Systems and methods for detecting and quantifying double-stranded RNA

Abstract

This article provides compositions comprising double-stranded RNA (dsRNA) binding domains linked to components of a complementary system. When a pair of dsRNA binding domains bind to dsRNA, a detectable complex of the complementary components is formed, and dsRNA can be detected / quantified.

Description

[0001] Cross-reference to related applications

[0002] This invention claims priority to U.S. Provisional Patent Application 63 / 506,502, filed June 6, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] The computer-readable sequence list text submitted in this paper, entitled “PRMG_41970_601_SequenceListing.xml”, was created on June 5, 2024, and is 3,253,101 bytes in size. It is incorporated into this paper in its entirety by reference. Technical Field

[0005] This article provides compositions comprising double-stranded RNA (dsRNA) binding domains linked to components of a complementary system. When a pair of dsRNA binding domains bind to dsRNA, a detectable complex of the complementary components is formed, and dsRNA can be detected / quantified. Background Technology

[0006] Double-stranded RNA (dsRNA) is a byproduct and contaminant of in vitro mRNA transcription, for example, during the production of mRNA-based therapeutics (vaccines, gene therapies). The FDA has issued a guidance document specifically focused on the development of RNA-based therapeutics, titled "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)." This guidance document provides recommendations for the development and characterization of RNA-based therapies, including consideration of the presence of dsRNA. Current guidance states that dsRNA should be measured for all in vitro transcribed RNA products. Currently, there are no specified limits on the amount of dsRNA in RNA drug products.

[0007] Currently, dsRNA (i.e., dsRNA-specific, not ssRNA or DNA-specific) quantification can be performed using two recognized methods: ELISA and dot blot. Enzyme-linked immunosorbent assay (ELISA) can be used to quantify dsRNA using specific antibodies that recognize it. Sensitivity can be an issue, as commercially available ELISA kits exhibit sensitivities of 2–5 ng / ml. Acceptable ELISA Ab clones are J2, K1, and K2. However, despite being the current "gold standard" for dsRNA detection, these clones still have recognized problems in this field. For example, clone J2 exhibits preferred binding to the ends of dsRNA oligomers, as well as an internal binding site, A2N9A3N9A2 (where adenine is present on one face of the helix, see Bonin et al., RNA, 2000). Therefore, this antibody is not sequence-independent and does not represent accurate quantification of dsRNA. Another clone, K1, shows altered binding kinetics when testing dsRNA from different sources. Therefore, these clones cannot provide true quantification of dsRNA. Dot blots can be used for dsRNA detection, although they are less sensitive or quantitative than ELISA, and they rely on the same defective antibody clones.

[0008] There is a need for a quantitative, sensitive (e.g., detection limit < 1 ng / ml), specific, rapid (< 2 hours assay time) and easy-to-use (add-mix-read) assay to quantify dsRNA. Summary of the Invention

[0009] This article provides compositions comprising double-stranded RNA (dsRNA) binding domains linked to components of a complementary system. When a pair of dsRNA binding domains bind to dsRNA, a detectable complex of the complementary components is formed, and dsRNA can be detected / quantified.

[0010] In some embodiments, this document provides a double-stranded RNA (dsRNA) detection system comprising: (a)(i) a first fusion of a first dsRNA-binding domain and (ii) a first component of a detectable complex; and (b)(i) a second fusion of a second dsRNA-binding domain and (ii) a second component of the detectable complex. In some embodiments, after the first and second dsRNA-binding domains bind to dsRNA, the first and second components of the detectable complex associate to form the detectable complex. In some embodiments, in the absence of a promoting effect of binding to dsRNA via the first and second dsRNA-binding domains, the first and second components of the detectable complex exhibit low affinity for each other.

[0011] 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 therebetween) 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 therebetween) 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 comprises the dsRNA binding motif of SEQ ID NO: 3063. In some embodiments, the dsRNA binding domain comprises a dsRNA binding motif having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA binding domain comprises a dsRNA binding motif having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA binding domain comprises the dsRNA binding motifs of SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA binding domain has at least 70% sequence similarity to SEQ ID NO: 3061 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween). In some embodiments, the dsRNA binding domain comprises SEQ ID NO: 3061.

[0012] In some embodiments, the detectable complex is capable of generating a detectable signal. In some embodiments, the amount of signal generated by the detectable complex can be correlated with the amount of dsRNA in the sample. 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 fragments of proteins capable of generating a detectable signal, and wherein the detectable complex is capable of generating a detectable signal upon association of the first and second components of the detectable complex.

