be used to detect binding events in each embodiment, quantitation is achieved by counting beads having both detectable labels or those beads that include a detectably labeled extension product, and quantitation is also achieved by integrated intensity, e.g., detection by integrating over the signal for all binding events. Therefore, a kit can be provided for use with the method described above that includes one or more of the following in one or more vials, containers, or compartments: (a) Magnetic or non-magnetic beads with capture reagent; (b) two detection reagents with distinguishable fluorescent labels; and (c) Optional buffers and / or diluents for assay protocol. Another kit that can be used with the method described above can include one or more of the following on one or more vials, containers, or compartments: (a) Magnetic or non-magnetic beads with capture reagent; (b) Two detection reagents modified with nucleic324919-2079-0411 , v 1acid probes (optionally, detection reagents are provided separately and proximity probes (1 and 2) are additionally provided with instructions to modify detection reagents with probes); and (c) fluorescently labeled probes; optional reagents required for modification of detection reagents with proximity probes; assay diluent, calibrator, circularization oligonucleotides, ligation mix or components thereof, e.g., ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; RCA mixture or components thereof, e.g., BSA, buffer, dNTP, Tween 20, Phi29 DNA polymerase.

[0104] In another embodiment, the methods described herein can be applied to a flowcell analyzed, bead-based format. In such a system, the bead used can be magnetic and the surface of the beads is modified to include one or more copies of a capture reagent. The detection reagents employed in this system are a pair of detection reagents modified with nucleic acid probes, as described WO2013070990, in which case, the immunoassay method includes an extension process, e.g., RCA-PLA to generate an amplified product indicative of the presence of each detection reagent that can be detected. The measurement step includes forming a sandwich complex on the beads, performing RCA-PLA and labeling the amplicon with ECL-labeled detection probes. The labeled beads are then introduced into the flow cell and the beads are captured in the flow cell. In particular, a magnetic field is applied to draw the magnetic particles, e.g., beads, to the electrode surface, which can comprise various metals, e.g., platinum. A voltage source is used to apply a voltage to an electrode and an emission light detector can be used to detect binding events; quantitation is achieved by counting beads having a detectably labeled extension product, and quantitation is also achieved by integrated intensity, e.g., detection by integrating over the signal for all binding events. A kit that can be used with the method described above can include one or more of the following on one or more vials, containers, or compartments: (a) Magnetic beads with capture reagent; (b) Two detection reagents modified with nucleic acid probes (optionally, detection reagents are provided separately and proximity probes (1 and 2) are additionally provided with instructions to modify detection reagents with probes): and (c) ECL labeled probes; optional reagents required for modification of detection reagents with proximity probes; assay diluent, calibrator, circularization oligonucleotides, ligation mix or components thereof, e.g., ligation buffer, ATP, BSA, Tween 20, T4 DNA ligase; RCA mixture or components thereof, e.g., BSA, buffer, dNTP, Tween 20, Phi29 DNA polymerase.334919-2079-0411 , v 1

[0105] In a specific embodiment of a flow-cell analyzed, bead-based format, a sample is incubated with a biotinylated monoclonal analyte-specific capture antibody and a mixture of monoclonal analyte-specific antibodies, each conjugated to oligonucleotides, which react to form a sandwich complex. After the addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interactions between biotin and streptavidin. A ligation mix is added to the mixture, and the mixture is incubated with the ligation mix, washed to remove excess circularization oligonucleotides, and incubated with RCA mixture. The mixture is washed and a mixture of biotin-labeled detection probes are added. To incorporate a suitable label, e.g., a luminescent, chemiluminescent, or electrochemiluminescence label, e.g., SULFO-TAG, the detection probe is synthesized with a terminal biotin label and prebound to SULFO-TAG labeled streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode, e.g., a metal electrode, such as a platinum electrode. Unbound substances are then removed with a suitable wash buffer, e.g., ProCell (TPA containing buffer). Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. The application of voltage and measurement of the resultant emission can be done in any suitable flow-cell, e.g., a Cobas and / or Elecsys instrument (available from Hoffmann-La Roche LTD.).

[0106] In yet another embodiment, the methods described herein can be applied to a bead-based format, with capillary flow to digitally count individual molecules. In such a system, the bead used can be magnetic and the surface of the bead is modified to include one or more copies of a capture reagent. The detection reagents employed in this system are a pair of detection reagents that include distinguishable fluorescent labels. The measurement step includes forming a sandwich complex including the capture reagent, analyte, and detection reagents, crosslinking detection reagents, eluting detection reagents and introducing the beads into a flow cell. An excitation light source and emission light detector for multi-color detection can be used to detect binding events, quantitation is achieved by correlating detection of two fluorophores in the flow cell, and quantitation is also achieved by integrated intensity, e.g., detection by integrating over the signal for all binding events. A kit that can be used with the method described above can include one or more of the following on one or more vials, containers, or compartments: (a) Magnetic beads with capture reagent; (b) Two cross-linkable detection reagents with distinguishable fluorescent labels; and (c) Optional buffers and / or diluents for assay protocol.344919-2079-0411 , v 1

