Systems and methods for the detection and quantification of double-stranded RNA

Abstract

This specification provides compositions comprising double-stranded RNA (dsRNA) binding domains bound to components of a complementary system. When a pair of dsRNA-binding domains bind to dsRNA, a detectable complex of complementary components is formed, allowing for the detection / quantification of dsRNA.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 506,502, filed on June 6, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List The text of the computer-readable sequence listing filed with this specification (titled "PRMG_41970_601_SequenceListing.xml", created on June 5, 2024, file size 3,253,101 bytes) is incorporated herein by reference in its entirety.

[0003] This specification provides compositions comprising double-stranded RNA (dsRNA) binding domains bound to components of a complementary system. When a pair of dsRNA-binding domains bind to dsRNA, a detectable complex of complementary components is formed, allowing for the detection / quantification of dsRNA. [Background technology]

[0004] Double-stranded RNA (dsRNA) is a by-product and contaminant of in vitro mRNA transcription that occurs during the manufacture of mRNA-based therapeutics (vaccines, gene therapies), for example. The FDA has issued a guideline titled "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)," which focuses particularly on the development of RNA-based therapeutics. This guideline provides recommendations for the development and characterization of RNA-based therapeutics, including considerations regarding the presence of dsRNA. Current guidelines stipulate that dsRNA must be measured for all in vitro transcription RNA products. Currently, there are no established limits on the amount of dsRNA in RNA pharmaceuticals.

[0005] Currently, quantification of dsRNA (i.e., quantification specific to dsRNA, rather than ssRNA or DNA) can be performed by two widely accepted methods: ELISA and dot blotting. Enzyme-linked immunosorbent assay (ELISA) can be used to quantify dsRNA using a specific antibody that recognizes dsRNA. Sensitivity can be an issue, as commercially available ELISA kits exhibit a sensitivity of 2–5 ng / ml. Accepted ELISA Ab clones are J2, K1, and K2. However, despite being the current "gold standard" for dsRNA detection, these clones have problems recognized in situ. For example, clone J2 shows preferred binding to the terminals of the dsRNA oligo, as well as to the internal binding site of A2N9A3N9A2 (adenine is presented on one side of the helix; see Bonin et al., RNA, 2000). Therefore, this antibody is sequence-independent and does not show accurate quantification of dsRNA. Another clone, K1, shows changes in binding kinetics when testing dsRNA from different sources. Therefore, these clones do not provide true quantification of dsRNA. Dot blots can be used for dsRNA detection, but they are not as sensitive or quantitative as ELISA and rely on the same flawed antibody clones.

[0006] What is needed is a quantitative, highly sensitive (e.g., detection limit <1 ng / ml), specific, rapid (<2 hour assay time), and easy-to-use (add-mix-measure) assay for quantifying dsRNA. [Overview of the Initiative]

[0007] This specification provides compositions comprising double-stranded RNA (dsRNA) binding domains bound to components of a complementary system. When a pair of dsRNA-binding domains bind to dsRNA, a detectable complex of complementary components is formed, allowing for the detection / quantification of dsRNA.

[0008] In some embodiments, a double-stranded RNA (dsRNA) detection system is provided, which comprises (a) a first fusion of (i) a first dsRNA-binding domain and (ii) a first component of a detectable complex, and (b) a second fusion of (i) a second dsRNA-binding domain and (ii) a second component of a detectable complex. In some embodiments, when the first dsRNA-binding domain and the second dsRNA-binding domain bind to dsRNA, the first and second components of the detectable complex associate to form a detectable complex. In some embodiments, the first and second components of the detectable complex exhibit low affinity for each other if there is no promotion via binding of the first and second dsRNA-binding domains to dsRNA.

[0009] In some embodiments, the first dsRNA-binding domain and the second dsRNA-binding domain contain different amino acid sequences. In some embodiments, the first dsRNA-binding domain and the second dsRNA-binding domain contain the same amino acid sequence. In some embodiments, the dsRNA-binding domain contains a dsRNA-binding motif having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence similarity to SEQ ID NO: 3062 and / or SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain contains a dsRNA-binding motif having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity to SEQ ID NO: 3062 and / or SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain contains the dsRNA-binding motif of SEQ ID NO: 3062. In some embodiments, the dsRNA-binding domain includes the dsRNA-binding motif of SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain includes the dsRNA-binding motif of SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain contains at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to sequence number 3061. In some embodiments, the dsRNA-binding domain contains at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to sequence number 3061.In some embodiments, the dsRNA-binding domain includes SEQ ID NO: 3061.

[0010] In some embodiments, the detectable complex can generate a detectable signal. In some embodiments, the amount of signal generated by the detectable complex can be correlated with the amount of dsRNA in a sample having the system. In some embodiments, the signal includes one or more of fluorescence, luminescence, enzyme activity, and ligand binding. In some embodiments, the first and second components of the detectable complex are protein fragments capable of generating a detectable signal, and the detectable complex can generate a detectable signal when the first and second components of the detectable complex associate.

[0011] In some embodiments, the detectable signal is fluorescence. In some embodiments, the first and second components of the detectable complex contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between them) sequence identity with the first and second fragments of the fluorescent protein. In some embodiments, the fluorescent protein is selected from yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, dansyl chloride, phycocyanin, and phycoerythrin.

[0012] In some embodiments, the detectable signal is enzyme activity. In some embodiments, the first and second components of the detectable complex contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between them) sequence identity with the first and second fragments of the enzyme. In some embodiments, the enzyme is selected from beta-lactamase, dihydrofolate reductase (DHFR), adhesion plaque kinase (FAK), Gal4, and horseradish peroxidase. In some embodiments, the detectable signal is luminescence in the presence of a substrate. In some embodiments, the first and second components of the detectable complex contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between them) sequence identity with the first and second fragments of the luciferase. In some embodiments, the luciferase is selected from Oplophorus luciferase, firefly luciferase, click beetle luciferase, Renilla luciferase, sea firefly luciferase, aequorin luminescent protein, and oberin luminescent protein. In some embodiments, the first and second components of the detectable complex together contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between thereof) sequence identity with SEQ ID NO: 3041. In some embodiments, the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3042, and the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3050. In some embodiments, the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3043, and the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3051.In some embodiments, the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3044, and the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3052. In some embodiments, the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3045, and the first component of the detectable complex includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3053. In some embodiments, the system further includes a substrate.

[0013] In some embodiments, the detectable signal is ligand binding. In some embodiments, the detectable complex is a modified dehalogenase complex, where the first and second components of the modified dehalogenase complex contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between them) sequence identity with the first and second fragments of the modified dehalogenase. In some embodiments, the modified dehalogenase includes SEQ ID NO: 1. In some embodiments, the system further includes a haloalkyl ligand for the modified dehalogenase. In some embodiments, the haloalkyl ligand includes an R-linker-AX, where R is the detectable moiety, X is a halogen, and AX is a substrate for the dehalogenase enzyme. In some embodiments, R is a fluorophore.

[0014] In some embodiments, a double-stranded RNA (dsRNA) detection system is provided herein, comprising (a) a first fusion of (i) a PKR-derived dsRNA-binding domain sequence and (ii) a peptide component of a bioluminescent complex, and (b) a second fusion of (i) a PKR-derived dsRNA-binding domain sequence and (ii) a polypeptide component of a bioluminescent complex, wherein when the PKR-derived dsRNA-binding domain sequence binds to dsRNA, a luminescent complex is formed by the structural complementarity of the peptide component and the polypeptide component, and the luminescence signal produced by the luminescent complex in the presence of dsRNA and a substrate for the luminescent complex is enhanced compared to the luminescence signal produced in the absence of dsRNA. In some embodiments, the system further comprises a substrate for the luminescent complex. In some embodiments, the substrate for the luminescent complex is an imidazopyrazine phosphophore. In some embodiments, the imidazopyrazine phosphophore is coelenterazine or flimazine. In some embodiments, the system further comprises dsRNA. In some embodiments, the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA-binding domain includes at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3061. In some embodiments, the dsRNA-binding domain contains at least 70% sequence identity with SEQ ID NO: 3061 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these). In some embodiments, the dsRNA-binding domain contains SEQ ID NO: 3061.In some embodiments, the peptide component has at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3038, and / or the polypeptide component has at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3037. In some embodiments, the peptide component has at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3038, and / or the polypeptide component has at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3037. In some embodiments, the peptide component includes SEQ ID NO: 3038, and the polypeptide includes SEQ ID NO: 3037.

[0015] In some embodiments, methods for detecting dsRNA in a sample are provided herein, comprising contacting the sample with a system described herein and detecting a signal from a detectable complex, wherein the amount of the detected signal correlates with the amount of dsRNA in the sample. In some embodiments, the sample comprises a single-stranded RNA-based therapeutic agent. In some embodiments, the sample further comprises dsRNA (e.g., a contaminant). [Brief explanation of the drawing]

[0016] [Figure 1] A cartoon illustrating the binding of protein kinase R (PKR) to dsRNA and the dimerization of two PKR molecules. [Figure 2] A cartoon illustrating an exemplary embodiment of the technology, in which the two components of the luminescence complex (LgBiT and SmBiT) are provided as a fusion with the dsRNA-binding domain of PKR. Binding of this dsRNA-binding domain to dsRNA results in the formation of the active luminescence complex. [Figure 3]Graph showing the increase in the luminescence signal with the increase in dsRNA in the presence of the exemplary system of FIG. 2. [Figure 4] Graph showing the increase in the fluorescence signal with the increase in dsRNA in the presence of an exemplary system containing a fusion of a split GFP construct and the dsRNA binding domain of PKR (PKR-LgGFP and PKR-SmGFP). **DETAILED DESCRIPTION OF THE INVENTION**

[0017] Definitions Any methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the embodiments described herein, but some preferred methods, compositions, devices, and materials are described herein. However, prior to describing these substances and methods, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, because these can be modified by routine experimentation and optimization. Also, it should be understood that the technical terms used herein are for the sole purpose of describing specific versions or embodiments and are not intended to limit the scope of the embodiments described herein.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the specification of the present invention, including definitions, will prevail. Accordingly, the following definitions apply with respect to the embodiments described herein.

[0019] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those of ordinary skill in the art, and the like.

[0020] As used herein, the term "and / or" includes any combination of the listed items, including any one of the listed items. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which should be considered as being individually described by the statement "A, B, and / or C".

[0021] As used herein, the term “comprise” and its linguistic variations mean that the described features, elements, method steps, etc. exist, and that the existence of additional features, elements, method steps, etc. is not excluded. Conversely, the term “consisting of” and its linguistic variations mean that the described features, elements, method steps, etc. exist, and that any undescribed features, elements, method steps, etc. are excluded, except for impurities that are usually present. The expression “consisting essentially of” means the described features, elements, method steps, etc., and any additional features, elements, method steps, etc. that do not substantially affect the fundamental nature of the composition, system, or method. Many embodiments herein are described using the non-restrictive term “comprise.” Such embodiments include a number of restrictive "consisting of" and / or "essentially consisting of" embodiments, which may be claimed or described using alternative language such as "consisting of" and / or "essentially consisting of."

[0022] As used herein, the term “substantially” means that the described properties, parameters, and / or values ​​do not need to be exactly achieved, and deviations or variations, including, for example, tolerances, measurement errors, measurement accuracy limits, and other factors known to those skilled in the art, may occur in an amount that does not preclude the effect that the feature is intended to provide. A substantially non-existent property or feature (e.g., substantially non-fluorescent) may be a property or feature that is within the range of noise, lower than the background, below the detection capability of the assay used, or is only a fraction of a significant property (e.g., fluorescence intensity of an active fluorophore) (e.g., less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.00001%, less than 0.000001%, less than 0.0000001%).