[0013] In some embodiments, the detectable signal is fluorescence. In some embodiments, the first and second components of the detectable complex have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) 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, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, cyanin, dansyl chloride, phycocyanin, and phycoerythrin.

[0014] In some embodiments, the detectable signal is enzyme activity. In some embodiments, the first and second components of the detectable complex have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with the first and second fragments of the enzyme. In some embodiments, the enzyme is selected from β-lactamases, dihydrofolate reductase (DHFR), focal adhesion 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 have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with the first and second fragments of a luciferase. In some embodiments, the luciferase is selected from spiny shrimp luciferase, firefly luciferase, click beetle luciferase, sea snail luciferase, sea firefly luciferase, jellyfish luciferin, and obelin luciferin. In some embodiments, the first and second components of the detectable complex together share at least 70% sequence identity with SEQ ID NO: 3041 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween). In some embodiments, the first component of the detectable complex shares at least 70% sequence identity with SEQ ID NO: 3042 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween), and the first component of the detectable complex shares at least 70% sequence identity with SEQ ID NO: 3050 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween). In some embodiments, the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO: 3043 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween), and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO: 3051 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween). In some embodiments, the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO: 3044 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween), and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO: 3052 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween).In some embodiments, the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO: 3045 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween), and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO: 3053 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween). In some embodiments, the system further includes a substrate.

[0015] In some embodiments, the detectable signal is ligand binding. In some embodiments, the detectable complex is a modified dehalogenase complex, wherein the first and second components of the modified dehalogenase complex have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with the first and second fragments of the modified dehalogenase. In some embodiments, the modified dehalogenase comprises SEQ ID NO: 1. In some embodiments, the system further comprises a haloalkyl ligand of the modified dehalogenase. In some embodiments, the haloalkyl ligand comprises R-connector-AX, wherein R is the detectable moiety, X is a halogen, and AX is a substrate for the dehalogenase. In some embodiments, R is a fluorophore.

[0016] In some embodiments, this document provides a double-stranded RNA (dsRNA) detection system comprising: (a)(i) a first fusion of a PKR-derived dsRNA binding domain sequence and (ii) a peptide component of a bioluminescent complex; and (b)(i) a second fusion of 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 the dsRNA, a bioluminescent complex is formed through structural complementarity between the peptide and polypeptide components; and wherein the bioluminescent signal generated by the bioluminescent complex is enhanced in the presence of the substrate of the dsRNA and the bioluminescent complex compared to the bioluminescent signal generated in the absence of dsRNA. In some embodiments, the system further comprises a substrate of the bioluminescent complex. In some embodiments, the substrate of the bioluminescent complex is an imidazopyrazine bioluminescent agent. In some embodiments, the imidazopyrazine bioluminescent agent is coelentrin or furimazine. In some embodiments, the system further comprises dsRNA. In some embodiments, the dsRNA binding domain comprises a dsRNA binding motif having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity to SEQ ID NO: 3062 and SEQ ID NO: 3063. In some embodiments, the dsRNA binding domain comprises a dsRNA binding motif having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity to SEQ ID NO: 3061. In some embodiments, the dsRNA-binding domain has at least 70% sequence identity with SEQ ID NO: 3061 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween). In some embodiments, the dsRNA-binding domain comprises SEQ ID NO: 3061. In some embodiments, the peptide component has at least 70% sequence similarity with SEQ ID NO: 3038 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween), and / or the polypeptide component has at least 70% sequence similarity with SEQ ID NO: 3037 (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween).In some embodiments, the peptide component has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 3038, and / or the polypeptide component has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 3037. In some embodiments, the peptide component comprises SEQ ID NO: 3038, and the polypeptide comprises SEQ ID NO: 3037.

[0017] In some embodiments, this document provides a method for detecting dsRNA in a sample, the method comprising contacting the sample with a system described herein and detecting a signal from a detectable complex, wherein the amount of signal detected is correlated with the amount of dsRNA in the sample. In some embodiments, the sample contains a single-stranded RNA-based therapeutic agent. In some embodiments, the sample also contains dsRNA (e.g., a contaminant). Attached Figure Description

[0018] Figure 1 A cartoon depicting the binding of protein kinase R (PKR) to dsRNA and the dimerization of the two PKRs.

[0019] Figure 2 A cartoon depicting an exemplary embodiment of the present technology, wherein two components of the luminescent complex (LgBiT and SmBiT) are provided as a fusion with the dsRNA binding domain of PKR; the binding of the dsRNA binding domain to dsRNA results in the formation of an active luminescent complex.

