Logic capable protease probes and uses thereof

Abstract

The present invention relates to recombinant proteins as logic capable protease probes. Also provided are methods for detecting proteases, recombinant polynucleotides encoding the recombinant proteins, recombinant host cells comprising the recombinant polynucleotides and / or expressing the recombinant proteins, and method for producing the recombinant proteins.

Description

[0001] LOGIC CAPABLE PROTEASE PROBES AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application No. 63 / 661,261, filed June 18, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes.

[0004] REFERENCE TO U.S. GOVERNMENT SUPPORT

[0005] This invention was made with government support under grant number 2220667 awarded by National Science Foundation (NSF). The United States has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2101715- 001301_SequenceListing.xml, created June 10, 2025, which is 18 KB in size. The information in XML file format of the Sequence Listing is incorporated herein by reference in its entirety.

[0008] FIELD OF THE INVENTION

[0009] The invention relates to recombinant proteins useful as logic capable protease probes for detecting proteases.

[0010] BACKGROUND OF THE INVENTION

[0011] Proteases are a diverse and highly influential class of enzymes that facilitate proteolysis - the cleavage of proteins. They serve as the executioners of many biological pathways through processes including, activating and deactivating proteins, modulating cell signaling / death / division, controlling the activity of other proteases or enzymes, and even physically remodeling tissue. Because of their potent role, their activity is tightly regulated at the epigenetic, transcriptional, and post transcriptional level, with added control pathways at the post-translational level through protease activation, deactivation, glycosylation and inhibitors. Proteases are clinically relevant in almost every pathology including cancer, viral infection, implant rejection, chronic wounds failing to heal, post-myocardial infarction remodeling, arthritis (osteo and rheumatoid), and many more examples. Despite the many layers of control, the dysregulation of proteases is a hallmark of many diseases with the specific dysregulation profile often serving as a molecular fingerprint that could be used as a diagnostic or prognostic marker. Additionally, non-endogenous proteases have significant clinical interest due to their foundational role for viral replication making them a common therapeutic target for antiviral therapies. Because most proteases are expressed as inactive zymogens and subject to inactivation, their expression doesn't directly correlate with their activity making direct activity essential for characterizing protease environments. For this reason activity assays are the current standard, these probes are largely limited to FRET peptides.

[0012] FRET peptides detect protease activity using a fluorophore-quencher pair linked by a cleavable substrate. When intact, the precise spacing of the fluorophore and quencher enables Forster Resonance Energy Transfer (FRET), suppressing the majority of fluorescence. Upon protease cleavage of the peptide, the fluorophore separates from the quencher, disrupting FRET and restoring fluorescence - indicating protease activity via measuring the increase.

[0013] There are some inherent limitations of probes that use the FRET phenomena. First, extremely strict spacing is required for the FRET efficiency, which is dictated by: efficiency = l / (l + (r / Ro) / K6) where r = distance between fluorophore and quencher and Ro = the forstor radius (50% efficiency). What this equation means is any slight deviation from the ideal distance reduces the efficiency of transfer to the sixth power, substantially dropping the FRET efficiency if the linker length is changed by even 1 nanometer or just 2-3 amino acids. This spacing requirement disallows any changes to the design that would enable the probe to detect multiple proteases simultaneously. Second, with ideal spacing FRET peptides typically have an efficiency of 70-80% often they are as low as 30-60% due to sub optimal spacing. What this means is even when the peptide should be "off" prior to cleavage they are still fluorescent reducing the difference between the on and off state because the off state isn't truly off. Third, FRET peptides far outperform recombinant FRET protein probes, which are mostly <50% efficiency and considerably dimmer. As a result, recombinant FRET protein probes are rarely used for in vitro assays. Additionally, the FRET peptides which are heavily used in in vitro assays are synthesized via solid state synthesis, which is an extremely costly and solvent intensive approach for producing the probes.

[0014] There remains a need for protease probes with low background, high dynamic range and logic capabilities.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention relates to recombinant splicing together split proteins (SplitFAST fragments) tethered as an input responsive manor platform and uses thereof as logic capable protease probes for detecting proteases.

[0017] The present invention provides a first recombinant protein. The first recombinant protein comprises a first linker, a first protein fragment on the N-terminus of the first linker, and a second protein fragment on the C-terminus of the first linker. The first protein fragment consists of an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 1. The second protein fragment consists of an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of GDSYWVFVKRV (SEQ ID NO: 2), GDSYWVFVKR (SEQ ID NO: 3), GDSYWVFVK (SEQ ID NO: 4), and GDSYWVFV (SEQ ID NO: 5). The first linker comprises a first cleavage site. The first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when the first cleavage site is not cleaved than the fluorescence generated when the first cleavage site is cleaved.

[0018] The present invention provides a second recombinant protein. The second recombinant protein is the first recombinant protein, in which the first linker further comprises a second cleavage site. The second cleavage site is different from the first cleavage site. The first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when the first linker cleavage site and the second cleavage site are not cleaved than the fluorescence generated when at least one of the first cleavage site and the second cleavage site is cleaved.

[0019] The present invention provides a third recombinant protein. The third recombinant protein is the second recombinant protein, in which the first linker further comprises a third cleavage site for a third protease. The third cleavage site is different from the first cleavage site and the second cleavage site. The first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when the first cleavage site, the second cleavage site, and the third cleavage site are not cleaved than the fluorescence generated when at least one of the first cleavage site, the second cleavage site and the third cleavage site is cleaved.

[0020] The present invention provides a fourth recombinant protein. The fourth recombinant protein is the first recombinant protein, which further comprises a first N- terminal leucine zipper on the N-terminus of the first protein fragment, a first C- terminal leucine zipper on the C-terminus of the second protein fragment, a second N- terminal leucine zipper on the N-terminus of the first N-terminal leucine zipper, a second C-terminal leucine zipper on the C-terminus of the first C-terminal leucine zipper, and a second linker between the first N-terminal leucine zipper and the first protein fragment or between the first C-terminal leucine zipper and the second protein fragment. The first N-terminal leucine zipper and the first C-terminus leucine zipper form a first pair leucine zipper complex. The second N-terminal leucine zipper and the second C-terminus leucine zipper form a second pair leucine zipper complex. The second linker comprises a second cleavage site. The second cleavage site is different from the first cleavage site. The first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when at least one of the first cleavage site and the second cleavage site is not cleaved than the fluorescence generated when the first linker cleavage site and the second cleavage site are cleaved.