[0107] Moreover, the methods described herein can be applied to a bead-based format that includes the separation of beads into individual nanowells. In such a system, the bead used can be magnetic and the surface of the bead is modified to include one or more copies of a capture reagent. The detection reagents employed in this system are a pair of detection reagents that include distinguishable enzyme labels. The measurement step includes forming a sandwich complex including the capture reagent, analyte, and detection reagents, and adding substrates for the two enzyme labels. The beads are then captured in individual nanowells. The assay can be multiplexed spectrally based on the identification of enzyme products with different spectral properties. An excitation light source and emission light detector for multi-color detection can be used to detect binding events, quantitation is achieved by counting nanowells that contain both enzyme products, and quantitation is also achieved by integrated intensity, e.g., detection by integrating over the signal for all nanowells. A kit that can be used with the method described above can include one or more of the following on one or more vials, containers, or compartments: (a) Magnetic beads with capture reagent; (b) Two detection reagents each modified with distinguishable enzyme labels, e.g., biotinylated detection reagent and a hapten-conjugated detection reagent; (c) Streptavidin-beta galactosidase, anti-hapten conjugated enzyme, resorufin-beta-d-galactopyranoside; (d) array, e.g., Quanterix DVD format array; (e) fluorocarbon oil; and (0 optional buffers and / or diluents for assay protocol. In this specific embodiment, the detectable signal is enhanced by combining the use of a nanowell high-sensitivity system with a proximity-based detection system. While this specific embodiment is illustrated using a particular proximity-based detection system, the skilled artisan will appreciate the fact that the other proximity-based detection systems described herein can also be used to enhance the detectable signal in the assay, e.g., FRET donor / acceptor system; luminescent labels that differ from one another with respect to spectral properties; or the use of first and second enzymes that are hydrolytic enzymes, as described above, and the appropriate accompanying substrates.

[0108] Still further, the methods described herein can be applied to a bead-array based platform. In such a system, the bead used can be non-magnetic and the surface of the bead is modified to include one or more copies of a capture reagent. The detection reagents employed in this system are a pair of detection reagents that include first and second nucleic acid probes. The measurement step includes forming a sandwich complex including the capture reagent, analyte, and detection reagents, extending one of the probes to form an extended sequence, wherein extension is dependent on co-localization of the first and second probes in the354919-2079-0411 , v 1sandwich complex, labeling the extended sequence with a fluorescent probe, and releasing the extended sequence from the surface into an eluent. An excitation light source and emission light detector for multi-color detection can be used to detect binding events, quantitation is achieved by counting individual detectably labeled extension products, and quantitation is also achieved by integrated intensity, e.g., detection by integrating over the signal for all binding events. A kit that can be used with the method described above can include one or more of the following on one or more vials, containers, or compartments: (a) Non-magnetic beads with capture reagent; (b) Two detection reagents modified with nucleic acid probes (optionally, detection reagents are provided separately and proximity probes (1 and 2) are additionally provided with instructions to modify detection reagents with probes); and (c) Fluorescently labeled probes; optional reagents required for modification of detection reagents with proximity probes; assay diluent, calibrator, circularization oligonucleotides, ligation mix or components thereof, e.g., ligation buffer, ATP, BS A, Tween 20, T4 DNA ligase; RCA mixture or components thereof, e.g., BSA, buffer, dNTP, Tween 20. Phi29 DNA polymerase.

[0109] Still further, certain methods described herein may utilize the subdivision (partitioning) of individual cells or subpopulations of cells into separate partitions. A partition may be, for example, a well, a microwell, a hole, a droplet (e.g., a droplet in an emulsion), a continuous phase of an emulsion, a test tube, a spot, a capsule, a surface of a bead, or any other suitable container for sequestering one cell or a subpopulation of cells. Each partition may also contain, or be contained within any other suitable partition. For example, a well or microwell may comprise a droplet (e.g., a droplet in an emulsion) or a bead, or a droplet may comprise a capsule, bead, or another droplet. These descriptions are merely illustrative, and all suitable combinations and pluralities are also envisioned.

[0110] In yet another embodiment, the methods described herein can be applied to cells within an intact tissue or tissue section of a recombinant, non-human animal capable of expressing one or more synthetic reporters. In certain embodiments, a tissue sample is obtained from a recombinant, non-human animal capable of expressing one or more synthetic reporters, the tissue sample is placed on a slide or other suitable surface, the tissue sample is contacted by detection reagents (e.g., antibodies) that specifically bind the synthetic reporters, and detecting the binding of the detection reagents and the synthetic reporters as described herein. Such methods permit, for example, the assessment of the presence or absence of one or more synthetic reporters in heterogeneous tissues in situ. The tissue sample may be, for example, a364919-2079-0411 , v 1fresh tissue sample, a frozen tissue sample, or a formalin-fixed, paraffin-embedded tissue sample.

[0111] The assays described herein can be performed using one or more kits including a set of components employed in the assay. For example, a kit may include, in one or more vials, containers, or compartments, an expression cassette encoding a synthetic reporter and a capture reagent for the synthetic reporter.

[0112] Another kit that can be used to perform the assays described herein includes, in one or more vials, containers, or compartments, an expression cassette encoding a synthetic reporter and a capture reagent (e.g., a first antibody) for the synthetic reporter; and a first detection reagent for the analyte comprising an antibody to an epitope on the synthetic reporter.

[0113] The kits described hereinabove can further include one or more of the following: one or more additional reagents or buffers. In addition, if the one or more detection reagents comprise a detectable label, the kit can also include a co-reactant for the detectable label employed in the kit. Alternatively, if the one or more detection reagents are components of a coupled enzyme reaction system, then each of the detection reagents comprise first and second enzymes and the kit further includes, in one or more containers, vials or compartments, one or more substrates for the coupled enzyme reaction system, and optionally, a labeled component configured to bind to a product of the coupled enzyme reaction system. For example, the first enzyme can be an oxidase, the second enzyme a peroxidase, and the kit further includes an oxidase substrate and a labeled tyramide derivative. In another embodiment, the first and second detectable reagents can be comprise components of a proximity-dependent detection system, e.g., a FRET donor and a FRET acceptor, or luminescent labels that differ from one another with respect to their spectral properties.IV. Definitions

[0114] Nucleic acids can include one or more transcription regulatory element operably linked to the open reading frame, where the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory374919-2079-0411 , v 1sequences, and the like. Expression control / regulatory elements can be obtained from the genome of any suitable organism.