[0020] Figure 3 Depicting in existence Figure 2 In the case of an exemplary system, a graph showing the increase in luminescence signal as dsRNA increases.

[0021] Figure 4 A graph depicting the increase in fluorescence signal with increasing dsRNA in the presence of an exemplary system containing a fusion of split GFP constructs and dsRNA-binding domains of PKR (PKR-LgGFP and PKR-SmGFP).

[0022] definition

[0023] While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the embodiments described herein, this document describes some preferred methods, compositions, apparatuses, and materials. However, before describing the materials and methods of the invention, it should be understood that the invention is not limited to the specific molecules, compositions, methods, or schemes described herein, as these can vary based on routine experimentation and optimization. It should also be understood that the terminology used in the description is for the purpose of describing a particular version or embodiment only and is not intended to limit the scope of the embodiments described herein.

[0024] Unless otherwise specified, 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 pertains. However, in case of conflict, this specification, including the definitions, shall prevail. Therefore, the following definitions apply in the context of the embodiments described herein.

[0025] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein and in the appended claims include plural indicators. Thus, for example, reference to “polypeptide” means one or more polypeptides and their equivalents known to those skilled in the art, etc.

[0026] As used herein, the term “and / or” includes any and all combinations of the listed items, including any one of the items listed individually. For example, “A, B and / or C” covers A, B, C, AB, AC, BC, and ABC, each of which should be considered as being described individually by the statement “A, B and / or C”.

[0027] As used herein, the term "comprising" and its variations indicate the presence of one or more listed features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term "consisting of" and its variations indicate the presence of one or more listed features, elements, method steps, etc., excluding any unlisted features, elements, method steps, etc., except for generally relevant impurities. The phrase "substantially consisting of" indicates the listed features, elements, method steps, etc., as well as any additional features, elements, method steps, etc., that do not substantially affect the fundamental properties of the composition, system, or method. Many embodiments described herein are described using the open-ended language of "comprising". Such embodiments encompass multiple closed-ended "consisting of" and / or "substantially consisting of" embodiments, which may alternatively use such language for claims or descriptions.

[0028] As used herein, the term "substantially" means that the characteristic, parameter, and / or value does not need to be precisely achieved, but deviations or variations, including, for example, tolerances, measurement errors, measurement accuracy limitations, and other factors known to those skilled in the art, may occur in an amount that does not preclude the effect intended to provide the characteristic. A substantially non-existent (e.g., substantially non-fluorescent) characteristic or feature may be a characteristic or feature that is within noise, below background, below the detection capability of the assay used, or a small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of a significant characteristic (e.g., the fluorescence intensity of an active fluorophore).

[0029] As used herein, when referring to an amino acid sequence or a position within an amino acid sequence, the phrase “corresponding to” refers to the relative position of an amino acid residue or segment to the mentioned sequence, and not necessarily to a specific identity of the amino acid at that position. For example, “the peptide corresponding to positions 36 to 48 of SEQ ID NO: 1” may have less than 100% sequence identity (e.g., >70% sequence identity) with positions 36 to 48 of SEQ ID NO: 1, but in the context of the described composition or system, the peptide refers to those positions.

[0030] As used herein, the term "system" refers to multiple components (e.g., devices, compositions, etc.) that can be used for a particular purpose. For example, two individual biomolecules, whether present in the same composition or not, can constitute a system if they can be used together for a common purpose.

[0031] As used herein, the term "complementary" refers to the characteristic that two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) can hybridize, dimerize, or otherwise form a complex. For example, "complementary peptides and polypeptides" can combine to form a complex. Complementary elements may require assistance (facilitation) to form a complex (e.g., from interacting elements), such as placing the elements in the correct complementary conformation, placing the elements in the correct complementary proximity, co-locating the complementary elements, lowering the complementary interaction energy, overcoming insufficient affinity between them, etc.

[0032] As used herein, the term "complex" refers to an assembly or aggregate of molecules (e.g., peptides, polypeptides, etc.) in direct and / or indirect contact with each other. In one respect, "contact," or more specifically "direct contact," means that two or more molecules are close enough that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, dominate the molecular interactions. In this respect, molecular complexes (e.g., peptides, polypeptides, etc.) are formed under measured conditions such that the complex is thermodynamically advantageous (e.g., compared to the non-aggregated or non-complexed state of its component molecules). As used herein, unless otherwise stated, the term "complex" refers to an assembly of two or more molecules (e.g., peptides, polypeptides, etc.). The molecules that assemble to form a complex are referred to herein as "components" or variations thereof.