[0021] The present invention provides a fifth recombinant protein. The fifth recombinant protein is the first recombinant protein, which further comprises a first N- terminal leucine zipper on the N-terminus of the first protein fragment, a first C- terminal leucine zipper on the C-terminus of the second protein fragment, a second N- terminal leucine zipper on the N-terminus of the first N-terminal leucine zipper, a second C-terminal leucine zipper on the C-terminus of the first C-terminal leucine zipper, a second linker between the first N-terminal leucine zipper and the first protein fragment, and a third linker between the first C-terminal leucine zipper and the second protein fragment. The first N-terminal leucine zipper and the first C-terminal leucine zipper form a first pair leucine zipper complex. The second N-terminal leucine zipper and the second C-terminal leucine zipper form a second pair leucine zipper complex. The second linker comprises a second cleavage site. The second cleavage site is different from the first cleavage site. The third linker comprises a third cleavage site. The third cleavage site is different from the first cleavage site and the second cleavage site. The first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when at least one of the second cleavage site and the third cleavage site is not cleaved than the fluorescence generated when the first linker cleavage site is cleaved and at least one of the second cleavage site and the third cleavage site is cleaved.

[0022] In the first, second, third, fourth or fifth recombinant protein, the second protein fragment may consist of an amino acid sequence at least 90% identical to the amino acid sequence of GDSYWVFVKR (SEQ ID NO: 3).

[0023] According to the first, second, third, fourth or fifth recombinant protein, the fluorogen may be selected from the group consisting of 4-hydroxy-3-methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4-hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM).

[0024] In the first, second, third, fourth or fifth recombinant protein, the first linker may comprise (G4S)n, wherein n is 1 to 3.

[0025] In the first, second, third, fourth or fifth recombinant protein, the first cleavage site may consist of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 7), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13), IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

[0026] A first method for detecting one or more target proteases in a target sample is provided. The first detection method comprises: (a) incubating the first recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the first recombinant protein by a control protease capable of cleaving the first cleavage site, whereby a first test sample is obtained; (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen; (c) incubating the first recombinant protein in the medium in the absence of the test sample under the conditions in step (a), whereby a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site.

[0027] A second method for detecting one or more target proteases in a target sample is provided. The second detection method comprises: (a) incubating the second recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the second recombinant protein by a first control protease capable of cleaving the first cleavage site and cleavage of the second cleavage site in the second recombinant protein by a second control protease capable of cleaving the second cleavage site, whereby a first test sample is obtained; (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen; (c) incubating the second recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site and / or the second cleavage site.

[0028] A third method for detecting one or more target proteases in a target sample is provided. The third detection method comprises: (a) incubating the third recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the third recombinant protein by a first control protease capable of cleaving the first cleavage site, cleavage of the second cleavage site in the third recombinant protein by a second control protease capable of cleaving the second cleavage site, and cleavage of the third cleavage site in the third recombinant protein by a third control protease capable of cleaving the third cleavage site, whereby a first test sample is obtained; (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen; (c) incubating the third recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site, the second cleavage site, and / or the third cleavage site.

[0029] A fourth method for detecting one or more target proteases in a target sample is provided. The fourth detection method comprises: (a) incubating the fourth recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the fourth recombinant protein by a first control protease capable of cleaving the first cleavage site and cleavage of the second cleavage site in the fourth recombinant protein by a second control protease capable of cleaving the second cleavage site, whereby a first test sample is obtained; (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen; (c) incubating the fourth recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site and the second cleavage site.

[0030] A fifth method for detecting one or more target proteases in a target sample is provided. The fifth detection method comprises: (a) incubating the fifth recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the fifth recombinant protein by a first control protease capable of cleaving the first cleavage site, cleavage of the second cleavage site in the fifth recombinant protein by a second control protease capable of cleaving the second cleavage site, and cleavage of the third cleavage site in the fifth recombinant protein by a third control protease capable of cleaving the third cleavage site, whereby a first test sample is obtained; (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen; (c) incubating the fifth recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and (d) making a -1- second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site, and at least one of the second cleavage site and the third cleavage site.

[0031] Each detection method may further comprise quantifying the one or more target proteases. For example, the first, second, third, fourth or fifth method may further comprise quantifying the one or more target proteases.

[0032] For each recombinant protein of the present invention, a recombinant polynucleotide is provided. The recombinant polynucleotide encodes the recombinant protein. For example, the recombinant polynucleotide encodes the first, second, third, fourth or fifth recombinant protein.

[0033] For each recombinant protein of the present invention, a recombinant host cell is provided. The recombinant host cell expresses the recombinant protein. For example, the recombinant host cell expresses the first, second, third, fourth or fifth recombinant protein.

[0034] For each recombinant protein of the present invention, a recombinant host cell is provided. The recombinant host cell comprises a recombinant polynucleotide encoding the recombinant protein. For example, the recombinant host cell comprises a recombinant polynucleotide encoding the first, second, third, fourth or fifth recombinant protein.

[0035] For each recombinant protein of the present invention, a method for producing the recombinant protein is provided. The production method comprises: (a) growing a recombinant host cell, wherein the host cell comprises a recombinant polynucleotide encoding the recombinant protein; and (b) expressing the recombinant protein by the recombinant host cell. The method may further comprise purifying the recombinant protein from the recombinant host cell.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1A-D show a single input SPLICE probe. (A) Simple design of the single input SPLICE probes, a flexible linker causes the forced proximity of the SplitFAST fragments which results in increased affinity and subsequent reconstitution and fluorescence in the presence of HMBR. Cleavage of the flexible linker abolishes the forced proximity reverting the affinity to the basal low levels (> luM) resulting in splitFAST de-complementation and the fluorescence turning off. (B) an SDS-PAGE denaturing protein gel shows that SPLICETEVpis cleaved at the expected size in the presence of TEVp and (C) shows that the cleavage results in the abolishment of fluorescence. As seen the off state is a small fraction of the on state, it also shows that the other single protease SPLICE variants are cleaved and lose fluorescence when incubated with Thrombin, TEVp, or MMP3cd. The fluorescence in the off state is very low, it is seen that FRET peptides have ~4x more background . Additionally, the dynamic range for each probe is listed beneath the protease and seen to dwarf the dynamic range of FRET this value is calculated as the average of the uncleaved probe divided by the average of the cleaved probe for both FRET and SPLICE done under identical reaction conditions. (D) the rate of cleavage is shown with a dynamic assay where the lower line rapidly loses fluorescence due to proteolytic activity while the upper line simply photobleaches at a rate consistent across all assays of variants (~1.2x over lhr).