[0115] A nucleic acid sequence is “operably linked’’ when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid encoding a synthetic reporter, or a nucleic acid directing expression of a synthetic reporter may include an inducible promoter or a disease-associated promoter. A nucleic acid operably linked to an expression control element can also be referred to as an expression cassette.

[0116] An “expression vector” is a nucleic acid that contains a protein-coding nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, transcription regulatory element (e.g., a promoter, enhancer), intron, ITR(s), and polyadenylation signal.

[0117] A “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and / or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and optionally other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.

[0118] An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level or tissue specificity of expression. It is capable of operating in either orientation (5’->3’ or 3’->5') and may be capable of functioning even when positioned either upstream or downstream of the promoter.

[0119] Promoters and / or enhancers may be derived in their entirety from a native gene or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments. A promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate / control effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.[0120| Synthetic reporters may possess insertions, deletions and / or substitutions of amino acids. Substitutions may be the exchange of one amino acid for another at one or more384919-2079-0411 , v 1sites within the protein. Substitutions may or may not be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine: phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Insertions may be the insertion of a signal peptide or affinity tag, or simply a single residue.

[0121] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth herein. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns.

[0122] As used herein, “AAV” refers to an adeno-associated virus vector. As used herein, “AAV” refers to any AAV serotype and variant, including but not limited to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO, AAVrh74, AAV9, AAV9P (also known as AAVMYO), and Myo- AAV vector, wherein the number following AAV indicates the AAV serotype. The term “AAV” can also refer to any known AAV (vector) system. The AAV vector may be a single-stranded AAV (ssAAV) or a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. Structurally, AAVs are small (25 nm), single-DNA stranded non-enveloped viruses with an icosahedral capsid. Naturally occurring or engineered AAV serotypes and variants that differ in the composition and structure of their capsid protein have varying tropism, i.e., ability to transduce different cell types.

[0123] A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence394919-2079-0411 , v 1of the native protein. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the disclosure will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%. to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to a disclosed nucleotide sequence.

[0124] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%.

[0125] The term “substantial identity” in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%.91%, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

[0126] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is404919-2079-0411 , v 1therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0127] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0128] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0129] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.V. Examples

[0130] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Reporter Scaffold Expression and Purification

[0131] Candidate protein scaffolds were identified by identifying independently folding domains of large proteins whose structure were available in the Protein Data Bank. Preference was given to candidates from bacterial and phage sources that lack disulfide bonds.

[0132] Candidate protein scaffolds were designed using criteria that included size, sequence homology, structural complexity, and predicted protein stability. Candidate protein scaffolds were analyzed using Foldseek software to identify scaffolds with structurally similar414919-2079-0411 , v 1folds as found in proteins known to exhibit a high degree of thermostability, such as proteins originating from thermophilic organisms. From among these candidate protein scaffolds, the scaffolds that exhibited molecular weights of less than 25,000 Da, were predicted to tolerate one or more regions for epitope insertion, and contained no disulfide bonds were selected. These sequences were then subject to BLAST in order to identify candidates protein scaffolds exhibiting the lowest sequence homology against human proteins. These candidate protein scaffolds were then subject to further analysis as follows.

[0133] The epitope insertion regions present within the independently folding domains are where the synthetic epitopes are inserted. In order to validate the candidate scaffolds, protein tags that are compatible with internal positioning within target proteins, and for which commercially available antibodies are available, were selected: E2-Tag (SEQ ID NO: 29) and T7-Tag (SEQ ID NO: 30).[0134| The 2N8O scaffold, which is 51 amino acids and 5,984 Da, was tested. This scaffold is a three-helix bundle, and thus has two regions available for epitope insertion. In order to test the effect of epitope insertion, the E2-Tag sequence was inserted in one epitope insertion region and the T7-Tag sequence was inserted in the other epitope insertion region. The resulting reporter (2N8O-E2-T7) was 73 amino acids and 8,229 Da. AlphaFold2 was used to predict the tertiary structure of the 2N8O-E2-T7 reporter, which indicated that the scaffold structure was unperturbed by epitope insertion.

[0135] The 5DMA scaffold, which is 53 amino acids and 5,756 Da, was also tested. This scaffold has a beta-sheet structure that provides two regions for epitope insertion. In order to test the effect of epitope insertion, the E2-Tag sequence was inserted in one epitope insertion region and the T7-Tag sequence was inserted in the other epitope insertion region. The resulting reporter (5DMA-E2-T7) was 64 amino acids and 7,092 Da. AlphaFold2 was used to predict the tertiary structure of the 5DMA-E2-T7 reporter, which indicated that the scaffold structure was unperturbed by epitope insertion.

[0136] To test the stability of various candidate scaffolds, nucleic acids encoding the scaffolds was in vitro translated using the PURExpress kit. The soluble fractions of the translation reactions were analyzed on 16% Tri cine SDS-PAGE gels to identify viable constructs. The tested scaffolds were 5DMA, 2N8O, 5JCV-A, PsbP123, 5JCV-B, and FimA120. The 2N8O-E2-T7 and 5DMA-E2-T7 constructs were also tested. As shown in FIG.424919-2079-0411 , v 17, only the 5DMA (Lane 1), 2N8O (Lane 3), PsbP123 (Lane 6), and FimA120 (Lane 8) scaffold were soluble. The addition of E2 and T7 tags results in an insoluble product for the 2N8O-E2-T7 (Lane 4) and 5DMA-E2-T7 (Lane 2) constructs.