[0023] When used herein, the phrase "corresponding to" refers to the relative position of an amino acid residue or amino acid segment with respect to the sequence being referred to, and does not necessarily refer to the specific identity of an amino acid at that position. For example, "a peptide corresponding to positions 36-48 of SEQ ID NO: 1" may include less than 100% sequence identity (e.g., more than 70% sequence identity) with positions 36-48 of SEQ ID NO: 1, but in the context of the composition or system described, the peptide is relevant to those positions.

[0024] As used herein, the term “system” refers to a set of components (e.g., a device, a composition, etc.) used for a particular purpose. For example, two distinct biomolecules, whether present in the same composition or not, may constitute a system if they are both useful for a common purpose.

[0025] As used herein, the term “complementary” refers to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that they can hybridize with each other, dimerize, or otherwise form complexes. For example, “complementary peptides and polypeptides” can assemble to form complexes. Complementary elements may require assistance (facilitation) to form complexes (e.g., from interacting elements), such as arranging elements in appropriate conformations for complementarity, arranging elements in appropriate proximity for complementarity, coexisting complementary elements, reducing interaction energy for complementarity, or compensating for insufficient affinity between them.

[0026] As used herein, the term “complex” refers to an assembly or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with one another. In one embodiment, “contact,” or more specifically “direct contact,” means that two or more molecules are in close proximity, and as a result, non-covalent attractive interactions, such as van der Waals forces, hydrogen bonds, ionic interactions, and hydrophobic interactions, occupy the interactions between the molecules. In such embodiments, the complex of molecules (e.g., peptides, polypeptides, etc.) is formed under assay conditions in which the complex is thermodynamically favorable (e.g., compared to the non-aggregated or non-complexed states of its component molecules). As used herein, the term “complex” refers to an assembly of two or more molecules (e.g., peptides, polypeptides, etc.) unless otherwise specified. The molecules that come together to form a complex are referred herein as “components,” or their linguistic variations thereof.

[0027] As used herein, the term "low affinity" describes intermolecular interactions between two or more entities that are too weak to result in significant complex formation between entities, unless the concentration is considerably higher than physiological or assay conditions (e.g., 2x, 5x, 10x, 100x, 1000x or more) or facilitated by the formation of a second complex of attached elements (e.g., interacting elements).

[0028] As used herein, the term “high affinity” describes an intermolecular interaction between two or more (e.g., three) entities that is strong enough to cause detectable complex formation without the need for the formation of a second complex of the attached elements (e.g., interacting elements) under physiological or assay conditions.

[0029] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, and unless otherwise specified, all of them are D and L stereoisomers if their structure allows for stereoisomerization.

[0030] The term "proteinogenesis amino acids" refers to 20 amino acids encoded in human genetic information, including alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). Selenocysteine ​​and pyrrolicin may also be considered proteinogenesis amino acids.

[0031] The term "non-proteinogenic amino acid" refers to an amino acid that is not naturally encoded or found in the genetic information of any organism and is not biosynthetically incorporated into proteins during translation. Non-proteinogenic amino acids may be "non-natural amino acids" (amino acids that do not exist in nature) or "naturally occurring non-proteinogenic amino acids" (e.g., norvaline, ornithine, homocysteine). Non-proteinogenic amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, and allo- Examples of non-proteinogenic compounds include, but are not limited to, hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-alkylglycines containing N-methylglycine, N-methylisoleucine, N-alkylpentylglycines containing N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine ("Norleu"), octylglycine, ornithine, pentylglycine, pipecolic acid, thioproline, homolysine, and homoarginine. Non-proteinogenic compounds also include the D-amino acid form of any amino acid as defined herein, as well as non-alpha amino acid forms of any amino acid as defined herein (such as beta-amino acids, gamma-amino acids, and delta-amino acids), all of which are within the scope of this specification and may be included in peptides as defined herein.

[0032] The term "amino acid analog" refers to an amino acid (e.g., natural or unnatural amino acids, proteolytic or non-proteolytic amino acids) in which one or more of its C-terminal carboxyl group, N-terminal amino group, and side-chain bioactive groups are reversibly or irreversibly chemically blocked or otherwise modified with another bioactive group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid, N-ethylglycine is an amino acid analog of glycine, and alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, and S-(carboxymethyl)-cysteine ​​sulfone.

[0033] As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to polymer compounds of two or more amino acids linked together via a peptide amide bond (--C(O)NH--) in their main chains. The term “peptide” usually refers to a short amino acid polymer (e.g., a chain with fewer than 30 amino acids), while “polypeptide” usually refers to a longer amino acid polymer (e.g., a chain with 30 or more amino acids).

[0034] As used herein, “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For the purposes of this disclosure, each of the following eight groups includes amino acids that are conservative substitutions with each other: 1) Alanine (A) and glycine (G); 2) Aspartic acid (D) and glutamic acid (E); 3) Asparagine (N) and glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), and valine (V); 6) Phenylalanine (F), tyrosine (Y), and tryptophan (W); 7) Serine (S) and threonine (T); and 8) Cysteine ​​(C) and methionine (M).

[0035] Based on the properties of their common side chains, amino acid residues can be classified into the following classes, for example: polar positive charge (or basic) (e.g., histidine (H), lysine (K), and arginine (R)); polar negative charge (or acidic) (e.g., aspartic acid (D), glutamic acid (E)); polar neutral (e.g., serine (S), threonine (T), asparagine (N), glutamine (Q)); nonpolar aliphatic (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); nonpolar aromatic (e.g., phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0036] In some embodiments, unless otherwise specified, conserved or semi-conserved amino acid substitutions may also include non-natural amino acid residues having similar chemical properties to natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include, but are not limited to, peptide mimes and other inverted or reversed amino acid moieties. Embodiments described herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0037] Non-conservative substitutions can involve the exchange of a member of one class for a member of another class.

[0038] As used herein, the term “sequence identity” refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same monomer subunit sequence composition. The term “sequence similarity” refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical properties and can be classified into families such as acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-charged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The "sequence identity percentage" (or "sequence similarity percentage") is calculated by (1) comparing two optimally aligned sequences across a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, or a specified window); (2) determining the number of positions containing identical (similar) monomers (e.g., positions where the same amino acid is found in both sequences, or positions where similar amino acids are found in both sequences) to obtain the number of matching positions; (3) dividing the number of matching positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, or a specified window); and (4) multiplying the result by 100 to obtain the sequence identity percentage or sequence similarity percentage. For example, if peptides A and B are both 20 amino acid long and all amino acids except one position are identical, peptides A and B have 95% sequence identity. If amino acids at non-identical positions share the same biophysical properties (e.g., both are acidic), peptides A and B will have 100% sequence similarity.As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some of the amino acids in peptide C, then peptide C and peptide D have 70% sequence identity, but peptide D has 93.3% sequence identity with respect to the optimal comparison window of peptide C. For the purposes of calculating “sequence identity percentage” (or “sequence similarity percentage”) as used herein, any gaps in the aligned sequences are treated as mismatches at that position.

[0039] Any peptide / polypeptide described herein as having a specific percentage of sequence identity or similarity (e.g., at least 70%) to a reference sequence number may be described as having the maximum number of substitutions (or terminal deletions) relative to its reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) to sequence number Z (e.g., 100 amino acids) may have up to X substitutions (e.g., 10) compared to sequence number Z, and therefore may also be described as having X or fewer substitutions (e.g., 10) compared to sequence number Z.

[0040] As used herein, the term “sample” is used in its broadest sense. In a sense, this term means specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and include liquids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Samples may also refer to cell lysates, or purified forms of enzymes, peptides, and / or polypeptides as described herein. Cell lysates may include cells lysed with a lysant, or lysates such as rabbit reticulocytes or wheat germ lysates. Samples may also include cell-free expression systems. Environmental samples include environmental substances such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples to which the present invention is applicable. Pharmaceutical samples include any therapeutic agent that is tested for the presence and / or concentration of dsRNA, such as RNA-based therapeutic agents.

[0041] As used herein, the terms “fusion,” “fusion polypeptide,” and “fusion protein” refer to a chimeric protein comprising a first protein or polypeptide of interest, which is bound to a second different peptide, polypeptide, or protein (e.g., an interacting element).

[0042] As used herein, the terms “conjugated” and “conjugation” refer to the covalent bond between two molecular entities (e.g., after synthesis and / or during synthetic manufacturing). Chemical (e.g., “chemically” bonded) or enzymatic attachment of a peptide or small molecule tag to a protein or small molecule is an example of conjugation.

[0043] As used herein, the term "modified dehalogenase" refers to a halogenase variant (artificial variant) having one or more mutations that prevent the release of substrate from the protein after halogen removal, resulting in a covalent bond between the substrate and the modified dehalogenase. Modified dehalogenases are not classical enzymes because they do not release substrate and therefore cannot undergo metabolic turnover. The HALOTAG system (Promega) is a commercially available modified dehalogenase and substrate system.

[0044] As used herein, the term "bioluminescence" refers to the generation and emission of light by chemical reactions catalyzed or enabled by enzymes, proteins, protein complexes, or other biomolecules (e.g., bioluminescent complexes). In a typical embodiment, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted into an unstable form by the bioluminescent entity, after which the substrate emits light.

[0045] As used herein, the term “Oplophorus luciferase” (“OgLuc”) refers to a luminescent polypeptide having remarkable sequence identity, structural conservation, and / or functional activity with the luciferase produced by or derived from the deep-sea shrimp Oplophorus gracilirostris. In particular, the OgLuc polypeptide refers to a luminescent polypeptide having remarkable sequence identity, structural conservation, and / or functional activity with SEQ ID NO: 3034 (NanoLuc), which contains the mature 19kDa subunit of the Oplophorus luciferase protein complex (e.g., without a signal sequence), e.g., 10 β-chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10), and which emits light using a substrate such as coelenterazine or a coelenterazine derivative.

[0046] As used herein, the term “β9-like peptide” refers to a peptide (or peptide tag) that exhibits remarkable sequence identity, structural conservation, and / or functional activity with respect to the β (beta) 9 chain of the OgLuc polypeptide. In particular, a β9-like peptide is a peptide that structurally complements an OgLuc polypeptide lacking a β9 chain, resulting in enhanced complex luminescence compared to the OgLuc polypeptide in the absence of the β9-like peptide. Other “βX-like peptides” may be similarly named (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

[0047] As used herein, the term “β10-like peptide” refers to a peptide (or peptide tag) that exhibits remarkable sequence identity, structural conservation, and / or functional activity with the β (beta) 10 chain of the OgLuc polypeptide. In particular, a β10-like peptide is a peptide that can structurally complement an OgLuc polypeptide lacking a β10 chain, resulting in enhanced complex luminescence compared to the OgLuc polypeptide in the absence of the β10-like peptide. Other “βX-like peptides” may be similarly named (e.g., β1-like, β2-like, β3-like, β4-like, β5-like, β6-like, β7-like, β8-like, β9-like).

[0048] When used herein, "β 1~8 The term "similar polypeptide" refers to polypeptides that have sequence and structural similarity to the β (beta) chains 1-8 of the OgLuc polypeptide, but lack β (beta) chains 9 and 10. Y~Z "Like polypeptides" can be named similarly (e.g., β 1~4 Polypeptides, β 2~8 Polypeptides, β 5~10 (e.g., various polypeptides).

[0049] As used herein, the term "NANOLUC" refers to an artificial luciferase or bioluminescent polypeptide commercially manufactured by Promega Corporation.

[0050] As used herein, the term "LgBiT" refers to a polypeptide corresponding to the β-polypeptide corresponding to SEQ ID NO: 3037, which is useful, for example, in binary complementarity for forming a bioluminescent complex. 1~9 It refers to a polypeptide corresponding to the β-like polypeptide.

[0051] As used herein, the term "SmBiT" refers to a peptide corresponding to the β-like peptide corresponding to SEQ ID NO: 3039, which is useful, for example, in binary complementarity for forming a bioluminescent complex, but has a low affinity for LgBiT (e.g., requires promotion for complex formation). 10 It refers to a peptide corresponding to the β-like peptide.