[0033] As used herein, the term “low affinity” describes an intermolecular interaction between two or more entities that is too weak to result in the formation of a significant complex between the entities unless the concentration is significantly higher than (e.g., 2, 5, 10, 100, 1000 or more times) physiological or analytical conditions, or promotes the formation of a second complex of attachment elements (e.g., interaction elements).

[0034] As used herein, the term “high affinity” describes an intermolecular interaction between two or more (e.g., three) entities that is strong enough to produce detectable complex formation under physiological or assay conditions without promoting the formation of a second complex with attachment elements (e.g., interaction elements).

[0035] The term "amino acid" refers to natural amino acids, non-natural amino acids, and amino acid analogs, which, unless otherwise stated, are D- and L-steroimeric forms if their structure allows for such stereoisomers.

[0036] The term "protein amino acid" refers to the 20 amino acids encoded in the human genetic code, 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 pyrrolidone can also be considered protein amino acids.

[0037] The term "non-protein amino acid" refers to an amino acid that is not naturally encoded or found in the genetic code of any organism, and is not incorporated into proteins through biosynthesis during translation. Non-protein amino acids can be "non-natural amino acids" (amino acids that do not exist in nature) or "naturally occurring non-protein amino acids" (e.g., valine, ornithine, homocysteine, etc.). Examples of non-proteinogenic amino acids include, but are not limited to, azacyclobutane carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmodium, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, isomeric-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodestapyrin, isomeric-isoleucine, N-methylalanine, N-alkylglycine (including N-methylglycine), N-methylisoleucine, and N-alkylpentylglycine (including N-methylpentylglycine). N-methylvaline, naphthylalanine, n-valine, n-leucine (“n-leucine”), octylglycine, ornithine, pentylglycine, piperidine, thioproline, high-lysine, and high-arginine. Non-proteinogenics also include the D-amino acid form of any amino acid described herein, as well as the non-α-amino acid forms (β-amino acids, γ-amino acids, δ-amino acids, etc.) of any amino acid described herein, all of which are within the scope of this document and may be included in the peptides described herein.

[0038] The term "amino acid analog" refers to an amino acid (e.g., natural or non-natural, proteogenic or non-proteogenic) in which one or more C-terminal carboxyl groups, N-terminal amino groups, and side-chain bioactive groups have been chemically blocked, reversibly or irreversibly modified, or otherwise modified to form another bioactive group. For example, aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine formamide 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.

[0039] As used herein, unless otherwise stated, the terms “peptide” and “polypeptide” refer to polymers of two or more amino acids linked by a peptide amide bond (-C(O)NH-) through the backbone. The term “peptide” generally refers to a short amino acid polymer (e.g., a chain with fewer than 30 amino acids), while the term “polypeptide” generally refers to a longer amino acid polymer (e.g., a chain with more than 30 amino acids).

[0040] 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 contains amino acids that are conserved in their substitution for each other:

[0041] 1) Alanine (A) and glycine (G);

[0042] 2) Aspartic acid (D) and glutamic acid (E);

[0043] 3) Asparagine (N) and glutamine (Q);

[0044] 4) Arginine (R) and lysine (K);

[0045] 5) Isoleucine (I), leucine (L), methionine (M), and valine (V);

[0046] 6) Phenylalanine (F), tyrosine (Y), and tryptophan (W);

[0047] 7) Serine (S) and threonine (T); and

[0048] 8) Cysteine ​​(C) and methionine (M).

[0049] Amino acid residues can be classified into several categories based on common side-chain characteristics, such as: polar positive (or basic) (e.g., histidine (H), lysine (K), and arginine (R)); polar negative (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-conserved" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid of the same class.

[0050] In some embodiments, unless otherwise specified, conserved or semi-conserved amino acid substitutions may also encompass non-naturally occurring amino acid residues having chemical properties similar to native residues. These non-natural residues are typically incorporated through chemical peptide synthesis rather than synthesis in biological systems. These include, but are not limited to, peptide mimics and other reversed or inverted forms of amino acid moieties. In some embodiments, the embodiments described herein may be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0051] Non-conservative substitution can involve replacing a member of one class with a member of another class.