[0137] To test the stability of various candidate scaffolds with heat treatment, scaffolds 5DMA, 2N8O, PsbP123, and FimA120 were subjected to heat treatment at 60°C for 30 minutes. All four scaffolds remained soluble after heat treatment (FIGS. 8A and 8B). Further testing of heat stability was performed in various buffers. Treatment with NH3SO4 mildly affected the soluble yield. However, the scaffolds remained soluble after heat treatment at 60°C for 30 minutes in each of CutSmart buffer (New England Biolabs), Diluent 100 (Meso Scale Diagnostics), and Read Buffer B (Meso Scale Diagnostics).

[0138] The melting temperature of each reporter was tested using a thermal shift assay.5DMA, FimA120, and PsbP123 all exhibited a melting temperature between 52-55C°. These data corroborate the gel-based thermostability assessment.

[0139] Given that the introduction of E2 and T7 epitopes reduced soluble expression of 2N8O and 5DMA, further work was performed to understand the sequence and positional dependence of epitope tolerance by scaffolds. First, Foldseek was used to identify protein folds that are structurally similar to each of 5DMA ([3-sheet), 2N8O (3 -heli bundle), PsbP123 (a-|B-a sandwich), FimA120 (P-sandwich), a 4-helix bundle, and a -barrrel. Twenty-four scaffolds were identified. Of these, twenty-two were successfully expressed and purified (FIG. 9). The best expressing scaffolds, 3H-1, 4H-3, PsbP-3, and FimA-3, were selected for additional stability screening. These four scaffolds were determined to remain stable in MSD electrochemiluminescence (ECL) assay buffers during heat treatment at 60°C for 30 minutes.Example 2 - Identification and Selection of Epitopes

[0140] Epitope sequences were designed that were orthogonal to each other and to natural peptides, as well as predicted to be compatible with the reporter scaffold structure. A Python script was used to generate randomized sequences that are ten amino acids in length, contain three of any combination of amino acids R, E, D, Q, and N; contain seven of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y; do not contain three or more consecutive amino acids of any combination of L, I, V, M, W, and F; and do not contain amino acids C, K, or P. The randomized sequences were screened by BLAST, and the majority were found to match natural sequences in eukaryotes. Sequences with lowest434919-2079-0411 , v 1identity and smallest sequence coverage to natural sequences were identified and further screened against mammalian and human databases. Nine candidate synthetic epitopes were identified (Epitopes 1-9 in Table 2).Example 3 - Testing of Epitope Positions in Synthetic Reporters

[0141] In the 3H-1 and 4H-3 scaffolds, the inter-helix regions provide the epitope positions. To identify epitope positions for the PsbP-3 and FimA-3 scaffolds, three regions that are (1) solvent exposed, (2) positioned distal to other epitope sites, and (3) not involved in protein stability were selected.

[0142] To test epitope positions for the 3H-1 scaffold, Epitopes 1-10 in Table 2 were inserted into either epitope insertion region 1 or epitope insertion region 2. To test epitope positions for the 4H-3, PsbP-3, and FimA-3 scaffolds, Epitopes 1-10 in Table 2 were inserted into either epitope insertion region 1, epitope insertion region 2, or epitope insertion region. The non-modified epitope insertion region(s) in each construct had the sequence of Epitope 11.[0143| The 110 candidates were expressed and purified. The 3H-1 scaffold was highly tolerant of all epitope sequences at both positions (FIG. 10A). The FimA-3 scaffold was tolerant of most epitope sequences at all positions (FIG. 10B). The PsbP-3 scaffold was sensitive to epitope sequence and position, with only a 30% success rate (FIG. 10C). The 4H-3 scaffold could not be assessed by SDS-PAGE due to overlap in molecular weight with Promega FluoroTect Greentys fluorescent label.

[0144] All of the top synthetic reporter candidates supported soluble expression and purification. In addition, all synthetic reporters were stable in buffers Diluent 100 (Meso Scale Diagnostics) and Read Buffer A (Meso Scale Diagnostics) during heat treatment at 60°C for 30 minutes.Example 4 - Testing of Synthetic Reporters in Immunoassays

[0145] To validate that epitopes presented by synthetic reporters are antibody-accessible and can support immunoassays, N-terminally Strep-tagged reporters having a FLAG epitope inserted in one of the epitope positions were screened by ELISA. The FLAG epitopes were antibody-accessible in each of the tested synthetic reporters (FIG. 11 A).444919-2079-0411 , v 1

[0146] The four reporter scaffolds were also tested with two internal epitopes. For this, N-terminally Strep-tagged reporters were generated with a FLAG epitope inserted into the epitope insertion region 1 epitope position of each reporter scaffold. Then, either an E2, T7, HA, or c-Myc tag was inserted into the epitope insertion region 2 epitope position. The reporters were screened by ELISA and were found to retain functionality in immunoassays even with two internal epitopes (FIG. 1 IB).Example 5 - Secretion of Synthetic Reporters from Cells

[0147] In order for the synthetic reporters to be secreted from cells, various signal peptides were added to the N-terminus of the reporters. More specifically, sequences encoding signal peptides El, El 1, E14, E17, and S3 (Table 2) were independently added to expression constructs encoding the FimA 3 scaffold such that the signal peptide would be at the N-terminus of the FimA 3 scaffold. The FimA 3 scaffold also contained a FLAG epitope at its second epitope insertion region. The expression constructs were transfected into Expi293 cells and cultured for four days. At harvest, the cells were centrifuged at 14,000 rpm for 5 min. Both the cell pellet and the supernatant were analyzed by SDS-PAGE for the presence of the synthetic reporter. All of the tested signal peptides (El, Ell, E14, E17, and S3) resulted in successful secretion of the FimA 3 reporter.Example 6 - Multiplexed Detection of Synthetic Reporters