[0052] As used herein, the term "HiBiT" refers to a peptide corresponding to the β-like peptide corresponding to SEQ ID NO: 3038, which is useful, for example, in binary complementarity for forming a bioluminescent complex and has a high affinity for LgBiT (e.g., does not require promotion for complex formation). 10 It refers to a peptide corresponding to the β-like peptide. An exemplary HiBiT peptide corresponds to SEQ ID NO: 3038.

[0053] As used herein, the term "LgTrip" refers to a polypeptide corresponding to the β-like polypeptide. 1-8 An exemplary LgTrip corresponds to SEQ ID NO: 3045 and is useful, for example, in ternary complementarity with β9-like and β-like peptides for forming a bioluminescent complex or in binary complementarity with β-like dipeptides for forming a bioluminescent complex. 10 It is useful in ternary complementarity with β-like peptides for forming a bioluminescent complex or in binary complementarity with β-like dipeptides for forming a bioluminescent complex. 9~10 It is useful in ternary complementarity with β-like peptides for forming a bioluminescent complex or in binary complementarity with β-like dipeptides for forming a bioluminescent complex.

[0054] As used herein, the term "SmTrip10" refers to a peptide corresponding to the β-like peptide that is useful, for example, in three-molecule complementarity for forming a bioluminescent complex. 10 It refers to a peptide corresponding to the β-like peptide.

[0055] As used herein, the term "SmTrip9" refers to a peptide corresponding to the β9-like peptide that is useful, for example, in three-molecule complementarity for forming a bioluminescent complex.

[0056] As used herein, the term “sp” refers to a polypeptide that has been split into two fragments at an internal site of the original polypeptide. The fragments of an sp polypeptide may reconstitute the activity of the original polypeptide if they are structurally complementary and can form an active complex.

[0057] Detailed explanation This specification provides compositions comprising double-stranded RNA (dsRNA) binding domains bound to components of a complementary system. When a pair of dsRNA-binding domains bind to dsRNA, a detectable complex of complementary components is formed, allowing for the detection / quantification of dsRNA.

[0058] Protein kinase R (PKR, Uniprot number P19525) is an intracellular dsRNA sensor containing a dsRNA-binding domain (Figure 1, blue and red). Dimerization of PKR on dsRNA activates the kinase domain (Figure 1, green), initiating a downstream signaling pathway that activates inflammatory pathways and halts protein translation. PKR binds to dsRNA in a manner independent of the target sequence and requiring only that the dsRNA be at least 30 base pairs long (no upper limit). PKR binds to the phosphate backbone of dsRNA and does not interact with nucleotide bases (see, for example, Nanduri et al., EMBO J, 1998, which is incorporated entirely by reference).

[0059] Polypeptide constructs are provided herein that utilize the dsRNA-binding function of PKR and use it to facilitate the formation of a detectable complex in the presence of dsRNA. In some embodiments, pairs of components of the detectable complex are fused to a PKR dsRNA-binding domain (or a variant thereof). When the dsRNA-binding domain binds to dsRNA (but not in the absence of dsRNA), the components of the detectable complex interact to form the detectable complex and generate a corresponding signal. The presence and / or amount of dsRNA in a sample (e.g., environmental samples, biological samples, pharmaceutical samples, etc.) can be detected / quantified based on the signal generated by the system herein.

[0060] During the development of the embodiments described herein, experiments were conducted to test exemplary systems within the scope of this specification. First, when the commercially available LgBiT and SmBiT components of the NanoBiT system (Promega Corp, Madison, WI) were fused to the dsRNA-binding domain of PKR (see, for example, Figure 2), luminescence was detected when the dsRNA concentration was increased in the presence of a flimazine substrate (Figure 3). Second, when split GFP (LgGFP and SmGFP) were fused to the dsRNA-binding domain of PKR, fluorescence was detected when the dsRNA concentration was increased (Figure 4). These experiments demonstrate that the dsRNA-binding domain of PKR can be used to promote structural complementarity between complementary peptides / polypeptides, forming detectable complexes and signals in a dsRNA concentration-dependent manner. This specification provides a system for binding any preferred component of a detectable complex to the dsRNA-binding domain, and the use of such a system for detecting dsRNA in a sample.

[0061] I.PKR dsRNA binding domain Interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase R (PKR) is an IFN-stimulating gene (integrated as a whole by reference to Gale, M. Jr., and Katze, MG (1998). Pharmacol. Ther. 78, 29-46. Peters, GA, Hartmann, R., Qin, J., and Sen, GC (2001). Mol. Cell. Biol. 21, 1908-1920.; Pendel, A., and Sadler, A. (2011). J. Interferon Cytokine Res. 31, 59-70.), and functions as a pathogen recognition receptor by recognizing dsRNA, a typical byproduct of viral infection, and inducing IFN (integrated as a whole by reference to reference to Gilfoy, FD, and Mason, PW (2007). J. Virol. 81, 11148-11158.). PKR consists of two functionally distinct domains: an N-terminal regulatory domain and a C-terminal catalytic kinase domain. The regulatory domain contains two dsRNA-binding motifs, and dsRNA binding induces dimerization of PKR, enabling exposure of the catalytic site, autophosphorylation, and kinase activation (integrated as a whole by reference: Wu, S., and Kaufman, RJ (1997). J. Biol. Chem. 272, 1291-1296.; Nanduri, et al. (2000). EMBO J. 19, 5567-5574.; Dar et al. (2005). Cell 122, 887-900; Dey et al. (2005). Cell 122, 901-913.).Activated PKR catalyzes the phosphorylation of the regulatory α subunit of eukaryotic translation initiation factor 2 (eIF2α; Meurs, et al. (1992). J. Virol. 66, 5805-5814.; Clemens, MJ, and Elia, A. (1997). J. Interferon Cytokine Res. 17, 503-524.; incorporated as a whole by reference), which in turn inhibits the initiation of mRNA translation, resulting in a complete halt to both cellular and viral protein synthesis, and potentially leading to apoptosis in response to viral infection (Balachandran, et al. (1998). EMBO J. 17, 6888-6902.; incorporated as a whole by reference).

[0062] In some embodiments, compositions comprising a fusion of a dsRNA-binding domain and a component of a detectable complex are provided herein. In some embodiments, the dsRNA-binding domain is capable of binding to dsRNA in a sequence-independent manner. In some embodiments, the dsRNA-binding domain does not preferentially bind to the ends of dsRNA or to specific RNA structures. In some embodiments, the dsRNA-binding domain binds to dsRNA but not to single-stranded RNA (ssRNA) or DNA (double-stranded or single-stranded). In some embodiments, the dsRNA-binding domain binds to ssRNA and / or DNA at a level low enough that any signal generated from such binding is within the background of the assay herein.

[0063] In some embodiments, the dsRNA-binding domain of the fusion or system herein corresponds to the PKR dsRNA-binding domain. In some embodiments, the dsRNA-binding domain of the fusion herein contains at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3061 (PKR dsRNA-binding domain). In some embodiments, the dsRNA-binding domain of the fusion herein contains at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence similarity with SEQ ID NO: 3061 (PKR dsRNA-binding domain).

[0064] In some embodiments, the dsRNA-binding domain of the fusion or system herein includes one or more parts corresponding to a portion of the PKR dsRNA-binding domain. In some embodiments, the dsRNA-binding domain of the fusion or system herein includes a portion corresponding to the first dsRNA-binding motif of the PKR dsRNA-binding domain (SEQ ID NO: 3062). In some embodiments, the whole or a portion of the dsRNA-binding domain of the fusion herein includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3062 (PKR dsRNA-binding motif 1). In some embodiments, the whole or a portion of the dsRNA-binding domain of the fusion herein includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence similarity with SEQ ID NO: 3062 (PKR dsRNA-binding motif 1). In some embodiments, the dsRNA-binding domain of the fusion or system herein includes a portion corresponding to the second dsRNA-binding motif of the PKR dsRNA-binding domain (SEQ ID NO: 3063). In some embodiments, all or part of the dsRNA-binding domain of the fusion herein includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence identity with SEQ ID NO: 3063 (PKR dsRNA-binding motif 2). In some embodiments, all or part of the dsRNA-binding domain of the fusion herein includes at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence similarity with SEQ ID NO: 3063 (PKR dsRNA-binding motif 2).In some embodiments, the dsRNA-binding domain of the fusion as described herein includes a first segment having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in between) with SEQ ID NO: 3062 (PKR dsRNA-binding motif 1), and a second segment having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in between) with SEQ ID NO: 3063 (PKR dsRNA-binding motif 2). In some embodiments, the dsRNA-binding domain of the fusion as described herein includes a first segment having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3062 (PKR dsRNA-binding motif 1), and a second segment having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) to SEQ ID NO: 3063 (PKR dsRNA-binding motif 2). In some embodiments, the segments corresponding to PKR dsRNA-binding motif 1 and PKR dsRNA-binding motif 2 (or their variants) are fused by a linker of 1 to 100 amino acids in length. In some embodiments, the linker includes a native linker present in the PKR dsRNA-binding domain.

[0065] II. Detectable Complexes In some embodiments, the system herein includes a pair of fusions, namely, a first fusion containing (1) a dsRNA-binding domain bound to a first component of a detectable complex, and (2) a second fusion containing a dsRNA-binding domain bound to a second component of a detectable complex. In some embodiments, the components of the complex have sufficiently low affinity for each other that facilitation is required to form the complex and produce a detectable signal (or the signal produced during facilitation is significantly louder than the unfacilitated signal, which is present in the background). In some embodiments, binding of the dsRNA-binding domain bound to the components of the detectable complex facilitates the formation of the detectable complex and the generation of a detectable signal.

[0066] In some embodiments, the complex may generate any suitable signal that enables the detection of the binding of the complementary fusion pair to dsRNA as described herein. For example, the detectable complex may, upon complementarity, generate any suitable signal such as fluorescence, luminescence, enzyme activity, or ligand binding. In some embodiments, the component corresponds to a fragment (or variant of such fragment) of an enzyme or other protein that can generate a detectable signal. In such embodiments, the component forms a complex analogous to the enzyme or other protein, facilitated by the binding of the dsRNA-binding domain to dsRNA, and the corresponding signal can be detected. In other embodiments, the component may form a detectable complex that does not correspond to an existing enzyme or complex.

[0067] In some embodiments, the detectable complex corresponds to a protein split into two fragments that can interact noncovalently to form a complex exhibiting the functional activity of the protein (e.g., incorporated as a whole by reference to Shekhawat & Ghosh. Curr Opin Chem Biol. 2011 Dec;15(6):789-797). In some embodiments, a suitable protein is one that has detectable activity that is reversibly eliminated by fragmenting the protein into two separate components, the activity of which is reconstituted when the fragments are noncovalently rejoined by the binding of dsRNA-binding domains fused to each fragment to dsRNA. Any protein that can be split into fragments that can associate (e.g., facilitated) to reconstitute the activity of the parent protein may be used in embodiments herein.Examples of such proteins include beta-lactamase (Galarneau et al. Nat. Biotech. 2002; 20(6): 619-622; incorporated as a whole by reference), ubiquitin (Johnsson & Varshavsky A. Proc Natl Acad Sci USA. 1994; 91: 10340-13044; incorporated as a whole by reference), dihydrofolate reductase (DHFR) (Pelletier et al. Proc Natl Acad Sci USA. 1998; 95: 12141-12146; incorporated as a whole by reference), adhesion plaque kinase (FAK), Gal4, GFP, and variants (Ghosh et al. J. Am. Chem. Soc. 2000; 122: 5658-5659; Hu & Kerppola.Nat.Biotechnol.2003;21:539-1545; incorporated as a whole by reference) (e.g., EGFP), horseradish peroxidase, infrared fluorescent protein, various luciferases (Remy & Michnick.Nat.Meth.2006;3(12):977-979.;Paulmurugan & Gambhir.Ana.Chem.2003;759(7):1584-1589; incorporated as a whole by reference) (e.g., recombinase-enhancing bimolecule luciferase, Gaussia princeps luciferase, firefly (Luker et al. Proc Natl Acad Sci USA.2004;101:12288-122893; incorporated as a whole by reference), tobacco ecchi disease virus (TEV) protease (Wehr et al. Examples include al.Nat.Meth.2006;3(12):985-993 (integrated as a whole by reference), thymidine kinase (Massoud et al.Nat.Med.2010;16(8):921-927; integrated as a whole by reference), and colismic acid mutase (Muller et al.Prot.Sci.2010;19(5):1000-1010; integrated as a whole by reference).In some embodiments, the components of the complex used in the embodiments herein are variants of protein fragments (e.g., less than 100% sequence identity) that can exhibit similar activity to the protein upon complex formation. Other examples of detectable complex components are listed below.