[0038] FIGS. 2A-D show NOR. (A) physical design change that results in NOR gating capabilities along with its respective logic nomenclature, (B) demonstration of the NOR- gating potential, this one single probe is able to respond to either Thrombin, TEVp OR MMP3cd with the same on off characteristics as the single input probe (C) SPLICEcovid responds to the covid relevant protease 3CLpro and PLpro (D) inhibition of 3CLpro (the 'main' covid protease) using a known inhibitor (GRL-0496), by looking at the inverse initial rates of change plotted against the inverse substrate concentration (referred to as Lineweaver-burke analysis), obtaining an IC50 of 35.7nM which is well within range of the literature reported 30nM.

[0039] FIGS. 3A-E show NAND & NAND-OR. (A) Design of SPLICENANDconsists of the addition of two leucine zippers on the n-terminus, and their respective partners on the c-terminus, the resulting structure results in strong association between the two pairs capable of forcing SplitFAST reconstitution even if the internal linker is cleaved. The protease substrate labile linker between cFAST and the c-terminal zippers allows for this association to be independently abolished via protease cleavage (in this case TEVp). (B) Change in fluorescence show clear NAND-gated response only in the presence of both proteases of interest (TEVp and MMP3cd), when one or neither of the proteases of interest are present there is a less than 1.5x change in fluorescence. (C) Logic behavior of SPLICENANDand SPLICENAND 0R, (D) change in design to achieve the mixed logic SPLICENAND-0Rprobe, the linker between the n- terminal leucine zippers and nFAST was modified to be protease labile by an orthogonal protease— in this case Thrombin. (E) Change in fluorescence (RFL) demonstrates clear NAND-OR behavior with a large signal change only occurring one of the two protease environments of interest is present (MMP3cd AND Thrombin) or (MMP3cd AND TEVp), otherwise the signal change is low.

[0040] DETAILED DESCRIPTION OF THE INVENTION The present invention relates to recombinant proteins useful as logic capable protease probes and uses thereof for detecting proteases in a sample. The invention is based on the inventors' surprisingly discovery of splicing together split proteins (SplitFAST fragments) with tethers for them to fold in a way that results in an input responsive sensor platform, providing Split Protein Interpretation of Cleavage Environments (SPLICE). Also provided by the present invention are methods for detecting proteases in samples, recombinant polynucleotides encoding the recombinant proteins, recombinant host cells for expressing the recombinant proteins, and methods for producing the recombinant proteins.

[0041] The inventors have developed a unique SPLICE system, which has no strict spacing requirements and allows for changing the design to probe multiple proteases with a single probe. The SPLICE system has a much lower background (or fluorescence in the 'OFF' state). The SPLICE system is a recombinant protein and therefore can simply be expressed in, for example, E. coli, instead of synthesized.

[0042] The inventors have developed a time point assay to show the inherent maximum change in fluorescence when a protease probe is 100% cleaved. The maximum dynamic range for FRET probes varies about 4-8x. The inventors have shown much higher maximum dynamic ranges for SPLICE probes, which vary depending on their logic or design but are consistently over 15x with some designs exceeding 40x. The inventors have conducted time point assays for all designs to highlight the inherently higher dynamic range for the SPLICE probes compared to FRET probes.

[0043] A dynamic assay is a common way of quantifying enzyme activity (classic FRET application). The inventors have carried out dynamic assays with several proteases. The inventors have set up an experiment to make what is called a "Lineweaver-burke" analysis plot based on the initial rate of change in fluorescence across a variety of conditions to quantify the enzymes response to a known inhibitor, and demonstrated that SPLICE can be used to characterize the inhibition of the COVID protease 3CLpro using the known inhibitor GRL-0496 and came to the same IC50 value that is reported in literature. The inventors have also showed that the rate of change is dependent on protease concentration, and inhibitor concentration.

[0044] The term "protease probe" as used herein refers to a molecule such as a protein capable of detecting a proteolytic activity and outputting a change in signal, for example, fluorescence, to reflect the measured proteolytic activity.

[0045] The term "logic capable" or "logic capability" as used herein refers to the ability of a protease probe to generate a single Boolean output in a proteolytic environment, which single Boolean output may be used in combination with the physical structure of the protease probe to characterize multiple proteases in the proteolytic environment. The term "linker" as used herein refers to a short linear chain of amino acids. The linker may have about 10-20, 10-30, 10-40, 10-40, 20-30, 20-40, 20-50, 30-40, 30-50 or 40-50 amino acids. The linker may comprise a cleavage site.

[0046] The term "cleavage site" or "cleavage site for a protease" as used herein refers to an amino acid sequence that is specifically recognized and cut by a protease. Exemplary cleavage sites including RPFSMIMG (SEQ ID NO: 6) for MMP3cd, ENLYFQS (SEQ ID NO: 7) for TEVp, LVPRGS (SEQ ID NO: 8) for thrombin, GSTSAVLQSGFRK (SEQ ID NO: 9) for 3CLpro, ATIMMQRG (SEQ ID NO: 10) for HIV-pl, PLGLAG (SEQ ID NO: 11) for MMP2 / 9, AENK (SEQ ID NO: 12) for 6-Secretase, DEVD (SEQ ID NO: 13) for caspase 3, IETD (SEQ ID NO: 14) for caspase 8, and ADVIPARRRG (SEQ ID NO: 15) for HCV NS3 / 4A protease.

[0047] The term "homology" or "homologous" as used herein refers to similarity between the amino acid sequence of a protein and a reference sequence. The similarity or homology may be at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40- 100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70- 100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100%.

[0048] The term "fluorogen" as used herein refers to a chemical compound that is not initially fluorescent but becomes fluorescent upon a chemical reaction. Examples of fluorogens include 4-hydroxy-3-methylbenzylidene rhodamine (HMBR) for producing green-yellow fluorescence, 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR- 3,5DOM) for providing orange-red fluorescence, and (4-hydroxy-3,5-dimethoxy- phenyl)allylidene-rhodanine (HPAR-3,5DOM) for providing near-infrared (NIR) fluorescence.