[0148] For screening four signaling pathways, a multiplexed format is employed using four different scaffolds, each containing a unique reporter epitope. This enables detection of multiple pathway activation events within the cell using the aforementioned immunoassay methods. In this example, four 3H-1 scaffolds are designed to contain T7, FLAG, HA, or c-Myc at one epitope insertion site resulting in four different reporters, all containing N-terminal Strep tags. Each construct containing the synthetic reporter gene of interest will have a unique promoter region upstream of the synthetic reporter gene that is responsive to transcription factors of interest. In this example, PDX-1, HMG 1(Y), MafA, and Beta2 promoter sequences are used. PDX-1 is a transcription factor for insulin gene expression, which at high concentrations of intracellular glucose is phosphorylated, effectively up-regulating insulin gene expression. In this case, a construct containing the PDX-1 promoter is placed upstream of the reporter gene for 3H-1 containing a T7 epitope, to evaluate up-regulation or down-regulation of the PDX-1 pathway. Similarly, HMG 1(Y), MafA, and Beta2 are also transcription factors454919-2079-0411 , v 1for insulin and pathway activation of these transcription factors is detected with synthetic reporter genes containing FLAG, HA, and c-Myc epitopes under control of HMG 1(Y), MafA, and Beta2 promoters, respectively.

[0149] The resulting four reporter constructs are transfected into pancreatic islet cells and the cells cultured for 24-48 hrs at 37°C in a humidified incubator with 5% CO2. A drug candidate compound is added to the various cell cultures at a series of concentrations and the cell cultures are incubated for an additional 24 hrs. No drug candidate is added to some cell cultures to serve as controls. The cells from each cell culture are lysed and supernatants are obtained. A 4-spot MSD plate coated with mouse anti-T7, FLAG, HA, and c-Myc antibodies at unique spots are prepared and cell lysates are added to each well. Next, rabbit anti-strep antibody and SULFO-tagged anti-rabbit antibody are added to each well to enable for ECL detection of the 3H-1 reports captured at each of the 4 spots on the MSD plate. ECL signal is obtained from each of the four spots (i.e., the anti-T7, FLAG, HA, and c-Myc antibody spots) for each cell lysate and is used to inform the extent of pathway activation or suppression for each of the respective transcription factors in cell cultures treated with candidate drug as compared to controls. Observation of an increase in ECL signal at the anti-T7 spot in candidatedrug treated cell lysates as compared to controls, and an observation of no significant change in ECL signal at the FLAG, HA, and c-Myc spots in candidate-drug treated cell lysates as compared to controls, indicates that the candidate drug affects the PDX-1 signaling pathway but does not affect the HMG 1(Y), MafA, and Beta2 signaling pathways.

[0150] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.464919-2079-0411 , v 1SEQUENCE LISTSEQ ID NO: 1 - three-helices scaffold AEIAAIEYEQAAIKEEIAAIKDKIAAIKEYIAAIXXXXXXXXXXXXXXXXXXXXEKIA AIKEEQAAIEEEIQAIKEEIAAIKYLIAQIXXXXXXXXXXXXXXXXXXXXAEIAAIKY KQAAIKNEIAAIKQEIAAIEQMIAAIX at positions 35, 36, 89, and 90 is any amino acidX at positions 37-54 and 91-108 is absent or any amino acidSEQ ID NO: 2 - four-helices scaffold AYTVMDKNMKEAYKLCGEYDKLLSKINNMSYEXXXXXXXXXXXXXXXXXXXXT QTLIKEVKKLNEICDKIIEYEEKAYAVXXXXXXXXXXXXXXXXXXXXEPYKEFIGM RLERHHLLKLWREAWIDQLEVLEKXXXXXXXXXXXXXXXXXXXXYLAATEIQTS MDELSNKTFQIKQEEEKFLAKHPDVAKIAREWEKIYEYHESSX at positions 33, 34, 81, 82, 134, and 135 is any amino acidX at positions 35-52, 83-100, and 136-153 is absent or any amino acidSEQ ID NO: 3 - a-P-a sandwich scaffold ATDYFLDPNNEYIYATLEGFEAYYDETEQSLTVFNXXXXXXXXXXXXXXXXXXXX NLFLNISKLSEXXXXXXXXXXXXXXXXXXXXDLTTLVQEIKKSLENSGFKILTTDER DFQKYKATTFETLSMQXXXXXXXXXXXXXXXXXXXXILVRVEFTIFEVPNRGFYMI LSAAQAESYDSLRMLIRAAKMMLMFIEX at positions 36, 37, 67, 68, 129, and 130 is any amino acidX at positions 38-55, 69-86, and 131-148 is absent or any amino acidSEQ ID NO: 4 - -sandwich scaffold 1 DLPFIYAGSTIEIRPDESREAEVMLRNLGGDVESAVIILNSDFITSDTALIESWXXXXXX XXXXXXXXXXXXXXGETKKVVLGLKADSXXXXXXXXXXXXXXXXXXXXGVYEA KIIVDYRSXXXXXXXXXXXXXXXXXXXXGEKGVAEVPVLVKIVX at positions 55, 56, 89, 90, 122, and 123 is any amino acidX at positions 57-74, 91-108, and 124-141 is absent or any amino acidSEQ ID NO: 5 - P-sandwich scaffold 2474919-2079-0411 , v 1DLPFNYAGSTIEIRPDESREAEVMLRNLGGDVESAVIILNSDFITSDTALIESWXXXXX XXXXXXXXXXXXXXXGETKKVQLGLKADSXXXXXXXXXXXXXXXXXXXXGVYE AKIIVDYRSXXXXXXXXXXXXXXXXXXXXGEKGVAEVQVLVKINX at positions 55, 56, 89, 90, 122, and 123 is any amino acidX at positions 57-74, 91-108, and 124-141 is absent or any amino acid484919-2079-0411 , v 1

Claims

CLAIMS1. A protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1; wherein positions 35-54 are a first peptide sequence; wherein positions 89-108 are a second peptide sequence; wherein the first peptide sequence and the second peptide sequence are each independently 2 to 20 amino acids in length.