[0068] A.HaloTag In some embodiments, fusions of a dsRNA-binding domain and a complementary peptide / polypeptide pair that can interact with each other (e.g., facilitated by the binding of the dsRNA-binding domain to a dsRNA) are provided to form an active modified dehalogenase complex that can form a covalent bond with a haloalkane ligand. In some embodiments, a first fusion is provided comprising a first complementary peptide or polypeptide fragment of a modified dehalogenase, and a second fusion is provided comprising a second complementary peptide or polypeptide fragment of a modified dehalogenase, which, upon interaction (e.g., facilitated by the binding of the dsRNA-binding domain to a dsRNA), form an active modified dehalogenase complex that can form a covalent bond with a haloalkane ligand. In some embodiments, the complementary peptide / polypeptide is a fragment of a split mutant dehalogenase.

[0069] In some embodiments, the peptide / polypeptide component capable of forming a modified dehalogenase complex is derived from a commercially available HALOTAG protein (Promega) and / or a fragment of a split mutant dehalogenase, such as the mutant dehalogenase disclosed in U.S. Publication Application 2006 / 0024808 (the disclosures of this U.S. Publication Application are incorporated herein by reference).

[0070] In some embodiments, the components of the compositions, systems, and methods herein are split-modified dehalogenases. In some embodiments, a first fragment of the mutant dehalogenase is fused to a first dsRNA-binding domain, and a second fragment of the mutant dehalogenase is fused to a second dsRNA-binding domain. In some embodiments, at least one of the mutant dehalogenase fragments has a substitution that forms a covalent bond with a haloalkane ligand when present in a fully-length modified dehalogenase (or the corresponding complex). In some embodiments, the first and second fragments of the mutant dehalogenase can interact (e.g., facilitated by the binding of the bound dsRNA-binding domain to dsRNA) to form an active modified dehalogenase complex.

[0071] HALOTAG is a 297-residue self-labeled polypeptide (33 kDa) derived from the bacterial hydrolase (dehalogenase) enzyme, modified to covalently bind to the haloalkane portion of the enzyme's ligand. The HALOTAG ligand can bind to solid surfaces (e.g., beads) or functional groups (e.g., fluorophores), and the HALOTAG polypeptide can fuse to various target proteins, thus allowing the covalent attachment of target proteins to solid surfaces or functional groups. The HALOTAG polypeptide is a genetically modified dehalogenase with an active site that specifically binds to the chloroalkane linker of the haloalkane ligand, enhancing and increasing the ligand binding rate (see Pries et al. The Journal of Biological Chemistry. 270(18):10405-11; incorporated as a whole). The reaction that forms a bond between the protein tag and the chloroalkane linker is rapid under physiological conditions and essentially irreversible (Waugh DS (June 2005). Trends in Biotechnology. 23(6):316-20; incorporated as a whole by reference). In the native hydrolase enzyme, nucleophilic attack of the chloroalkane-reactive linker leads to the substitution of the halogen with an amino acid residue, resulting in the formation of a covalent alkyl enzyme intermediate. This intermediate is then hydrolyzed by amino acid residues within the wild-type hydrolase (Chen et al. (February 2005). Current Opinion in Biotechnology. 16(1):35-40; incorporated as a whole by reference). This regenerates the enzyme after the reaction. However, in the modified haloalkane dehalogenase HALOTAG, the reaction intermediate cannot be hydrolyzed due to the enzyme mutation and therefore cannot proceed through the second reaction. As a result, the intermediate persists as a stable covalent adduct without the associated reverse reaction (it is incorporated as a whole, as referenced by Marks et al. (August 2006) Nature Methods. 3(8):591-6).Various HALOTAG ligands, functional groups, fusions, assays, modifications, and uses are described in U.S. Patents 8,748,148, 9,593,316, 10,246,690, 8,742,086, 9,873,866, 10,604,745, U.S. Patent Application 2009 / 0253131, U.S. Patent Application 2010 / 0273186, 20130337539, U.S. Patent Application 2012 / 0258470, U.S. Patent Application 2012 / 0252048, U.S. Patent Application 2011 / 0201024, and U.S. Patent Application 2014 / 0322794. These are incorporated as a whole by reference, respectively.

[0072] In some embodiments, the modified dehalogenase fragments, complementary peptides, complementary polypeptides, etc. described herein constitute a HALOTAG-based complementarity system. In some embodiments, the modified dehalogenase fragments, complementary peptides, complementary polypeptides, etc. described herein correspond to sequences within the HALOTAG protein (e.g., sequence identity, sequence similarity, 3D structure, etc.). In some embodiments, the modified dehalogenase complexes described herein, comprising two or more peptide or polypeptide components, correspond to the HALOTAG protein and can be bound to a halcoalkyl ligand in a similar manner.

[0073] In some embodiments, extensive experiments have been conducted to demonstrate the feasibility of generating fragments of HALOTAG (and its variants) that can interact to form modified dehalogenase complexes capable of binding to haloalkyl ligands, as described in U.S. Provisional Application No. 63 / 338,323 and PCT Application No. PCT / US23 / 20959 (both incorporated herein by whole reference), and of optimizing variants of HALOTAG fragments for desired properties. Embodiments are not limited to HALOTAG sequences, as described herein. In some embodiments, what is provided herein is a split-modified dehalogenase (e.g., as a fusion with a dsRNA-binding domain) with a sequence different from HALOTAG (SEQ ID NO: 1).

[0074] In some embodiments, compositions and systems are provided that include components of a split-modified dehalogenase, such as a split-halotag ("spHT") or a variant thereof (e.g., as a fusion with a dsRNA-binding domain). In some embodiments, the systems and compositions herein include spHT peptides and polypeptides (e.g., as part of the fusions described herein).

[0075] In some embodiments, compositions (e.g., fusions) and systems (e.g., multiple fusions with suitable ligands and substrates) are provided, comprising polypeptides, peptides, fragments, and combinations thereof derived from the modified dehalogenase sequence of Sequence ID No. 1 (HALOTAG) below. MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETF QAFRTTDVGRKLIIDQNVFIEGTLMGVVRPLTEEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG.

[0076] In some embodiments, the spHT peptides and polypeptides herein (e.g., as part of a fusion with a dsRNA-binding domain) contain at least 70% sequence identity with a portion of SEQ ID NO: 1 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In some embodiments, the spHT peptides and polypeptides (e.g., as part of a fusion with a dsRNA-binding domain) contain 100% sequence identity with all or a portion of SEQ ID NO: 1. In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of a fusion with a dsRNA-binding domain) have at least 70% sequence similarity to all or part of SEQ ID NO: 1 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). In some embodiments, the spHT peptides and polypeptides described herein (e.g., as part of a fusion with a dsRNA-binding domain) have 100% sequence similarity to all or part of SEQ ID NO: 1.

[0077] In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains A at the position corresponding to position 2 of SEQ ID NO: 1. In other embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains S at the position corresponding to position 2 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains V at the position corresponding to position 47 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains T at the position corresponding to position 58 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains G at the position corresponding to position 78 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains F at the position corresponding to position 88 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains M at the position corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) contains F at the position corresponding to position 128 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) contains T at the position corresponding to position 155 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) contains K at the position corresponding to position 160 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) contains V at the position corresponding to position 167 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains T at the position corresponding to position 172 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains M at the position corresponding to position 175 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains G at the position corresponding to position 176 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains N at the position corresponding to position 195 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains E at the position corresponding to position 224 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains D at the position corresponding to position 227 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as defined herein (e.g., as part of a fusion with a dsRNA-binding domain) contains K at the position corresponding to position 257 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as defined herein (e.g., as part of a fusion with a dsRNA-binding domain) contains A at the position corresponding to position 264 of SEQ ID NO: 1. In some embodiments, the peptide or polypeptide as defined herein contains N at the position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as defined herein (e.g., as part of a fusion with a dsRNA-binding domain) contains L at the position corresponding to position 273 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as defined herein (e.g., as part of a fusion with a dsRNA-binding domain) contains S at the position corresponding to position 291 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains T at the position corresponding to position 292 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains E at the position corresponding to position 294 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains I at the position corresponding to position 295 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains S at the position corresponding to position 296 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as specified herein (e.g., as part of a fusion with a dsRNA-binding domain) contains G at the position corresponding to position 297 of SEQ ID NO: 1.

[0078] In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain S at the position corresponding to position 2 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain L at the position corresponding to position 47 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain S at the position corresponding to position 58 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain D at the position corresponding to position 78 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain Y at the position corresponding to position 88 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain L at the position corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain C at the position corresponding to position 128 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain A at the position corresponding to position 155 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain E at the position corresponding to position 160 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain A at the position corresponding to position 167 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain A at the position corresponding to position 172 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain K at the position corresponding to position 175 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain C at the position corresponding to position 176 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain K at the position corresponding to position 195 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain A at the position corresponding to position 224 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain N at the position corresponding to position 227 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain E at the position corresponding to position 257 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain T at the position corresponding to position 264 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain H at the position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain Y at the position corresponding to position 273 of SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain P at the position corresponding to position 291 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain A at the position corresponding to position 292 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at the position corresponding to position 294 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at the position corresponding to position 295 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at the position corresponding to position 296 of SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide as used herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at the position corresponding to position 297 of SEQ ID NO: 1.

[0079] In some embodiments, the sp dehalogenase comprises two peptide and / or polypeptide components that together have at least 70% sequence similarity or identity with all or part of Sequence ID No. 1 (e.g., more than 70% sequence similarity or identity, more than 75% sequence similarity or identity, more than 80% sequence similarity or identity, more than 85% sequence similarity or identity, more than 90% sequence similarity or identity, more than 95% sequence similarity or identity, more than 96% sequence similarity or identity, more than 97% sequence similarity or identity, more than 98% sequence similarity or identity, more than 99% sequence similarity or identity). For example, the first peptide / polypeptide component of the sp polypeptide corresponds to the first portion of SEQ ID NO: 1 (e.g., with at least 70% sequence similarity or sequence identity with the first portion), and the second peptide / polypeptide component of the sp polypeptide corresponds to the second portion of SEQ ID NO: 1 (e.g., with at least 70% sequence similarity or sequence identity with the second portion). In some embodiments, the sp dehalogenase (e.g., spHT) comprises two fragments that together have 100% sequence similarity or sequence identity with all or part of SEQ ID NO: 1. For example, the first fragment of the sp polypeptide has 100% sequence similarity or sequence identity with the first portion of SEQ ID NO: 1, and the second fragment of the sp polypeptide has 100% sequence similarity or sequence identity with the second portion of SEQ ID NO: 1.