[0049] The present invention provides a first recombinant protein. The first recombinant protein may comprise a first linker, a first protein fragment on the N- terminus of the first linker, and a second protein fragment on the C-terminus of the first linker. The first protein fragment may comprise an amino acid sequence homologous to the amino acid sequence of FAST residues 1-114 (SEQ ID NO: 1). The second protein fragment may comprise an amino acid sequence homologous to the amino acid sequence of FAST residues 115-125 (SEQ ID NO: 2), FAST residues 115- 124 (SEQ ID NO: 3), FAST residues 115-123 (SEQ ID NO: 4) or FAST residues 115-122 (SEQ ID NO: 5). The first linker may comprise a first cleavage site. The first protein fragment and the second protein fragment in the first recombinant protein may generate more fluorescence, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times more fluorescence, in the presence of a fluorogen when the first cleavage site is not cleaved than the fluorescence generated when the first cleavage site is cleaved.

[0050] The first protein fragment may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40- 60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50- 80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60- 99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80- 99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 1. The first protein fragment may consist of an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40- 100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70- 100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 1. The first protein fragment may consist of SEQ ID NO: 1.

[0051] The second protein fragment may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 2, 3, 4 or 5. The first protein fragment may consist of an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 2, 3, 4 or 5. The second protein fragment may consist of SEQ ID NO: 2, 3, 4 or 5. For example, second protein fragment may consist of SEQ ID NO: 3.

[0052] For the first recombinant protein, the fluorogen may be 4-hydroxy-3- methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4-hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM). In the first recombinant protein, the first linker may comprise of n repeats of the 5 amino acid sequence of gly-gly-gly-gly-ser (GGGGS) (SEQ ID NO: 16), also known as (G4S)n, wherein n is 1 to 3. The first cleavage site may consist of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 6), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13) IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

[0053] For each first recombinant protein, a first method for detecting one or more target proteases is provided. The first detection method may comprise (a) incubating the first recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the first recombinant protein by a control protease so that a first test sample is obtained. The control protease is capable of cleaving the first cleavage site. The first detection method may also comprise (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen. The first detection method may further comprise (c) incubating the first recombinant protein in the medium in the absence of the test sample under the conditions in step (a) so that a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen. An increase from the first fluorescence measurement to the second fluorescence measurement by, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times, may indicate that the target sample comprises one or more target proteases capable of cleaving the first cleavage site.

[0054] The present invention provides a second recombinant protein. The second recombinant protein may be the first recombinant protein, in which the first linker further comprises a second cleavage site. The second cleavage site is different from the first cleavage site. The first protein fragment and the second protein fragment in the second recombinant protein may generate more fluorescence, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times more fluorescence, in the presence of a fluorogen when the first linker cleavage site and the second cleavage site are not cleaved than the fluorescence generated when at least one of the first cleavage site and the second cleavage site is cleaved.

[0055] For the second recombinant protein, the fluorogen may be 4-hydroxy-3- methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4-hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM).

[0056] In the second recombinant protein, each of the first linker and the second linker may comprise (G4S)n, wherein n is 1 to 3. Each of the first linker and the second linker may consist of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 6), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13) IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

[0057] For each second recombinant protein, a second method for detecting one or more target proteases is provided. The second detection method may comprise (a) incubating the second recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the second recombinant protein by a first control protease and cleavage of the second cleavage site in the second recombinant protein by a second control protease so that a first test sample is obtained. The first control protease is capable of cleaving the first cleavage site. The second control protease is capable of cleaving the second cleavage site. The second detection method may also comprise (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen. The second detection method may further comprise (c) incubating the second recombinant protein in the medium in the absence of the test sample under the conditions in step (a) so that a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen. An increase from the first fluorescence measurement to the second fluorescence measurement by, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times, may indicate that the target sample comprises one or more target proteases capable of cleaving the first cleavage site and / or the second cleavage site.

[0058] The present invention provides a third recombinant protein. The third recombinant may be the second recombinant protein, in which the first linker further comprises a third cleavage site for a third protease. The third cleavage site is different from the first cleavage site and the second cleavage site. The first protein fragment and the second protein fragment in the third recombinant protein may generate more fluorescence, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times more fluorescence, in the presence of a fluorogen when the first cleavage site, the second cleavage site, and the third cleavage site are not cleaved than the fluorescence generated when at least one of the first cleavage site, the second cleavage site and the third cleavage site is cleaved.

[0059] For the third recombinant protein, the fluorogen may be 4-hydroxy-3- methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4-hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM). In the third recombinant protein, each of the first linker, the second linker and the third linker may comprise (G4S)n, wherein n is 1 to 3. Each of the first linker, the second linker and the third linker may consist of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 6), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13) IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

[0060] For each third recombinant protein, a third method for detecting one or more target proteases is provided. The third detection method may comprise (a) incubating the third recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the third recombinant protein by a first control protease, cleavage of the second cleavage site in the third recombinant protein by a second control protease, and cleavage of the third cleavage site in the third recombinant protein by a third control protease so that a first test sample is obtained. The first control protease is capable of cleaving the first cleavage site. The second control protease is capable of cleaving the second cleavage site. The third control protease is capable of cleaving the third cleavage site. The third detection method may also comprise (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen. The third detection method may further comprise (c) incubating the third recombinant protein in the medium in the absence of the test sample under the conditions in step (a) so that a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen. An increase from the first fluorescence measurement to the second fluorescence measurement by, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times, may indicate that the target sample comprises one or more target proteases capable of cleaving the first cleavage site, the second cleavage site, and / or the third cleavage site.

[0061] The present invention provides a fourth recombinant protein. The fourth recombinant protein may be the first recombinant protein further comprising a first N- terminal leucine zipper on the N-terminus of the first protein fragment, a first C- terminal leucine zipper on the C-terminus of the second protein fragment, a second N- terminal leucine zipper on the N-terminus of the first N-terminal leucine zipper, a second C-terminal leucine zipper on the C-terminus of the first C-terminal leucine zipper, and a second linker between the first N-terminal leucine zipper and the first protein fragment or between the first C-terminal leucine zipper and the second protein fragment. The first N-terminal leucine zipper and the first C-terminus leucine zipper form a first pair leucine zipper complex. The second N-terminal leucine zipper and the second C-terminus leucine zipper form a second pair leucine zipper complex. The first linker comprises a first cleavage site. The second linker comprises a second cleavage site. The second cleavage site is different from the first cleavage site. The first protein fragment and the second protein fragment in the fourth recombinant protein may generate more fluorescence, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times more fluorescence, in the presence of a fluorogen when at least one of the first cleavage site and the second cleavage site is not cleaved than the fluorescence generated when the first linker cleavage site and the second cleavage site are cleaved.