2. The protein of claim 1 comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 1.

3. The protein of claim 1 comprising the amino acid sequence of SEQ ID NO: 1.

4. The protein of any one of claims 1 to 3, wherein the amino acid sequence of at least one of the first peptide sequence and the second peptide sequence is:(a) 8 to 12 amino acids in length;(b) contains at least 2 but no more than 4 of any combination of amino acids R, E, D, Q, and N;(c) contains at least 5 but no more than 9 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

5. The protein of any one of claims 1 to 4, wherein the amino acid sequence of at least one of the first peptide sequence and the second peptide sequence is:(a) 10 amino acids in length;(b) contains 3 of any combination of amino acids R, E, D, Q, and N;(c) contains 7 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;4919-2079-0411 , v. 1(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

6. The protein of any one of claims 1 to 3, wherein the amino acid sequences of the first peptide sequence and the second peptide sequence are each independently identical to the amino acid sequence of one of SEQ ID NOs: 6-30.

7. The protein of claim 6, wherein the amino acid sequence of at least one of the first peptide sequence and the second peptide sequence is identical to the amino acid sequence of one of SEQ ID NOs: 6-14.

8. The protein of claim 6, wherein the amino acid sequence of at least one of the first peptide sequence and the second peptide sequence is identical to the amino acid sequence of SEQ ID NO: 16.

9. The protein of any of claims 1 to 8, further comprising an affinity tag sequence.

10. The protein of claim 9, wherein the affinity tag sequence comprises a streptavidin- binding peptide sequence (SEQ ID NO: 28).

11. The protein of any of claims 1 to 10, further comprising a signal peptide sequence.

12. The protein of claim 11 , wherein the signal peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 37-48.

13. A protein comprising the amino acid sequence of SEQ ID NO: 2; wherein positions 33- 52 are a first peptide sequence; wherein positions 81-100 are a second peptide sequence; wherein positions 134-153 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length; and wherein at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is at least 6 amino acids in length.

14. The protein of claim 13, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:504919-2079-0411 , v 1(a) 8 to 12 amino acids in length;(b) contains at least 2 but no more than 4 of any combination of amino acids R, E, D, Q, and N;(c) contains at least 5 but no more than 9 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

15. The protein of claim 13 or 14, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:(a) 10 amino acids in length;(b) contains 3 of any combination of amino acids R, E, D, Q, and N;(c) contains 7 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

16. The protein of claim 13 or 14, wherein the amino acid sequences of the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently identical to the amino acid sequence of one of SEQ ID NOs: 6-30.

17. The protein of claim 16, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NOs: 6-14.

18. The protein of claim 16, wherein the amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third514919-2079-0411 , v 1peptide sequence is identical to the amino acid sequence of one of SEQ ID NOs: 17- 19.

19. The protein of any of claims 13 to 18, further comprising an affinity tag sequence.

20. The protein of claim 19, wherein the affinity tag sequence comprises a streptavidin- binding peptide sequence (SEQ ID NO: 28).

21. The protein of any of claims 13 to 20, further comprising a signal peptide sequence.

22. The protein of claim 21 , wherein the signal peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 37-48.

23. A protein comprising the amino acid sequence of SEQ ID NO: 4, wherein positions 55- 74 are a first peptide sequence; wherein positions 89-108 are a second peptide sequence; wherein positions 122-141 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length; and wherein at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is at least 6 amino acids in length.

24. The protein of claim 23, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is: (a) 8 to 12 amino acids in length;(b) contains at least 2 but no more than 4 of any combination of amino acids R, E, D, Q, and N;(c) contains at least 5 but no more than 9 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

25. The protein of claim 23 or 24, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:524919-2079-0411 , v 1(a) 10 amino acids in length;(b) contains 3 of any combination of amino acids R, E, D, Q, and N;(c) contains 7 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T. V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

26. The protein of claim 23 or 24, wherein the amino acid sequences of the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently identical to the amino acid sequence of one of SEQ ID NOs: 6-30.

27. The protein of claim 26, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NOs: 6-14.

28. The protein of claim 26, wherein the amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NO: 23-25.

29. The protein of any of claims 23 to 28, further comprising an affinity tag sequence.

30. The protein of claim 29, wherein the affinity tag sequence comprises a streptavidin- binding peptide sequence (SEQ ID NO: 28).

31. The protein of any of claims 23 to 30, further comprising a signal peptide sequence.

32. The protein of claim 31, wherein the signal peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 37-48.

33. A protein comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 3, wherein positions 36-55 are a first peptide sequence; wherein positions 67-86 are a second peptide sequence; wherein positions 129-148 are a third peptide sequence; wherein the first peptide sequence, the second peptide534919-2079-0411 , v 1sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length.

34. The protein of claim 33 comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 3.

35. The protein of claim 33 comprising the amino acid sequence of SEQ ID NO: 3.

36. The protein of any one of claims 33 to 35, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:(a) 8 to 12 amino acids in length;(b) contains at least 2 but no more than 4 of any combination of amino acids R, E, D, Q, and N;(c) contains at least 5 but no more than 9 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

37. The protein of any one of claims 33 to 36, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:(a) 10 amino acids in length;(b) contains 3 of any combination of amino acids R, E, D, Q, and N;(c) contains 7 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and544919-2079-0411 , v 1(e) does not contain amino acids C, K, or P.

38. The protein of any one of claims 33 to 35, wherein the amino acid sequences of the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently identical to the amino acid sequence of one of SEQ ID NOs: 6-30.