[0080] In some embodiments, the sp dehalogenase (e.g., as part of a fusion with a dsRNA-binding domain) includes an sp site. The sp site is an internal position in the parent sequence that defines the C-terminus of the first component or fragment of the sp dehalogenase and the N-terminus of the second component or fragment. For example, if a theoretical 100-amino acid polypeptide is split at an sp site between residues 57 and 58 of the parent polypeptide (referred to herein as the sp site at 57), the first component polypeptide may correspond to positions 1-57, and the second component polypeptide may correspond to positions 58-100. In some embodiments herein, the sp site in SEQ ID NO: 1 may occur at any position from position 5 to position 290 of SEQ ID NO: 1. In some embodiments, SEQ ID NOs: 2-577 are exemplary components of an spHT polypeptide having 100% sequence identity with SEQ ID NO: 1. In some embodiments, an active SPHT complex is formed between two fragments collectively containing amino acids corresponding to each position in SEQ ID NO: 1. For example, a polypeptide having the sequence of SEQ ID NO: 26 and a peptide having the sequence of SEQ ID NO: 27 both contain the amino acids corresponding to each position in SEQ ID NO: 1. Any pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NOs: 2-577, both containing the amino acids corresponding to each position in SEQ ID NO: 1 (with or without positional deletion or duplication), are used in the embodiments herein. In some embodiments, the spHT dehalogenase comprises any of the following pairs of fragments (e.g., fused to a dsRNA-binding domain): SEQ ID NOs: 2 and 3, 4 and 5, 6 and 7, 8 and 9, 10 and 11, 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 30 and 31, 32 and 33, 34 and 35, 36 and 37, 38 and 39, 40 and 41, 42 and 43, 44 and 45, 46 and 4 7, 48 and 49, 50 and 51, 52 and 53, 54 and 55, 56 and 57, 58 and 59, 60 and 61, 62 and 63, 64 and 65, 66 and 67, 68 and 69, 70 and 71, 72 and 73, 74 and 75, 76 and 77, 78 and 79, 80 and 81, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91,92 and 93, 94 and 95, 96 and 97, 98 and 99, 100 and 101, 102 and 103, 104 and 105, 106 and 107, 108 and 109, 110 and 111, 112 and 113, 114 and 115, 116 and 117, 118 and 119, 120 and 121, 121, 122 and 123, 124 and 125, 126 and 127, 128 and 129, 130 and 131, 132 and 133, 134 and 135, 136 and 137, 138 and 139, 140 and 141, 142 and 143, 144 and 145, 146 and 147, 148 and 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 172 and 173, 174 and 175, 176 and 177, 178 and 179, 180 and 181, 182 and 183, 184 and 185, 186 and 187, 188 and 189, 190 and 191, 192 and 193, 194 and 195, 196 and 197, 198 and 199, 200 and 201, 202 and 203, 204 and 205, 206 and 207, 208 and 209, 190 and 211, 212 and 2 13, 214 and 215, 216 and 217, 218 and 219, 220 and 221, 222 and 223, 224 and 225, 226 and 227, 228 and 229, 300 and 301, 302 and 303, 304 and 305, 306 and 307, 308 and 309, 310 and 311, 312 and 313, 314 and 315, 316 and 317, 318 and 319, 320 and 321, 322 and 323, 324 and 325, 326 and 327, 328 and 329, 330 and 331, 332 and 333, 334 and 335, 336 and 337, 338 and 339, 340 and 341, 342 and 343, 344 and 345, 346 and 347, 348 and 349, 350 and 351, 352 and 353, 354 and 355, 356 and 357, 358 and 359, 360 and 361, 362 and 363, 364 and 365, 366 and 367, 368 and 369, 370 and 371, 372 and 373, 374 and 375, 376 and 377, 378 and 379, 380 and 381, 382 and 383, 384 and 385, 386 and 387, 388 and 389, 390 and 391, 392 and 393,394 and 395, 396 and 397, 398 and 399, 400 and 401, 402 and 403, 404 and 405, 406 and 407, 408 and 409, 410 and 411, 412 and 413, 414 and 415, 416 and 417, 418 and 419, 420 and 421, 422 and 423, 424 and 425, 426 and 427, 428 and 429, 430 and 431, 432 and 433, 434 and 435, 436 and 437, 438 and 439, 4 40 and 441, 442 and 443, 444 and 445, 446 and 447, 448 and 449, 450 and 451, 452 and 453, 454 and 455, 456 and 457, 458 and 459, 460 and 461, 462 and 463, 464 and 465, 466 and 467, 468 and 469, 470 and 471, 472 and 473, 474 and 475, 476 and 477, 478 and 479, 480 and 481, 482 and 483, 484 and 485, 48 6 and 487, 488 and 489, 490 and 491, 492 and 493, 494 and 495, 496 and 497, 498 and 499, 500 and 501, 502 and 503, 504 and 505, 506 and 507, 508 and 509, 510 and 511, 512 and 513, 514 and 515, 516 and 517, 518 and 519, 520 and 521, 522 and 523, 524 and 525, 526 and 527, 528 and 529, 530 and 531, 532 and 533, 534 and 535, 536 and 537, 538 and 539, 540 and 541, 542 and 543, 544 and 545, 546 and 547, 548 and 549, 550 and 551, 552 and 553, 554 and 555, 556 and 557, 558 and 559, 560 and 561, 562 and 563, 564 and 565, 566 and 567, 568 and 569, 570 and 571, 572 and 573, 574 and 575, and 576 and 577.

[0081] In some embodiments, spHT comprises a pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NOs: 2-577, where both contain amino acids corresponding to each position in SEQ ID NO: 1, but with a deletion of up to 40 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or in between) at one or both C-terminuses or N-terminuses of the fragment. For example, the pair corresponding to SEQ ID NOs: 7 and 28 both correspond to the positions in SEQ ID NO: 1 but with a deletion of 11 residues. In some embodiments, any pair of SEQ ID NOs: 2-577 that both correspond to the sequence of SEQ ID NO: 1 but with a deletion of up to 40 amino acids is within the range of spHT as used herein. In some embodiments, the deletion is adjacent to the splitting site. In some embodiments, the deletion corresponds to the N-terminus or C-terminus of SEQ ID NO: 1.

[0082] In some embodiments, spHT comprises a pair of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NOs: 2-577, where the pair both contain amino acids corresponding to each position in SEQ ID NO: 1, but with up to 40 amino acid lengths overlapping at one or both C-terminuses or N-terminuses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or in between). For example, the pair corresponding to SEQ ID NOs: 6 and 29 both correspond to the positions in SEQ ID NO: 1 but with an 11-residue overlap. In some embodiments, any pair of SEQ ID NOs: 2-577 that both correspond to the sequence of SEQ ID NO: 1 but with up to 40 amino acid overlaps is within the scope of spHT as defined herein. In some embodiments, the overlap is adjacent to the splitting site. In some embodiments, the overlap corresponds to the N-terminus or C-terminus of SEQ ID NO: 1.

[0083] For example, a fragment utilizing any sp site corresponding to a position between positions 5 and 290 of sequence number 1 is readily conceivable and falls within the scope of this specification.

[0084] In some embodiments, spHT is located at position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 313, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 48, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 1 79, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 21 0, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 ,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,It includes sp portions corresponding to 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290.

[0085] In some embodiments, spHT comprises sp portions corresponding to positions between 5 and 13, 36 and 51, 63 and 72, 84 and 92, 104 and 130, 142 and 148, 160 and 174, 186 and 189, 311 and 313, 221 and 229, or 269 and 290 of sequence number 1.

[0086] In some embodiments, the spHT peptides and polypeptides described herein (e.g., fused to a dsRNA-binding domain) include one or more substitutions or deletions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, or more) for one of SEQ ID NOs. 2 to 557. In some embodiments, sp peptides and polypeptides (e.g., fused to a dsRNA-binding domain) are provided that have 70% to 100% sequence identity with one of sequence numbers 2 to 557 (e.g., more than 70% sequence identity, more than 75% sequence identity, more than 80% sequence identity, more than 85% sequence identity, more than 90% sequence identity, more than 95% sequence identity, more than 96% sequence identity, more than 97% sequence identity, more than 98% sequence identity, more than 99% sequence identity). In some embodiments, sp peptides and polypeptides (e.g., fused to a dsRNA-binding domain) are provided that have 70% to 100% sequence similarity (e.g., over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, over 96%, over 97%, over 98%, over 99%) to one of SEQ ID NOs: 2 to 557.

[0087] In some embodiments, pairs of sp peptides and / or polypeptides (e.g., fused to a dsRNA-binding domain) are provided that can form an active sp dehalogenase complex (active spHT complex). In some embodiments, such pairs have at least 70% sequence identity or similarity with two of SEQ ID NOs: 2-557, and together contain residues corresponding to 100% of the positions in SEQ ID NO: 1, and allow up to 40 deletions or duplications at the C-terminus or N-terminus of the peptide / polypeptide.

[0088] In some embodiments, the first fragment of the spHT complementary pair (e.g., fused to the dsRNA binding domain) is located at positions 1 through 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 6 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 313, 104, 105, 106 ,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 31 0, 311, 312, 313, 314, 315, 316, 317, 318, 319, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 ,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,This corresponds to 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290.

[0089] In some embodiments, the second fragment of the spHT complementary pair (e.g., fused to the dsRNA binding domain) is located at positions 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 313, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 1 40, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 1 71, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 310, 311, 31 2, 313, 314, 315, 316, 317, 318, 319, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233 ,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,Positions 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290 correspond to position 294.

[0090] In some embodiments, the overlap portion of the spHT complementary pair is 1 to 40 amino acid lengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or a range between those).

[0091] In some embodiments, the deletion portion of the spHT complementary pair is 1 to 40 amino acid lengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 31, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or a range between those).

[0092] The exemplary spHT fragment sequences of SEQ ID NOs. 2-577 contain 100% sequence identity with a portion of SEQ ID NO. 1, and these sequences contain no portions that do not match SEQ ID NO. 1 with 100% sequence identity. However, as described herein, spHT peptides and polypeptides may have less than 100% sequence identity with SEQ ID NO. 1 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, but less than 100%). Accordingly, peptides and polypeptides having less than 100% sequence identity with one of SEQ ID NOs: 2-577 (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, but less than 100% sequence identity) are provided herein and are found to be used in complementary pairs and complexes herein.

[0093] In some embodiments, the spHT complementary pair as used herein comprises a peptide corresponding to SEQ ID NO: 578 and a polypeptide corresponding to SEQ ID NO: 1188. SEQ ID NOs. 578 and 1188 are fragments of SEQ ID NO: 1 and have 100% sequence identity with a portion of SEQ ID NO: 1. In some embodiments, the spHT complementary pair comprises a peptide having 100% sequence identity with SEQ ID NO: 578, and such peptides are referred to herein as "SmHT". In some embodiments, the spHT complementary pair comprises a polypeptide having 100% sequence identity with SEQ ID NO: 1188, and such polypeptides are referred to herein as "LgHT". Extensive experiments were conducted to analyze variants of SmHT and LgHT. SEQ ID NOs. 579–1187 correspond to peptide variants in which at least one position and up to all positions of SEQ ID NO: 588 are substituted. Each peptide of SEQ ID NOs. 578–1187 was synthesized and tested for various properties, including the ability to form an active complex with a complementary LgHT variant polypeptide. Sequence IDs 1189-3033 correspond to polypeptide variants with one or more substitutions compared to Sequence ID 1188. Each polypeptide of Sequence ID 1188-3033 was synthesized and tested for various properties, including its ability to form an active complex with complementary SmHT variant peptides.

[0094] In some embodiments, the Specified Reference Instruments (SEQ ID NO: 578-1187) provides an SmHT peptide or SmHT variant peptide (e.g., fused to a dsRNA-binding domain) having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range between these) sequence similarity (e.g., conserved or semi-conserved similarity). In some embodiments, the peptide (e.g., fused to a dsRNA-binding domain) corresponds to SmHT (SEQ ID NO: 578), but has one or more substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or a range between these) of one or more SEQ ID NOs from SEQ ID NOs from 588-1187. In some embodiments, the SmHT variant (e.g., fused to a dsRNA-binding domain) has 1 to 8 non-conservative substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or in between) for one of sequence numbers 578 to 1187.