[0062] For the fourth recombinant protein, the fluorogen may be 4-hydroxy-3- methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4-hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM).

[0063] In the fourth recombinant protein, each of the first linker and the second linker may comprise (G4S)n, wherein n is 1 to 3. Each of the first linker and the second linker may consist of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 6), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13) IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

[0064] For each fourth recombinant protein, a fourth method for detecting one or more target proteases is provided. The fourth detection method may comprise (a) incubating the fourth recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the fourth recombinant protein by a first control protease and cleavage of the second cleavage site in the fourth recombinant protein by a second control protease so that a first test sample is obtained. The first control protease is capable of cleaving the first cleavage site. The second control protease is capable of cleaving the second cleavage site. The fourth detection method may also comprise (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen. The fourth detection method may further comprise (c) incubating the fourth recombinant protein in the medium in the absence of the test sample under the conditions in step (a) so that a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen. An increase from the first fluorescence measurement to the second fluorescence measurement by, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times, may indicate that the target sample comprises the one or more target proteases capable of cleaving the first cleavage site and the second cleavage site.

[0065] The present invention provides a fifth recombinant protein. The firth recombinant protein may be the first recombinant protein further comprising a first N- terminal leucine zipper on the N-terminus of the first protein fragment, a first C- terminal leucine zipper on the C-terminus of the second protein fragment, a second N- terminal leucine zipper on the N-terminus of the first N-terminal leucine zipper, a second C-terminal leucine zipper on the C-terminus of the first C-terminal leucine zipper, a second linker between the first N-terminal leucine zipper and the first protein fragment, and a third linker between the first C-terminal leucine zipper and the second protein fragment. The first N-terminal leucine zipper and the first C-terminus leucine zipper form a first pair leucine zipper complex. The second N-terminal leucine zipper and the second C-terminus leucine zipper form a second pair leucine zipper complex. The first linker comprises a first cleavage site. The second linker comprises a second cleavage site. The second cleavage site is different from the first cleavage site. The third linker comprises a third cleavage site. The third cleavage site is different from the first cleavage site and the second cleavage site. The first protein fragment and the second protein fragment in the firth recombinant protein may generate more fluorescence, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times more fluorescence, in the presence of a fluorogen when at least one of the second cleavage site and the third cleavage site is not cleaved than the fluorescence generated when the first linker cleavage site is cleaved and at least one of the second cleavage site and the third cleavage site is cleaved .

[0066] For the fifth recombinant protein, the fluorogen may be 4-hydroxy-3- methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4-hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM).

[0067] In the fifth recombinant protein, each of the first linker, the second linker and the third linker may comprise (G4S)n, wherein n is 1 to 3. Each of the first linker, the second linker and the third linker may consist of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 6), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13) IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

[0068] For each fifth recombinant protein, a fifth method for detecting one or more target proteases is provided. The fifth detection method may comprise (a) incubating the fifth recombinant protein in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the fifth recombinant protein by a first control protease, cleavage of the second cleavage site in the fifth recombinant protein by a second control protease, and cleavage of the third cleavage site in the fifth recombinant protein by a third control protease so that a first test sample is obtained. The first control protease is capable of cleaving the first cleavage site. The second control protease is capable of cleaving the second cleavage site. The third control protease is capable of cleaving the third cleavage site. The fifth detection method may also comprise (b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen. The fifth detection method may further comprise (c) incubating the fifth recombinant protein in the medium in the absence of the test sample under the conditions in step (a) so that a second test sample is obtained; and (d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen. An increase from the first fluorescence measurement to the second fluorescence measurement by, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 times, may indicate that the target sample comprises one or more target proteases capable of cleaving the first cleavage site, and at least one of the second cleavage site and the third cleavage site.

[0069] For each detection method according to the present invention, the detection method may further comprise quantifying the one or more target proteases. For example, the first, second, third, fourth or fifth detection method may further comprise quantifying the one or more target proteases.

[0070] For each recombinant protein according to the present invention, a recombinant polynucleotide is provided. The recombinant polynucleotide may encode the recombinant protein. The recombinant polynucleotide may encode the first, second, third, fourth or fifth recombinant protein.

[0071] For each recombinant protein according to the present invention, a recombinant host cell is provided. The recombinant host cell may express the recombinant protein. The recombinant host may express the first, second, third, fourth or fifth recombinant protein.

[0072] For each recombinant polynucleotide according to the present invention, a recombinant host cell is provided. The recombinant host cell may comprise the recombinant polynucleotide. The recombinant host cell may comprise a recombinant polynucleotide encoding the first, second, third, fourth or fifth recombinant protein.

[0073] For each recombinant protein of the present invention, a method for producing the recombinant protein is provided. The production method may comprise growing a recombinant host cell. The host cell may comprise a recombinant polynucleotide encoding the recombinant protein. The production method may further comprise expressing the recombinant protein by the recombinant host cell. The production method may further comprise purifying the recombinant protein from the recombinant host cell.

[0074] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ± 10%, more preferably ±5%, even more preferably ± 1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

[0075] Example 1. Logic capable protease probes

[0076] In this study, recombinant proteins were prepared as logic capable proteases and used for detecting proteases in samples.

[0077] Materials. HMBR was obtained from MedChemExpress (MCE), and 3,5-DOMHBR was obtained from the twinklefactory. TEVp and human Thrombin were obtained from NEB.

[0078] Plasmid Generation. These plasmids were cloned in DH5a using a pET24a (Kan resistance, ITPG inducible t7 promoter). SPLICE(TEVp) was made with a gBIock obtained from Integrated DNA Technologies (IDT) cloned into the pET24a backbone using Gibson assembly, this served as the template for SPLICE(MMP3), SPLICE(Thrombin), and SPLICENOR. Using EcoRI and Agel restriction enzymes obtained from NEB, the TEVp substrate was digested out of the plasmid and replaced with annealed oligo sets to clone in an MMP3 substrate, Thrombin substrate and the three substrates (for NOR) with T4 ligase obtained from NEB. SPLICENANDwas cloned using a gblock from IDT and the pET24a backbone with standard Gibson assembly and used as the template for SPLICENAND-ORwhich using Gibson assembly was modified to insert the thrombin substrate into the linker. Plasmid and oligonucleotide sequences are listed in the supplemental.