39. The protein of claim 38, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NOs: 6-14.

40. The protein of claim 38, wherein the amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NO: 20-22.

41. The protein of any of claims 33 to 40, further comprising an affinity tag sequence.

42. The protein of claim 41, wherein the affinity tag sequence comprises a streptavidin- binding peptide sequence (SEQ ID NO: 28).

43. The protein of any of claims 33 to 42, further comprising a signal peptide sequence.

44. The protein of claim 43, wherein the signal peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 37-48.

45. A protein comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 5: wherein positions 55-74 are a first peptide sequence; wherein positions 89-108 are a second peptide sequence; wherein positions 122-141 are a third peptide sequence; wherein the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently 2 to 20 amino acids in length.

46. The protein of claim 45 comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 5.

47. The protein of claim 45 or 46 comprising the amino acid sequence of SEQ ID NO: 5.554919-2079-0411 , v 148. The protein of any one of claims 45 to 47, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:(a) 8 to 12 amino acids in length;(b) contains at least 2 but no more than 4 of any combination of amino acids R, E, D, Q, and N;(c) contains at least 5 but no more than 9 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

49. The protein of any one of claims 45 to 48, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is:(a) 10 amino acids in length;(b) contains 3 of any combination of amino acids R, E, D, Q, and N;(c) contains 7 of any combination of amino acids A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, and Y;(d) does not contain 3 consecutive amino acids of any combination of L, I, V, M, W, and F; and(e) does not contain amino acids C, K, or P.

50. The protein of any one of claims 45 to 47, wherein the amino acid sequences of the first peptide sequence, the second peptide sequence, and the third peptide sequence are each independently identical to the amino acid sequence of one of SEQ ID NOs: 6-30.564919-2079-0411 , v 151. The protein of claim 50, wherein the amino acid sequence of at least one of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NOs: 6-14.

52. The protein of claim 50, wherein the amino acid sequence of at least one, but not more than two, of the first peptide sequence, the second peptide sequence, and the third peptide sequence is identical to the amino acid sequence of one of SEQ ID NO: 20-22.

53. The protein of any of claims 45 to 52, further comprising an affinity tag sequence.

54. The protein of claim 53, wherein the affinity tag sequence comprises a streptavidin- binding peptide sequence (SEQ ID NO: 28).

55. The protein of any of claims 45 to 54, further comprising a signal peptide sequence.

56. The protein of claim 55, wherein the signal peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 37-48.

57. The protein of any one of claims 1 to 56, wherein the protein is soluble.

58. The protein of any one of claims 1 to 57, wherein the protein is soluble after treatment at 60°C for 30 minutes.

59. A polynucleotide comprising a nucleotide sequence encoding the protein of any one of claims 1 to 58.

60. An expression construct comprising a promoter sequence operably linked to a polynucleotide of claim 59.

61. The expression construct of claim 60, wherein the promoter sequence is an inducible promoter sequence.

62. An expression vector comprising one or more expression constructs of claim 60 or 61.

63. The expression vector of claim 62, further comprising an antibiotic resistance element.

64. The expression vector of claim 62 or 63, wherein the expression vector is a viral vector.574919-2079-0411 , v 165. The expression vector of claim 64, wherein the viral vector is an adeno-associated virus viral vector.

66. A recombinant cell comprising the expression construct of any one of claims 62 to 65.

67. The recombinant cell of claim 66, where the expression construct is a first expression construct, and the recombinant cell further comprises at least a second expression construct of claim 60 or 61, wherein the promoter of the first expression construct differs from the promoter of the second expression construct, and further wherein the polynucleotide comprising the nucleotide sequence encoding the protein of any of claim 1 to 58 of the first expression construct differs from the polynucleotide comprising the nucleotide sequence encoding the protein of any of claim 1 to 58 of the second expression construct.

68. The recombinant cell of claim 67, further comprising a third expression construct of claim 60 or 61 and a fourth expression construct of claim 60 or 61 , wherein the promoter and the polynucleotide comprising the nucleotide sequence encoding the protein of any of claim 1 to 58 of the third expression construct differs from the promoter and polynucleotide comprising the nucleotide sequence encoding the protein of any of claim 1 to 58 of the first expression construct, the second expression construct, and the fourth expression construct.

69. A recombinant cell comprising a plurality of different expression constructs according to claim 60 or 61, wherein each different expression construct has a different promoter and encodes a different protein from the other different expression constructs in the plurality of different expression constructs.

70. The recombinant cell of claim 69, wherein the recombinant cell comprises from one to fifty different expression constructs.

71. A recombinant, non-human animal, comprising the polynucleotide of claim 59, the expression construct of claim 60 or 61, the expression vector of any one of claims 62 to 65, or the recombinant cell of any one of claims 66 to 70.

72. A polypeptide comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,584919-2079-0411 , v 1SEQ ID NO: 13, or SEQ ID NO: 14 or an amino acid that is 80% or 90% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

73. A method of detecting an effect of a stimulus on a signal transduction pathway, the method comprising (a) contacting the recombinant cell of any one of claims 66-70 or the recombinant, non-human animal of claim 71 with the stimulus; and (b) detecting a change in expression of a synthetic reporter.

74. The method of claim 73, wherein the synthetic reporter comprises an affinity tag, wherein the synthetic reporter is detected using an antibody that binds to the affinity tag.

75. The method of claim 73, wherein the synthetic reporter comprises an affinity tag, wherein the synthetic reporter is detected using an antibody that binds to the affinity tag and an antibody that binds to an epitope of the synthetic reporter.

76. The method of claim 73, wherein the synthetic reporter is detected using an antibody that binds to an epitope of the synthetic reporter.