[0095] In some embodiments, this specification provides SmHT peptides or SmHT variant peptides (e.g., those fused to a dsRNA-binding domain), which SmHT peptides or SmHT variant peptides are X1X2X3X4X5(F / W / Y / M / H)X7(F / W / Y / D / R)X9X 10 X 11 (F / W / Y / M / H / R)(V / I / L / M / A / C)X 14 (V / I / L / A / C / MI / L / F / W)X 16 X 17 (Sequence ID 3034), and / or X1X2X3X4X5(F / W / Y)X7(F / W / Y)X9X 10 X 11 (F / W / Y)(V / I / L / M)X 14 (V / I / L)X 16 X 17 (Sequence ID 3035) Includes, Each X is any amino acid (for example, a protein synthesis amino acid).

[0096] In some embodiments, the Specified Information Provides LgHT polypeptides or LgHT variant polypeptides (e.g., fused to a dsRNA-binding domain) having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in between) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NOs. 1188-3033. In some embodiments, the polypeptide (e.g., one within a fusion as described herein, or one as a standalone reporter or tag) corresponds to LgHT (SEQ ID NO: 1188), but has one or more substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more, or in between) of one or more SEQ ID NOs. 1188. In some embodiments, an LgHT variant (e.g., fused to a dsRNA-binding domain) has at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or in between) with one of sequence numbers 1188-3033.

[0097] B.NanoLuc In some embodiments, fusions of a dsRNA-binding domain and a complementary pair of peptides / polypeptides that can interact with each other (e.g., facilitated by the binding of the dsRNA-binding domain to a dsRNA) are provided herein to form a luminescence complex that can interact with a luminescent substrate to produce luminescence. In some embodiments, the luminescence complex can produce significantly enhanced luminescence upon interaction with the luminescent substrate compared to the case of either complementary peptide / polypeptide alone, or the case of the luminescent substrate without the other peptide / polypeptide of the complementary pair.

[0098] In some embodiments, a first fusion is provided comprising a first complementary peptide or polypeptide fragment of luciferase, and a second fusion is provided comprising a second complementary peptide or polypeptide fragment of luciferase, and upon interaction (e.g., facilitated by the binding of a dsRNA-binding domain to dsRNA), the complementary peptide(s) / polypeptide(s)

[0099] In some embodiments, the peptide / polypeptide component capable of forming a luciferase complex is a fragment of a split luciferase, such as one derived from commercially available NANOLUC luciferase (Promega), including but not limited to NANOBIT(Promega), SMBIT peptide(Promega), and LGBIT polypeptide(Promega), which can promote the formation of a luminescent complex. In some embodiments, the peptide / polypeptide component capable of forming a luciferase complex is any of the peptide and polypeptide components described in U.S. Patent No. 9,797,889 and / or U.S. Application No. 16 / 439,565 (incorporated as a whole by reference).

[0100] In some embodiments, compositions (e.g., fusion polypeptides) and systems (e.g., multiple complementary fusion polypeptides, substrates, etc.) are provided herein, which comprise peptide / polypeptide fragments that can interact (e.g., facilitated by the binding of dsRNA-binding domains fused to the compositions and systems to dsRNA) to form an active luminescence complex capable of generating luminescence using a suitable substrate.

[0101] In some embodiments, fusion polypeptides and systems thereof (e.g., multiple complementary fusion polypeptides, substrates, etc.) are provided herein, which include a dsRNA-binding domain fused to a complementary peptide / polypeptide fragment that can interact (e.g., facilitated by the binding of a dsRNA-binding domain fused to the fusion polypeptide and system to a dsRNA) to form an active bioluminescent complex capable of generating luminescence upon interaction with a suitable luminescent substrate. In some embodiments, a first fusion is provided comprising a peptide / polypeptide fragment of a luminescent protein, and a second fusion is provided comprising a complementary peptide / polypeptide fragment of a luminescent protein, which, upon interaction (e.g., facilitated by the binding of a dsRNA-binding domain fused to the fragment to a dsRNA), form an active bioluminescent complex capable of generating luminescence upon interaction with a suitable luminescent substrate. In some embodiments, the complementary peptide(s) / polypeptide(s) is a fragment of a split luminescent protein (e.g., luciferase).

[0102] In some embodiments, what is provided herein is a pair of fusions of a dsRNA-binding domain and components of a binary complementary system capable of forming a luminescence complex. In other embodiments, a tertiary or multicomplementary system (e.g., three or more components) is utilized in the fusions of the dsRNA-binding domain and the system herein. For example, a fusion of a dsRNA-binding domain with three or more components of a system may be provided. Alternatively, the system may comprise two fusions of the dsRNA-binding domain with components of a luminescence complex and one or more additional components of the luminescence complex as isolated components.

[0103] Natural Oplophorus gracilirostris luciferase (OgLuc) and commercially available NANOLUC luciferase (Promega Corporation) each contain polypeptides with 10 beta chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10). U.S. Patent No. 9,797,889 (which is incorporated herein by reference in its entirety) describes the development and use of complementary systems containing β1-β9-like polypeptides and β10-like peptides (the specific OgLuc / NANOLUC-based polypeptide and peptide sequences in U.S. Patent No. 9,797,889 differ from the corresponding sequences in NANOLUC and wild-type natural OgLuc). Similarly, U.S. Patent Application No. 16 / 439,565 (which is incorporated herein by whole reference) describes the development and use of complementary systems comprising two or more OgLuc / NANOLUC peptides and / or polypeptides (the specific OgLuc / NANOLUC-based polypeptide and peptide sequences in U.S. Patent No. 16 / 439,565 differ from the corresponding sequences in NANOLUC and wild-type natural OgLuc).

[0104] In some embodiments, the fusion with the dsRNA-binding domain provided herein is a peptide component of a two-component bioluminescent complex having more than 40% (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, 100%) sequence identity with SEQ ID NO: 3036, and when the peptide component of the two-component bioluminescent complex comes into contact with a polypeptide comprising SEQ ID NO: 3037 in the presence of a substrate of the bioluminescent complex (e.g., facilitated by the binding of the dsRNA-binding domain to dsRNA), a detectable bioluminescent signal is generated (e.g., stronger luminescence than the components of the complex in the presence of the substrate). In some embodiments, the peptide (e.g., within the dsRNA-binding domain fusion) has less than 100% sequence identity with SEQ ID NO: 3036. In some embodiments, when the peptide component of a binary bioluminescent complex comes into contact with the polypeptide component of a binary bioluminescent complex having more than 40% sequence identity with SEQ ID NO: 3037 (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, 100%) (e.g., facilitated by the binding of a dsRNA-binding domain to dsRNA), a detectable bioluminescent signal is generated. In certain embodiments, when the peptide associates with a polypeptide containing or consisting of SEQ ID NO: 3037, a detectable bioluminescent signal is generated or significantly increased. In preferred embodiments, the peptide exhibits one or more changes (e.g., enhancement) in properties compared to the peptide of SEQ ID NO: 3038 or 3039, the properties being selected from affinity to the polypeptide comprising SEQ ID NO: 3037, expression, intracellular solubility, intracellular stability, and bioluminescent activity when conjugated with the polypeptide comprising SEQ ID NO: 3037 (e.g., within the context of the dsRNA-binding domain fusion as described herein).

[0105] Exemplary sequences of peptide components of the binary bioluminescent complex used in the embodiments herein (for example, as part of a fusion with a dsRNA-binding domain fusion) are described, for example, in U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference). The peptide components of the binary bioluminescent complex (for example, in a fusion herein) are not limited to these sequences, but in some embodiments, the peptide components of the binary bioluminescent complex herein may be selected from the amino acid sequences SEQ ID NOs. 3-438 and 2162-2365 described in U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference).

[0106] In some embodiments, the Spectrum provides a peptide component of a two-component bioluminescent complex (e.g., as a fusion with a dsRNA-binding domain) having more than 40% sequence identity with SEQ ID NO: 3038 (e.g., greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, 100%), wherein when the peptide component of the two-component bioluminescent complex comes into contact with a polypeptide comprising SEQ ID NO: 3037 (e.g., fused to a dsRNA-binding domain) in the presence of a substrate of the bioluminescent complex (e.g., facilitated by the binding of the dsRNA-binding domain fused to the dsRNA), a detectable bioluminescent signal is generated (e.g., stronger luminescence than the components of the complex in the presence of the substrate). In some embodiments, the peptide has less than 100% sequence identity with SEQ ID NO: 3036. In some embodiments, when the peptide component of a binary bioluminescent complex (e.g., fused to a dsRNA-binding domain) comes into contact with the polypeptide component of a binary bioluminescent complex (e.g., fused to a dsRNA-binding domain) having more than 40% sequence identity with SEQ ID NO: 3037 (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, 100%), a detectable bioluminescent signal is generated. In certain embodiments, when the peptide associates with a polypeptide containing or consisting of SEQ ID NO: 3037, a detectable bioluminescent signal is generated or significantly increased.

[0107] Exemplary sequences of peptide components of the two-component bioluminescent complex used in the embodiments herein (for example, as a fusion with a dsRNA-binding domain) are described, for example, in U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference). The peptide components of the two-component bioluminescent complex herein are not limited to these sequences, but in some embodiments, the peptide components of the two-component bioluminescent complex herein may be selected from the amino acid sequences SEQ ID NOs. 441-2156 described in U.S. Patent No. 9,797,889 (which is incorporated in its entirety by reference).

[0108] In some embodiments, this specification provides a fusion of a dsRNA-binding domain and a polypeptide component of a two-component bioluminescent complex having more than 40% (e.g., more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, 100%) sequence identity with SEQ ID NO: 3037, wherein when the polypeptide comes into contact with a peptide consisting of SEQ ID NO: 3036 or 3038 (e.g., within the fusion as described herein) in the presence of a substrate of the bioluminescent complex, a detectable bioluminescent signal is generated (e.g., stronger luminescence than the components of the complex in the presence of the substrate).

[0109] In some embodiments, this specification provides pairs of fusions, each containing a dsRNA-binding domain, wherein the first fusion comprises a first component of a bioluminescent complex having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these) with a first fragment of SEQ ID NO: 3041, and the second fusion comprises a second component of a bioluminescent complex having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these) with a complementary portion of SEQ ID NO: 3041. In some embodiments, the first component includes 40% or more sequence identity with SEQ ID NO: 3042 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these), and the complementary component includes 40% or more sequence identity with SEQ ID NO: 3050 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these). In some embodiments, the first component includes 40% or more sequence identity with SEQ ID NO: 3043 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these), and the complementary component includes 40% or more sequence identity with SEQ ID NO: 3051 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these).In some embodiments, the first component includes 40% or more sequence identity with SEQ ID NO: 3044 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these), and the complementary component includes 40% or more sequence identity with SEQ ID NO: 3052 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these). In some embodiments, the first component includes 40% or more sequence identity with SEQ ID NO: 3045 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these), and the complementary component includes 40% or more sequence identity with SEQ ID NO: 3053 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these). In some embodiments, the bioluminescent signal is substantially increased when the first component associates with the complementary component (e.g., facilitated by the binding of the fused dsRNA-binding domain to the dsRNA).

[0110] Exemplary sequences of peptide and polypeptide components of two-component or multi-segmented bioluminescent complexes used for fusion with dsRNA-binding domains in embodiments of this specification are described, for example, in U.S. Patent Application No. 16 / 439,565 (which is incorporated in its entirety by reference). While the peptide and polypeptide components of the two-component or multi-segmented bioluminescent complexes of the present invention are not limited to these sequences, in some embodiments, the peptide or polypeptide components of the two-component or multi-segmented bioluminescent complexes of the present invention may be selected from the amino acid sequences of SEQ ID NOs: 1 to 804 of U.S. Patent Application No. 16 / 439,565 (which is incorporated in its entirety by reference).

[0111] In some embodiments, β6-7-like peptides comprising SEQ ID NOs. 3054 and 3055 (e.g., fusions described herein) are provided herein. In some embodiments, β6-7-like peptides having sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) of SEQ ID NOs. 3054 and 3055 are provided herein.