[0079] Protein Generation & Purification. Expression of SPLICE probes were done in BL21(DE3) E. Coli at 25c (18c for NAND, and NAND-OR) in 50-250mL of TB media with lOOug / mL of kanamycin, they were induced at an OD of 0.5 using 1 mM of IPTG for 16 hours. Cells were harvested by centrifugation, and the pellets were either immediately purified or stored at -80 c. The pellet was resuspended at 5 mL / gram of pellet using a resuspension buffer (pH 7.4, 300mM NaCI, 25mM imidazole, 50mM Sodium phosphate) and then mechanically lysed using a sonicator microtip. The lysate was clarified via centrifugation (15minutes 15,000g). The soluble lysate was then purified via standard his-purification protocol with 500uL of Ni-NTA resin was used in a lOmL column. The soluble lysate was run through the column and then washed (pH 7.4, 300mM NaCI, 75mM Imidazole, 50mM Sodium phosphate) until detectable levels of protein no longer came off the column— typically lOmL. Finally, the proteins were eluted off the column with elution buffer (pH 7.4, 300mM NaCI, 350mM Imidazole, 50mM Sodium phosphate). The imidazole was dialyzed out over 16hours into IL PBS using SpectraPor 3.5-5kDa dialysis membranes (at 4 c) and then dialyzed a second time for Ihr to ensure complete removal of imidazole. Protein expression and purification was confirmed with SDS PAGE size analysis and the addition of HMBR and measurement of fluorescence.

[0080] Time point assay. End point assays to explore the dynamic range of each probe's design were done in protease specific buffers for protease specific time scales - ranging from 1-2 hours. Complete cleavage was confirmed with SDS PAGE validating the fluorescence result showing complete abolishment of fluoresence. TEVp and TEVp buffer were obtained from NEB [pH 7.5, ImM DTT, 0.5mM EDTA, 50mM Tris-HCL], (cat# P8112S) and the reaction was run at room temperature. Human thrombin protease was obtained from SigmaAldrich (cat# GE27-0846-01), and the buffer was prepared [pH 8, 500mM Tris-base, lOOmM CaCI2, 1500mM NaCI] and the digest was done at room temperature. Lastly MMP3cd was recombinantly expressed and his- purified in a similar protocol as outlined in the protein generation section, the digest was done for 2 hours at room temperature due to the comparatively lower protease activity in [pH 7.5, 50mM Tricine, 50mM NaCI, lOmM CaCI2, 5uM ZnCI2, and 0.05% Tween-20] . SPLICEN0Rwas tested in the protease buffer compatible with the protease tested under the same conditions outlined above for the single input designs. SPLICENAND ORwas digested with each protease alone following the above protocol, and when MMP3cd specifically was used with TEVp, we first digested the probe with MMP3cd and for two hours in MMP3cd buffer and then added the TEVp— because the TEVp stock solution contained EDTA which inactivates metalloproteinases such as MMP3cd. All other combinations were done simultaneously. The HMBR was added immediately prior to measurement (500nM) on the plate reader [excitation = 481nm, emission = 521nm] at a constant sensitivity (70) across all experiments including FRET comparisons, the probe concentration was held constant at 250nM for all single input designs and SPLICEN0R. 500nM was used for both SPLICENANDand SPLICENAND 0Rwith 600nM HMBR.

[0081] Dynamic assay. Dynamic assays were done at varying concentrations of probe and HMBR. We identified 300nM SPLICE + 400nM HMBR as having optimal properties, as the excess of HMBR is increased the rate of photobleaching is significantly reduced but the OFF state goes up reducing the dynamic range, alternatively increasing the concentration of the probe increases the rate of change in fluorescence while reducing photobleaching but substantially increases the OFF state. We found that the presence of photobleaching does not impact enzyme characterization because the rate is consistent and reliant on [HMBR] and imaging frequency and therefore equally impacts the control and experimental condition canceling out its influence. Each dynamic assay was done in its respective buffer.

[0082] Inhibition assay. The inhibition assays were procedurally similar to the above dynamic assay protocol with the addition of another component, inhibitor. We introduced an inhibitor [GRL-0496] and looked at the initial rates of change in fluorescence from the covid protease 3CLpro. We added 40nM of GRL-0496 and compared the rate of change in fluorescence to samples that lacked inhibitor. We compared the difference at different concentrations of SPLICE [0.05 - 0.4uM] to enable the standard quantification approach of lineweaver-burke analysis.

[0083] Results

[0084] To characterize the properties of a protease inhibitor, we used the absolute value of the initial rates to construct a lineweaver-burke plot, the response was consistent with traditional probes. We used the Lineweaver-burke plot to obtain the Ki value of the inhibitor (GRL-0496) on inhibiting 3CLpro. Because the Ki is unreported, we used the Cheng-Prussof equation which relates Ki to IC50 at a known concentration, and we obtained an IC50 of 37.7nM which was consistent with literature.

[0085] A. Single input SPLICE probes

[0086] SplitFAST is a fluorescent protein that has been split, nFAST and cFASTIO have a kd of 0.95 uM meaning that they have a low affinity for each other and will not associate with each other at lower concentrations. All variants revolve around using flexible protein linkers (G4S)n to increase the affinity between these two fragments resulting in them reconstituting so when you add the small molecule HMBR they fluoresce. Importantly this linker contains a protease cut sites so that this linker can be cleaved by a protease of interest. This direction of losing fluorescence due to cleavage is the inverse of normal FRET peptides which gain fluorescence when cleaved, but this directionality is unimportant because the output is a measurable change in signal. These cutsites are listed on page two along with options for future cut sites. In FIG. IB, lane 1 is the ladder confirming the single SPLICE protein (lane 2) is the expected size, lane 3 shows that when you introduce TEVp it cleaves SPLICETEVpinto the two expected pieces. While already accepted within the protein engineering space, this demonstrates that these proteases cleave at the expected site. Next, I show the inherent dynamic range of several SPLICE variants, first 250nM SPLICETEVpwas incubated with and without protease the green bars represent the on state (uncleaved for SPLICE and cleaved for FRET) and the red shows off state (cleaved for SPLICE and uncleaved for FRET). When compared to FRET peptides we see that FRET has a higher background fluorescence (Red bar in FIG. 1C) and as a result the dynamic range (5.7x) is much lower than SPLICE (16-26x). 5.7x is consistent with literature where FRET peptides typically achieve dynamic ranges of 3-8x. Importantly all these proteases use different buffers and the results indicate that SPLICE can be used across a range of buffers, temperatures and pHs. Next the dynamic assay FIG. ID shows the live activity of the protease on the probe, this was done with TEVp (as shown) and the other proteases. Notably there is photobleaching in the upper curve that lacks protease while significant, it was later shown (next figure) that this photobleaching does not interfere with any of the quantification that protease activity probes are used for. Additionally, photobleaching is a property that all fluorescent proteins / dyes have so considering SPLICE starts in the on state, photobleaching was expected.