77. The method of any one of claims 73 to 76, wherein the recombinant cell or recombinant, non-human animal comprises two or more expression constructs each having a different promoter and encoding a different synthetic reporter.

78. The method of claim 77, wherein the recombinant cell or recombinant, non-human animal comprises ten or more expression constructs each having a different promoter and encoding a different synthetic reporter.

79. The method of claim 77 or 78, wherein each synthetic reporter is detected using an antibody that specifically binds to an epitope that is unique to the synthetic reporter.

80. The method of claim 79, wherein the antibody for each synthetic reporter is present at a spatially discrete location on a microarray, wherein each discrete location only has one type of antibody to detect one of the synthetic reporters.594919-2079-0411 , v 181. The method of claim 80, wherein detecting a signal at a discrete location indicates that the signaling pathway associated with that synthetic reporter was activated.

82. The method of any one of claims 73 to 81, wherein the method is further defined as a method for screening compounds to detect effects on cell signaling.

83. A method of detecting an effect of at least 3 stimuli on signal transduction pathways, the method comprising: (a) contacting cells comprising expression cassettes for at least three different synthetic reporters, wherein each synthetic reporter is under the control of a different regulatory element; and (b) detecting a change in expression for each of the synthetic reporters.

84. The method of claim 83, wherein each synthetic reporter comprises a first epitope site and a second epitope site, wherein each of the first epitope site and the second epitope site is independently disposed between an alpha helix and a beta sheet, a first alpha helix and a second alpha helix, or a first beta sheet and a second beta sheet, wherein each synthetic reporter has a unique first epitope and / or wherein each synthetic reporter has a unique second epitope.

85. The method of claim 83 or 84, wherein the cells comprising expression cassettes for at least ten different synthetic reporters, wherein each synthetic reporter is under the control of a different regulatory element.

86. The method of any one of claims 83 to 85, wherein each synthetic reporter comprises a unique affinity tag, wherein each synthetic reporter is detected using an antibody that binds to its affinity tag.

87. The method of any one of claims 83 to 85, wherein each synthetic reporter comprises a unique affinity tag, wherein each synthetic reporter is detected using an antibody that binds to the affinity tag and an antibody that binds to an epitope of the synthetic reporter.

88. The method of any one of claims 83 to 85, wherein each of the synthetic reporters is detected using an antibody that binds to the unique first and / or second epitope of the synthetic reporter.604919-2079-0411 , v 189. The method of any one of claims 85 to 88, wherein the antibody for each synthetic reporter is present at a spatially discrete location on a microarray, wherein each discrete location only has one type of antibody to detect one of the synthetic reporters.

90. The method of claim 89, wherein detecting a signal at a discrete location indicates that the signaling pathway associated with that synthetic reporter was activated.

91. The method of any one of claims 83 to 90, wherein the method is further defined as a method for screening compounds to detect effects on cell signaling.

92. A method for detecting a microRNA in a sample, the method comprising:(a) contacting the sample with a first capture probe and a first detection probe under conditions to allow hybridization between the microRNA and the first capture probe and the first detection probe to form a binding complex if the microRNA is present in the sample, wherein the first capture probe comprises a first single stranded nucleotide segment having a sequence that is complementary to a first portion of the microRNA and a capture moiety, wherein the detection probe comprises (i) a second single stranded nucleotide segment having a sequence that is complementary to a second portion of the microRNA, (ii) a double stranded nucleotide segment that encodes a synthetic reporter, and (iii) a promoter, wherein the promoter is operably linked to the double stranded nucleotide segment that encodes the synthetic reporter;(b) capturing the binding complex on a solid support coated with a binding partner for the capture moiety;(c) releasing the binding complex from the solid support;(d) subjecting the released sample to a cell-free protein synthesis reaction to produce the synthetic reporter encoded by the detection probe; and (e) detecting the synthetic reporter, thereby detecting the microRNA.

93. The method of claim 92, further comprising washing the solid support between steps (b) and (c).

94. The method of claim 92 or 93, wherein the capture moiety is biotin.614919-2079-0411 , v 195. The method of any one of claims 92 to 94, wherein the synthetic reporter comprises a first epitope site and a second epitope site, wherein each of the first epitope site and the second epitope site is independently disposed between an alpha helix and a beta sheet, a first alpha helix and a second alpha helix, or a first beta sheet and a second beta sheet, wherein each synthetic reporter has a unique first epitope and / or wherein each synthetic reporter has a unique second epitope.

96. The method of any one of claims 92 to 95, wherein the synthetic reporter is the protein of any one of claims 1 to 5.

97. The method of any one of claims 92 to 96, wherein the synthetic reporter comprises an affinity tag, wherein the synthetic reporter is detected using an antibody that binds to the affinity tag.

98. The method of any one of claims 92 to 96, wherein the synthetic reporter comprises an affinity tag, wherein the synthetic reporter is detected using an antibody that binds to the affinity tag and an antibody that binds to an epitope of the synthetic reporter.

99. The method of any one of claims 92 to 96, wherein the synthetic reporter is detected using an antibody that binds to an epitope of the synthetic reporter.

100. The method of any one of claims 92 to 99, wherein the sample comprises two or more microRNAs and wherein step (a) comprises a detection probe and a capture probe for each microRNA, wherein each detection probe encodes a unique synthetic reporter.

101. The method of claim 100, wherein each synthetic reporter is detected using an antibody that specifically binds to an epitope that is unique to the synthetic reporter.

102. The method of claim 101 , wherein the antibody for each synthetic reporter is present at a spatially discrete location on a microarray, wherein each discrete location only has one type of antibody to detect one of the synthetic reporters.

103. The method of claim 102, wherein detecting a signal at a discrete location indicates that the microRNA associated with that synthetic reporter was present.624919-2079-0411 , v 1

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