[0112] In some embodiments, β7-8-like peptides (e.g., fusions described herein) comprising SEQ ID NOs. 3055 and 3056 are provided herein. In some embodiments, β7-8-like peptides (e.g., fusions described herein) having sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) with SEQ ID NOs. 3055 and 3056 are provided herein.

[0113] In some embodiments, β8-9-like peptides (e.g., within the fusion described herein) comprising SEQ ID NO: 3056 / 3059 or 3056 / 3060 are provided herein. In some embodiments, β8-9-like peptides (e.g., within the fusion described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) with SEQ ID NO: 3056 / 3059 or 3056 / 3060 are provided herein.

[0114] In some embodiments, β9-10-like peptides (e.g., within the fusion described herein) comprising SEQ ID NOs. 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058 are provided herein. In some embodiments, β8-9-like peptides (e.g., within the fusion described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) of SEQ ID NOs. 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058 are provided herein.

[0115] In some embodiments, β6-8-like peptides or polypeptides (e.g., fusions described herein) comprising SEQ ID NOs.3054-3056 are provided herein. In some embodiments, β6-8-like peptides or polypeptides (e.g., fusions described herein) having sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) of SEQ ID NOs.3054-3056 are provided herein.

[0116] In some embodiments, β7-9-like peptides or polypeptides (e.g., fusions described herein) comprising SEQ ID NOs. 3055 / 3056 / 3059 or 3055 / 3056 / 3060 are provided herein. In some embodiments, β7-9-like peptides or polypeptides (e.g., fusions described herein) having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) of SEQ ID NOs. 3055 / 3056 / 3059 or 3055 / 3056 / 3060 are provided herein.

[0117] In some embodiments, β8-10-like peptides or polypeptides (e.g., fusion bodies described herein) comprising SEQ ID NOs: 3056 / 3059 / 3057, 3056 / 3059 / 3058, 3056 / 3060 / 3057, or 3056 / 3060 / 3058 are provided herein. In some embodiments, β7-9-like peptides or polypeptides (e.g., fusion bodies described herein) having sequence identity of 40% or more (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or in between) with SEQ ID NOs. 3056 / 3059 / 3057, 3056 / 3059 / 3058 are provided herein.

[0118] C. Fluorescent Protein In some embodiments, fusions of a dsRNA-binding domain and a complementary peptide / polypeptide pair that can interact with each other (e.g., facilitated by the binding of the dsRNA-binding domain to dsRNA) are provided herein, which, when excited at a suitable wavelength (excitation spectrum), can emit fluorescence within a detectable range (emission spectrum). In some embodiments, the fluorescent complex can produce fluorescence that is significantly enhanced compared to either of the complementary peptides / polypeptides alone.

[0119] In some embodiments, a first fusion is provided comprising a first complementary peptide or polypeptide fragment of a fluorescent protein, and a second fusion is provided comprising a second complementary peptide or polypeptide fragment of a fluorescent protein, wherein upon interaction (e.g., facilitated by the binding of a dsRNA-binding domain to dsRNA), the complementary peptide(s) / polypeptide(s)(s)(s) form an active fluorescent complex that, when excited at a suitable wavelength(s) (excitation spectrum), fluoresces within a detectable range (emission spectrum). In some embodiments, the complementary peptide(s) / polypeptide(s)(s)(s)(s) is a fragment of a fluorescent protein.

[0120] In some embodiments, fragments of fluorescent proteins are provided for use in the embodiments herein. Exemplary fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, dansyl chloride, phycocyanin, and phycoerythrin. Table 1 provides examples of existing split fluorescent proteins that may be used in the embodiments herein (e.g., two components fused to a dsRNA-binding domain). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0121] As an exemplary embodiment, the components of split GFP (the peptide of SEQ ID NO: 3070 and the polypeptide of SEQ ID NO: 3068) were fused to a dsRNA-binding domain (SEQ ID NO: 3061) using the linker of SEQ ID NO: 3064. In some embodiments, this specification provides pairs of fusions, each containing a dsRNA-binding domain, wherein the first fusion comprises a first component of a fluorescent complex having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these) with a first fragment of SEQ ID NO: 3068, and the second fusion comprises a second component of a bioluminescent complex having 40% or more sequence identity (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%), or a range between these) with a complementary portion of SEQ ID NO: 3070.

[0122] III. Fusion In some embodiments, the fusion according to this specification includes a dsRNA-binding domain directly bound to a component of the detectable complex. In some embodiments, the dsRNA-binding domain includes two directly bound dsRNA-binding motifs. However, in other embodiments, the dsRNA-binding domain and the component of the detectable complex, and / or the two dsRNA-binding motifs within the dsRNA-binding domain, are fused via a linker. Such a linker may be of any suitable sequence and may be up to 100 amino acids long (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 445, 50, 60, 70, 80, 90, 100, or in the range between them). In some embodiments, the fusion according to this specification includes a linker that facilitates an optimized shape for complementarity, ligand / substrate binding, dsRNA binding, etc.

[0123] In some embodiments, the components of the fusion described herein (e.g., dsRNA-binding domains and / or motifs, detectable complex components, linkers, etc.) may be positioned in any suitable direction to enable dsRNA binding, structural complementarity to the detectable complex, and detectable signal / activity from the detectable complex. In some embodiments, the C-terminus of the dsRNA-binding domain is fused to the N-terminus of a component of the detectable complex (e.g., directly or via a linker). In some embodiments, the N-terminus of the dsRNA-binding domain is fused to the C-terminus of a component of the detectable complex (e.g., directly or via a linker). In some embodiments, the C-terminus of a first dsRNA-binding motif is fused to the N-terminus of a component of the detectable complex (e.g., directly or via a linker), and the N-terminus of a second dsRNA-binding motif is fused to the C-terminus of a component of the detectable complex (e.g., directly or via a linker).

[0124] IV. Substrates, Ligands, and Cofactors In some embodiments, the detectable complex used in the embodiments herein generates a detectable signal using substrates, ligands, cofactors, etc. In such embodiments, systems and methods are provided that include suitable substrates, cofactors, and / or ligands for the detectable complex and its components.

[0125] In some embodiments, the ligand / substrate of the present invention is permeable to the cell plasma membrane (i.e., it can pass from outside the cell (e.g., eukaryotic cells, prokaryotic cells) into the cell without chemical, enzymatic, or mechanical disruption of the cell membrane).

[0126] In some embodiments, the ligands herein include cleavable linkers, such as those described in U.S. Patent No. 10,618,907 (which is incorporated herein by reference in its entirety).

[0127] In the systems and methods herein that utilize various split enzymes as detectable complexes, suitable substrates and / or cofactors for use in such systems are provided. For example, dihydrofolate, ATP, tetramethylbenzidine, chorismite, etc., are provided so that the assembled detectable complex can generate a detectable signal.

[0128] A. Haloalkyl ligand In embodiments in which the detectable complex comprises a modified dehalogenase complex (e.g., split HALOTAG), a system and method comprising a haloalkane ligand are provided. The modified dehalogenase complex (e.g., formed by structural complementarity facilitated by binding of the fusion to dsRNA herein) binds to the haloalkane ligand, and the functional group bound to the haloalkane enables detection. In some embodiments, the haloalkane ligand is of formula (I):R-linker-AX, where R is a detectable functional group, the linker is a polyatomic linear or branched chain containing C, N, S, or O, or a group containing one or more rings, e.g., saturated or unsaturated rings, e.g., one or more aryl rings, heteroaryl rings, or any combination thereof, where AX is a ligand for a modified dehalogenase (e.g., HALOTAG) (e.g., A is (CH2) 4-20 (where X is a halide (e.g., Cl or Br)). Suitable ligands are described, for example, in U.S. Patents 11,072,812, 11,028,424, 10,618,907, and 10,101,332, which are incorporated by reference in whole. In certain embodiments, X in formula (I) is not a halide but a methylsulfonamide or trifluoromethylsulfonamide, and such embodiments result in interchangeable ligands that reversibly bind to modified dehalogenases (e.g., HALOTAG). Such ligands are described, for example, in Kompa et al. J.Am.Chem.Soc.2023,145,5,3075-3083, which are incorporated by reference in whole.

[0129] In some embodiments, R is one or more functional groups (such as fluorophores, biotin, chromophores, or fluorescent or luminescent molecules). Examples of functional groups used in the present invention include amino acids, proteins (e.g., enzymes, antibodies, or other immunogenic proteins), radionuclides, nucleic acid molecules, drugs, lipids, biotin, avidin, streptavidin, magnetic beads, solid supports, electron-impermeable molecules, chromophores, MRI contrast agents, dyes (e.g., xanthene dyes), calcium-sensitive dyes (e.g., 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2'-amino-5'- Examples include, but are not limited to, tylphenoxy)ethane-N,N,N',N'-tetraacetic acid (fluor-3), sodium-sensitive dyes (e.g., 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofranzyl)]bis(PBFI)), NO-sensitive dyes (e.g., 4-amino-5-methylamino-2',7'-difluorescein), or other fluorophores. In one embodiment, the functional group is an immunogenic molecule, i.e., a molecule that is bound by an antibody specific to that molecule.

[0130] In some embodiments, the ligand includes a fluorescent functional group (R). Suitable fluorescent functional groups include stilbazolium derivatives (Marquesa et al. Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling. ChemRxiv.).Cambridge: Cambridge Open Engage;2021; the entire text is incorporated by reference), xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), prene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porfin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA This includes, but is not limited to, FLOUR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, phycobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), and quantum dot nanocrystals.

[0131] In some embodiments, the ligand includes a fluorescence-generating functional group (R). The fluorescence-generating functional group generates and enhances a fluorescence signal upon binding of the ligand to a target (e.g., binding of a haloalkane to a modified dehalogenase). The problem of background signaling is mitigated by generating a significantly increased fluorescence (e.g., 10-fold, 31-fold, 50-fold, 100-fold, 310-fold, 500-fold, 100-fold or more) upon engagement with the target. Exemplary fluorescence-generating dyes used in embodiments herein include the JANELIA FLUOR family of fluorophores, as listed below. JANELIA FLUOR 549, SE: [ka] JANELIA FLUOR 646, SE: [ka] JANELIA FLUOR 585, SE: [ka] JANELIA FLUOR 635, SE: [ka] JANELIA FLUOR 669, SE: [ka] (See, for example, U.S. Patents 9,933,417, 10,018,624, 10,161,932, and 10,495,632, each incorporated as a whole by reference). In some embodiments, exemplary conjugates of JANELIA FLUOR 549 and JANELIA FLUOR 646 with haloalkane ligands for modified dehalogenases (e.g., HALOTAG) are commercially available (Promega Corp.). The use and design of the fluorescence-generating functional groups, dyes, probes, and ligands are described, for example, in Grimm et al. Nat Methods. 3117 Oct;14(10):987-994.; and in Wang et al. Nat Chem. 3120 Feb;12(2):165-172, which are incorporated as a whole by reference.

[0132] B. Bioluminescent substrates In some embodiments, the systems and methods described herein include a luminescence complex that utilizes a luminescent phore to generate a detectable luminescence signal. In some embodiments, a suitable luminescent phore is selected to pair with the luminescence complex.

[0133] In some embodiments, the systems and methods described herein (including bioluminescent complexes and / or components thereof) utilize imidazopyrazine phosphonate substrates to produce bioluminescence. Such embodiments include those utilizing NANOLUC-based, NANOBIT-based, and NANOTRIP-based luminescent complexes. In some embodiments, the substrate is coelenterazine. [ka] That is the case.

[0134] In some embodiments, the substrates are coelenterazine derivatives such as furimazine, furimazine analogs (e.g., fluoroflimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine, as further disclosed in WO2003 / 040100, U.S. Patent Application No. 12 / 056,073 (paragraph

[0086] ), U.S. Patent No. 8,669,103, and U.S. Provisional Application No. 63 / 379,573. These disclosures are incorporated herein by reference in their entirety.

[0135] In some embodiments, the substrate is frimazine [ka] That is the case.

[0136] In some embodiments, the substrate is fluoroflimazine. [ka] That is the case.