[0087] B. SPLICEN0R(NOR)

[0088] The original SPLICEN0Rdesign is shown in FIG. 2A and was done by increasing the length of the linker and introducing protease substrates for orthogonal proteases (Thrombin, MMP3cd, and TEVp), the linker increased in length by 1.4x, something that is not possible with FRET probes because FRET probes have very stringent spacing requirements and introducing 1-3 amino acids can substantially reduce FRET efficiency, this initial design introduces 14 amino acids in order to include all three cut sites. We incubated SPLICEN0Rin several buffers with and without proteases of interest to test for a change in fluorescence and found that it responds to all three proteases as expected. The introduction of logic did not change the background fluorescence or the dynamic range (which was 20.3x for thrombin, 18.4x for TEVp, and 20.4x for MMP3cd) again outperforming FRET peptides which again are in the 3-8 range for synthetic peptides and 1.5-4x for protein-based FRET. Next, we designed a NOR gate that can respond to both of SARs-CoV-2 protease's 3CLpro(main protease) and PLpr° (papain-like protease). We wanted to evaluate the inhibition of these proteases using SPLICEC0VIDusing two known inhibitors, GRL-0496 for 3CLpro and HY-17542 for PLpro. To date, used this probe to quantify the inhibiton of 3CLpro using GRL-0496. The result of the inhibition assay shown in FIG. 2D uses what is called a Lineweaver-burke analysis, where we vary the [SPLICEC0VID] at 400nM, 300nM, 200nM, lOOnM, 50nM both with and without GRL-0496 at 40nM. we then took the initial slope (first 20min) of all these curves. With this data we plotted the absolute value of the inverse rate of change in fluorescence against the inverse of SPLICE concentration resulting in two highly linear curves. The only non-traditional step in the above process was taking the absolute value of slope, this was done because with FRET peptides the slope is positive and with SPLICE it is negative, otherwise it was a traditional Lineweaver-Burk analysis. With the two lines (with and without inhibitor) we then calculated the x-intercept to obtain the kmand km' which we used to calculate the Ki (=35.7nM) I then used the Cheng-Prussof equation to convert Ki to IC50 which is a value that has been calculated for GRL-0496 inhibiting 3CLprowith this, we calculated the ICso to equal 35.7-35.9nM which matches the reported ICso of 30 ± lOnM found in literature (compound 10 in Ghosh et al., Bioorg Med Chem Lett. 2008 Aug 28;18(20):5684-5688. doi: 10.1016 / j.bmcl.2008.08.082).

[0089] FRET cannot have the increased linker length required to incorporate the NOR logic outlined above. This logic allows the use of one probe to detect several proteases of interest, additionally it was shown that despite SPLICE having an inverse response compared to FRET (abolishing fluorescence instead of increasing it) that the platform can clearly be used for the same quantification applications that FRET is often used for. Meaning it has the same application capabilities AND has novel logic capabilities with improved dynamic range.

[0090] C. SPLICENAND(NAND) and SPLICENAND-°R(NAND-OR)

[0091] Initial NAND-gate consisted of just 1 leucine zipper pair but the MMP3cd still had a signal change, because of this we introduced a second pair of zippers which reduced the background by 10% (from 1.64x down to 1.47x (shown in FIG. 3B and 3C)). For a change in fluorescence for SPLICENANDboth proteases of interest are required to be present, we used MMP3cd and TEVp because we had both proteases already, but because of the demonstrated modularity any protease with a known cut site could be used by simply swapping the recognition substrate. FIG. 3B shows that there is no change in fluorescence when neither protease or TEVp is present, and there is a negligible change in fluorescence (1.47x) when only MMP3cd is present, this small signal change is dwarfed by the ~30x change that results from detecting the specific proteolytic environment its designed to detect). This NAND-gated response has a lot of potential in screening for co-elevated protease environments associated with cancer / chronic wounds / etc to screen out unrelated single protease upregulation events. This logic gating behavior is depicted in traditional logic gate format in FIG. 3C. Next we wanted to design the probe so that it can respond to more than one proteolytic environment without sacrificing any specificity, the change in design is shown in FIG. 3D and the result is shown in FIG. 3E where you can see there is a change in fluorescence if there is (MMP3cd + thrombin) or (MMP3cd + TEVp) but no large change in fluorescence with (TEVp + Thrombin) or only one of the three proteases. This SPLICENAND 0Rnot only clearly worked, but it also demonstrates the modularity of this SPLICE platform. Table 1. Sequences

[0092] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED:

1. A recombinant protein comprising a first linker, a first protein fragment on the N-terminus of the first linker, and a second protein fragment on the C-terminus of the first linker, wherein the first protein fragment consists of an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 1, the second protein fragment consists of an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of GDSYWVFVKR.V (SEQ ID NO: 2), GDSYWVFVKR (SEQ ID NO: 3), GDSYWVFVK (SEQ ID NO: 4), and GDSYWVFV (SEQ ID NO: 5), the first linker comprises a first cleavage site, and the first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when the first cleavage site is not cleaved than the fluorescence generated when the first cleavage site is cleaved .

2. The recombinant protein of claim 1, wherein the first linker further comprises a second cleavage site, wherein the second cleavage site is different from the first cleavage site, and wherein the first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when the first linker cleavage site and the second cleavage site are not cleaved than the fluorescence generated when at least one of the first cleavage site and the second cleavage site is cleaved.

3. The recombinant protein of claim 2, wherein the first linker further comprises a third cleavage site for a third protease, wherein the third cleavage site is different from the first cleavage site and the second cleavage site, and wherein the first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when the first cleavage site, the second cleavage site, and the third cleavage site are not cleaved than the fluorescence generated when at least one of the first cleavage site, the second cleavage site and the third cleavage site is cleaved .

4. The recombinant protein of claim 1, further comprising a first N-terminal leucine zipper on the N-terminus of the first protein fragment, a first C-terminal leucine zipper on the C-terminus of the second protein fragment, a second N-terminal leucine zipper on the N-terminus of the first N-terminal leucine zipper, a second C-terminal leucine zipper on the C-terminus of the first C-terminal leucine zipper, and a second linker between the first N-terminal leucine zipper and the first protein fragment or between the first C-terminal leucine zipper and the second protein fragment, whereinthe first N-terminal leucine zipper and the first C-terminus leucine zipper form a first pair leucine zipper complex, the second N-terminal leucine zipper and the second C-terminus leucine zipper form a second pair leucine zipper complex, the second linker comprises a second cleavage site, the second cleavage site is different from the first cleavage site, and the first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when at least one of the first cleavage site and the second cleavage site is not cleaved than the fluorescence generated when the first linker cleavage site and the second cleavage site are cleaved.

5. The recombinant protein of claim 1, further comprising a first N-terminal leucine zipper on the N-terminus of the first protein fragment, a first C-terminal leucine zipper on the C-terminus of the second protein fragment, a second N-terminal leucine zipper on the N-terminus of the first N-terminal leucine zipper, a second C-terminal leucine zipper on the C-terminus of the first C-terminal leucine zipper, a second linker between the first N-terminal leucine zipper and the first protein fragment, and a third linker between the first C-terminal leucine zipper and the second protein fragment, wherein the first N-terminal leucine zipper and the first C-terminal leucine zipper form a first pair leucine zipper complex, the second N-terminal leucine zipper and the second C-terminal leucine zipper form a second pair leucine zipper complex, the second linker comprises a second cleavage site, the second cleavage site is different from the first cleavage site, the third linker comprises a third cleavage site, the third cleavage site is different from the first cleavage site and the second cleavage site, and the first protein fragment and the second protein fragment generate fluorescence in the presence of a fluorogen at least 10 times greater when at least one of the second cleavage site and the third cleavage site is not cleaved than the fluorescence generated when the first linker cleavage site is cleaved and at least one of the second cleavage site and the third cleavage site is cleaved.

6. The recombinant protein of any one of claims 1-5, wherein the second protein fragment consists of an amino acid sequence at least 90% identical to the amino acid sequence of GDSYWVFVKR. (SEQ ID NO: 3).

7. The recombinant protein of any one of claims 1-5, wherein the fluorogen is selected from the group consisting of 4-hydroxy-3-methylbenzylidene rhodamine (HMBR), 4-hydroxy-3,5-dimethoxybenzylidene rhodamine (HBR-3,5DOM), and (4- hydroxy-3,5-dimethoxy-phenyl)allylidene-rhodanine (HPAR-3,5DOM).

8. The recombinant protein of any one of claims 1-5, wherein the first linker comprises (G4S)n, wherein n is 1 to 3.

9. The recombinant protein of any one of claims 1-5, wherein the first cleavage site consists of an amino acid selected from the group consisting of RPFSMIMG (SEQ ID NO: 7), ENLYFQS (SEQ ID NO: 7), LVPRGS (SEQ ID NO: 8), GSTSAVLQSGFRK (SEQ ID NO: 9), ATIMMQRG (SEQ ID NO: 10), PLGLAG (SEQ ID NO: 11), AENK (SEQ ID NO: 12), DEVD (SEQ ID NO: 13), IETD (SEQ ID NO: 14), and ADVIPARRRG (SEQ ID NO: 15).

10. A method for detecting one or more target proteases in a target sample, comprising :(a) incubating the recombinant protein of claim 1 in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the recombinant protein by a control protease capable of cleaving the first cleavage site, whereby a first test sample is obtained;(b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen;(c) incubating the recombinant protein in the medium in the absence of the test sample under the conditions in step (a), whereby a second test sample is obtained; and(d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site.

11. A method for detecting one or more target proteases in a target sample, comprising :(a) incubating the recombinant protein of claim 2 in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the recombinant protein by a first control protease capable of cleaving the first cleavage site and cleavage of the second cleavage site in the recombinant protein by a second control protease capable of cleaving the second cleavage site, whereby a first test sample is obtained;(b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen;(c) incubating the recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and(d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site and / or the second cleavage site.

12. A method for detecting one or more target proteases in a target sample, comprising :(a) incubating the recombinant protein of claim 3 in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the recombinant protein by a first control protease capable of cleaving the first cleavage site, cleavage of the second cleavage site in the recombinant protein by a second control protease capable of cleaving the second cleavage site, and cleavage of the third cleavage site in the recombinant protein by a third control protease capable of cleaving the third cleavage site, whereby a first test sample is obtained;(b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen;(c) incubating the recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and(d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site, the second cleavage site, and / or the third cleavage site.

13. A method for detecting one or more target proteases in a target sample, comprising :(a) incubating the recombinant protein of claim 4 in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the recombinant protein by a first control protease capable of cleaving the first cleavage site and cleavage of the second cleavage site in the recombinant protein by a second control protease capable of cleaving the second cleavage site, whereby a first test sample is obtained;(b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen;(c) incubating the recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and(d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site and the second cleavage site.

14. A method for detecting one or more target proteases in a target sample, comprising :(a) incubating the recombinant protein of claim 5 in a medium in the presence of a target sample under conditions permitting cleavage of the first cleavage site in the recombinant protein by a first control protease capable of cleaving the first cleavage site, cleavage of the second cleavage site in the recombinant protein by a second control protease capable of cleaving the second cleavage site, and cleavage of the third cleavage site in the recombinant protein by a third control protease capable of cleaving the third cleavage site, whereby a first test sample is obtained;(b) making a first fluorescence measurement of the first test sample in the presence of the fluorogen;(c) incubating the recombinant protein in the medium in the absence of the target sample under the conditions in step (a), whereby a second test sample is obtained; and(d) making a second fluorescence measurement of the second test sample in the presence of the fluorogen, wherein an at least 10-fold increase from the first fluorescence measurement to the second fluorescence measurement indicates that the target sample comprises one or more target proteases capable of cleaving the first cleavage site, and at least one of the second cleavage site and the third cleavage site.

15. The method of any one of claims 10-14, further comprising quantifying the one or more target proteases.

16. A recombinant polynucleotide encoding the recombinant protein of any one of claims 1-5.

17. A recombinant host cell expressing the recombinant protein of any one of claims 1-5.

18. A recombinant host cell comprising the recombinant polynucleotide encoding the recombinant protein of any one of claims 1-5.

19. A method for producing the recombinant protein of any one of claims 1- 5, comprising:(a) growing a recombinant host cell, wherein the host cell comprises a recombinant polynucleotide encoding the recombinant protein of any one of claims 1-9; and(b) expressing the recombinant protein by the recombinant host cell.

20. The method of claim 19, further comprising purifying the recombinant protein from the recombinant host cell.

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