[0137] Suitable luminescent phosphonates for use in the systems or methods described herein will be understood. For example, firefly luciferin has a structure [ka] It possesses a structure and is a luciferin found in many Lampyridae species, and is a substrate for beetle luciferase. Latia luciferin has a structure [ka] It has and is derived from the freshwater snail Latia neritoides. The bacterium luciferin has structure [ka] It possesses and is used as a substrate for many bacterial luciferases. Coelenterazine is [ka] It has the structure and is found in radiolarians, ctenophores, cnidarians, squid, brittle stars, copepods, chaetognaths, fish, and shrimp, and is a luminescent substrate for the luciferases of these organisms. Variants and derivatives of coelenterazine, such as flimazine and fluoroflimazine, are used in the embodiments described herein (for example, together with bioluminescent complexes derived from Oplophorus). Other luminescent substrates include those of dinoflagellates. [ka] Valgurin (cypridina luciferin) [ka] N.nambi [ka] Examples include:

[0138] The appropriate pairing of bioluminescent proteins or complexes with luminescent phosphotes is well understood in the art.

[0139] V. Method In some embodiments, methods are provided herein for detecting the presence of dsRNA in a sample and / or quantifying its amount, the method comprising (a) contacting the sample with a sufficient concentration of the system described herein (e.g., a fusion of a dsRNA-binding domain and a pair of components of the detectable complex) and any necessary substrate, ligand, cofactor, etc., and (b) detecting and / or quantifying the signal generated by the detectable complex. In some embodiments, the amount of the signal (e.g., intensity) correlates with the amount of dsRNA in the sample. In some embodiments, the signal is compared to a signal from a control sample having a known concentration of dsRNA. In some embodiments, the signal is compared to an established value corresponding to a known dsRNA concentration in the sample.

[0140] In some embodiments, the sample is any suitable sample type that may contain biological samples, environmental samples, pharmaceutical samples (e.g., RNA (e.g., ssRNA) therapeutics), or dsRNA (e.g., as a contaminant). In some embodiments, the sample contains therapeutic RNA, and the method is performed as a quality control test to ensure that the amount of dsRNA in the sample is sufficiently low. [Examples]

[0141] Example 1 Split bioluminescent protein Fusions of the PKR dsRNA-binding domain with LgBiT and SmBiT (PKR-LgBiT and PKR-SmBiT) were expressed in E. coli and purified with a His tag. The proteins were well expressed and purified. Experiments conducted during the development of the embodiments herein demonstrate that adding PKR-LgBiT and PKR-SmBiT components (25 ng / ml each) together with flimazine to a sample containing poly(I:C) (synthetic dsRNA analog) enables highly sensitive (detection limit <0.1 ng / ml) and rapid (1 hour assay time) dsRNA quantification in an easy-to-use (add-mix-measure) assay format (Figure 3). The optimal incubation time was determined to be 1 hour. The exemplary assay is quantitative within a linear range of approximately 2 logs.

[0142] Example 2 Split fluorescent protein Fusions of the PKR dsRNA binding domain with LgGFP and SmGFP (PKR-LgGFP and PKR-SmGFP) were prepared. Experiments conducted during the development of the embodiments described herein demonstrate that when the PKR-LgBiT and PKR-SmBiT components are added to a sample containing dsRNA and exposed to the excitation wavelength of GFP, highly sensitive and rapid quantification of dsRNA is possible (Figure 4).

[0143] References The following references are incorporated herein by reference in their entirety. Cheng et al. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation.Virology. 2015 (PMID: 26351203). Monsion et al. Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 protein.Front Plant Sci.2018.(PMID: 29449856)

Claims

1. A double-stranded RNA (dsRNA) detection system, (a) (i) a first fusion of a first dsRNA-binding domain and (ii) a first component of the detectable complex, (b) A second fusion of (i) a second dsRNA-binding domain and (ii) a second component of the detectable complex, The system including the above.

2. The system according to claim 1, wherein when the first dsRNA-binding domain and the second dsRNA-binding domain bind to dsRNA, the first component and the second component of the detectable complex associate to form the detectable complex.

3. The system according to claim 2, wherein the first and second components of the detectable complex exhibit low affinity for each other in the absence of enhancement through binding of the first dsRNA-binding domain and the second dsRNA-binding domain to the dsRNA.

4. The system according to claim 1, wherein the first dsRNA-binding domain and the second dsRNA-binding domain comprise different amino acid sequences.

5. The system according to claim 1, wherein the first dsRNA-binding domain and the second dsRNA-binding domain contain the same amino acid sequence.

6. The system according to claim 1, wherein the dsRNA-binding domain comprises a dsRNA-binding motif having at least 70% sequence similarity to SEQ ID NO: 3062 and / or SEQ ID NO: 3063.

7. The system according to claim 6, wherein the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence identity with SEQ ID NO: 3062 and / or SEQ ID NO: 3063.

8. The system according to claim 7, wherein the dsRNA-binding domain includes the dsRNA-binding motif of Sequence ID No. 3062.

9. The system according to claim 7, wherein the dsRNA-binding domain includes the dsRNA-binding motif of Sequence ID No. 3063.

10. The system according to claim 6, wherein the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence similarity to SEQ ID NO: 3062 and SEQ ID NO: 3063.

11. The system according to claim 10, wherein the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence identity with SEQ ID NO: 3062 and SEQ ID NO: 3063.

12. The system according to claim 11, wherein the dsRNA-binding domain includes the dsRNA-binding motifs of SEQ ID NO: 3062 and SEQ ID NO: 3063.

13. The system according to claim 1, wherein the dsRNA-binding domain has at least 70% sequence similarity to sequence number 3061.

14. The system according to claim 13, wherein the dsRNA-binding domain has at least 70% sequence identity with sequence number 3061.

15. The system according to claim 14, wherein the dsRNA binding domain includes sequence number 3061.

16. The system according to claim 1, wherein the detectable complex can generate a detectable signal.

17. The system according to claim 16, wherein the amount of signal generated by the detectable complex can be correlated with the amount of dsRNA in a sample having the system.

18. The system according to claim 16, wherein the signal comprises one or more of fluorescence, luminescence, enzyme activity, and ligand binding.

19. The system according to claim 1, wherein the first and second components of the detectable complex are fragments of a protein capable of generating a detectable signal, and the detectable complex can generate the detectable signal when the first and second components of the detectable complex associate.

20. The system according to claim 18, wherein the detectable signal is fluorescence.

21. The system according to claim 20, wherein the first and second components of the detectable complex have at least 70% sequence identity with the first and second fragments of the fluorescent protein.

22. The system according to claim 21, wherein the fluorescent protein is selected from yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, dansyl chloride, phycocyanin, and phycoerythrin.

23. The system according to claim 22, wherein the fluorescent protein is GFP.

24. The system according to claim 23, wherein the first component contains at least 70% sequence identity with respect to sequence number 3068, and the second component contains at least 70% sequence identity with respect to sequence number 3070.

25. The system according to claim 19, wherein the detectable signal is enzyme activity.

26. The system according to claim 25, wherein the first and second components of the detectable complex have at least 70% sequence identity with the first and second fragments of the enzyme.

27. The system according to claim 26, wherein the enzyme is selected from beta-lactamase, dihydrofolate reductase (DHFR), adhesion plaque kinase (FAK), Gal4, and horseradish peroxidase.

28. The system according to claim 19, wherein the detectable signal is luminescence in the presence of a substrate.

29. The system according to claim 28, wherein the first and second components of the detectable complex have at least 70% sequence identity with the first and second fragments of luciferase.

30. The system according to claim 29, wherein the luciferase is selected from Oplophorus luciferase, firefly luciferase, click beetle luciferase, Renilla luciferase, sea firefly luciferase, aequorin luminescent protein, and oberin luminescent protein.

31. The system according to claim 28, wherein the first and second components of the detectable complex together have at least 70% sequence identity with sequence number 3041.

32. The system according to claim 31, wherein the first component of the detectable complex contains at least 70% sequence identity with sequence number 3042, and the first component of the detectable complex contains at least 70% sequence identity with sequence number 3050.

33. The system according to claim 31, wherein the first component of the detectable complex contains at least 70% sequence identity with sequence number 3043, and the first component of the detectable complex contains at least 70% sequence identity with sequence number 3051.

34. The system according to claim 31, wherein the first component of the detectable complex contains at least 70% sequence identity with sequence number 3044, and the first component of the detectable complex contains at least 70% sequence identity with sequence number 3052.

35. The system according to claim 31, wherein the first component of the detectable complex contains at least 70% sequence identity with sequence number 3045, and the first component of the detectable complex contains at least 70% sequence identity with sequence number 3053.

36. The system according to claim 38, further comprising the aforementioned substrate.

37. The system according to claim 19, wherein the detectable signal is ligand binding.

38. The system according to claim 38, wherein the detectable complex is a modified dehalogenase complex, and the first and second components of the modified dehalogenase complex have at least 70% sequence identity with the first and second fragments of the modified dehalogenase.

39. The system according to claim 38, wherein the modified dehalogenase includes SEQ ID NO:

1.

40. The system according to claim 38, further comprising a haloalkyl ligand for the modified dehalogenase.

41. The system according to claim 40, wherein the haloalkyl ligand comprises R-linker-A-X, where R is a detectable moiety, X is a halogen, and A-X is a substrate for a dehalogenase enzyme.

42. The system according to claim 41, wherein R is a fluorophore.

43. A double-stranded RNA (dsRNA) detection system, (a) (i) a first fusion of a PKR-derived dsRNA-binding domain sequence and (ii) a peptide component of a bioluminescent complex, (b) A second fusion of (i) the PKR-derived dsRNA binding domain sequence and (ii) the polypeptide component of the bioluminescent complex, Includes, When the PKR-derived dsRNA-binding domain sequence binds to dsRNA, a luminescent complex is formed due to the structural complementarity between the peptide component and the polypeptide component. The system wherein the luminescence signal generated by the luminescence complex in the presence of the dsRNA and the substrate for the luminescence complex is enhanced compared to the luminescence signal generated in the absence of the dsRNA.

44. The system according to claim 43, further comprising the substrate for the luminescent complex.

45. The system according to claim 44, wherein the substrate for the luminescent complex is an imidazopyrazine luminescent phosphodiol.

46. The system according to claim 44, wherein the imidazopyrazine luminescent phosphodiester is coelenterazine or frimazine.

47. The system according to claim 43, further comprising the dsRNA.

48. The system according to claim 43, wherein the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence similarity to SEQ ID NO: 3062 and SEQ ID NO: 3063.

49. The system according to claim 48, wherein the dsRNA-binding domain includes a dsRNA-binding motif having at least 70% sequence identity with SEQ ID NO: 3062 and SEQ ID NO: 3063.

50. The system according to claim 48, wherein the dsRNA-binding domain has at least 70% sequence similarity to sequence number 3061.

51. The system according to claim 50, wherein the dsRNA-binding domain has at least 70% sequence identity with sequence number 3061.

52. The system according to claim 50, wherein the dsRNA binding domain includes sequence number 3061.

53. The system according to claim 43, wherein the peptide component has at least 70% sequence similarity to SEQ ID NO: 3038, and / or the polypeptide component has at least 70% sequence similarity to SEQ ID NO: 3037.

54. The system according to claim 53, wherein the peptide component has at least 70% sequence identity with SEQ ID NO: 3038, and / or the polypeptide component has at least 70% sequence identity with SEQ ID NO: 3037.

55. The system according to claim 43, wherein the peptide component comprises SEQ ID NO: 3038, and the polypeptide comprises SEQ ID NO: 3037.

56. A method for detecting dsRNA in a sample, comprising: contacting the sample with a system according to one of claims 1 to 55; and detecting a signal from the detectable complex, wherein the amount of the detected signal correlates with the amount of dsRNA in the sample.

57. The method according to claim 56, wherein the sample comprises a single-stranded RNA-based therapeutic agent.

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