[0052] As used herein, the term "sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have a continuous composition of identical monomeric subunits. 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 characteristics and can be grouped into multiple 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 uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “sequence identity percentage” (or “sequence similarity percentage”) is calculated by: (1) comparing two best-aligned sequences within a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specific window); (2) determining the number of positions containing the same (or similar) monomers (e.g., the same amino acid appears in both sequences, similar amino acids appear in both sequences) to produce the number of matching positions; (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specific window); and (4) multiplying the result by 100 to produce the sequence identity percentage or sequence similarity percentage. For example, if peptide A and peptide B are both 20 amino acids long and have the same amino acid at all but one position, then peptide A and peptide B have 95% sequence identity. If the amino acids at different positions have the same biophysical properties (e.g., both are acidic), then peptide A and peptide 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 a portion of the amino acids in peptide C, then peptide C and peptide D have 70% sequence identity. However, the optimal comparison window for peptide D and peptide C has 93.3% sequence identity. For the purpose of calculating the “percentage of sequence identity” (or “percentage of sequence similarity”) in this paper, any gaps in the aligned sequences are treated as mismatches at that position.

[0053] Any peptide / peptide described herein that has a specific percentage sequence identity or similarity (e.g., at least 70%) to the reference sequence ID number may also be expressed as having the maximum number of substitutions (or terminal deletions) relative to the reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) to SEQ ID NO:Z (e.g., 100 amino acids) may have up to X substitutions (e.g., 10) relative to SEQ ID NO:Z, and thus may also be expressed as "having X (e.g., 10) or fewer substitutions relative to SEQ ID NO:Z".

[0054] As used herein, the term "sample" is used in its broadest sense. In one sense, it is intended to include samples or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, etc. A sample may also refer to cell lysates or purified forms of enzymes, peptides, and / or polypeptides described herein. Cell lysates may include cells or lysates that have been lysed with a lysing agent, such as rabbit reticulocytes or wheat germ lysates. Samples may also include cell-free expression systems. Environmental samples include environmental materials such as surface substances, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention. Pharmaceutical samples include any therapeutic agent for which the presence and / or concentration of dsRNA to be tested is, such as RNA-based therapeutic agents.

[0055] As used herein, the terms “fusion product,” “fusion polypeptide,” and “fusion protein” refer to a chimeric protein containing a first protein or polypeptide of interest linked to a second peptide, polypeptide, or protein (e.g., an interacting element).

[0056] As used herein, the terms “conjugated” and “conjugation” refer to the covalent connection of two molecular entities (e.g., post-synthesis and / or during synthetic production). Chemical (e.g., “chemical” conjugation) or enzymatic attachment of peptide or small molecule tags to proteins or small molecules is an example of conjugation.

[0057] As used herein, the term "modified dehalogenase" refers to a dehalogenase variant (artificial variant) with one or more mutations that prevent the release of the substrate from the protein after halogen removal, resulting in a covalent bond between the substrate and the modified dehalogenase. Because the modified dehalogenase does not release the substrate, it cannot be converted and is not a classical enzyme. The HALOTAG system (Promega) is a commercially available modified dehalogenase and substrate system.

[0058] As used herein, the term "bioluminescence" refers to the generation and emission of light by a chemical reaction catalyzed or facilitated by an enzyme, protein, protein complex, or other biomolecule (e.g., a bioluminescent complex). 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; the substrate then emits light.

[0059] As used herein, the term "OgLuc" refers to a luminescent polypeptide exhibiting significant sequence identity, structural conservation, and / or functional activity with luciferases produced and derived from deep-sea shrimp (Oplophorus gracilirostris). Specifically, an OgLuc polypeptide refers to a luminescent polypeptide exhibiting significant sequence identity, structural conservation, and / or functional activity with a mature 19 kDa subunit of an Oplophorus luciferase protein complex (e.g., without a signal sequence), such as SEQ ID NO: 3034 (NanoLuc), which comprises 10 β chains (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10) and is produced using substrates such as coelentrin or coelentrin derivatives or analogs.

[0060] As used herein, the term "β9-like peptide" refers to a peptide (or peptide tag) that contains a functionally active β9 chain of an OgLuc polypeptide with significant sequence identity, structural conservation, and / or similarity to the β9 chain. Specifically, a β9-like peptide is a peptide capable of structural complementation to an OgLuc polypeptide lacking a β9 chain, resulting in enhanced luminescence of the complex compared to an OgLuc polypeptide without a 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).

[0061] As used herein, the term "β10-like peptide" refers to a peptide (or peptide tag) that contains a functionally active β10 chain of an OgLuc polypeptide with significant sequence identity, structural conservation, and / or similarity. Specifically, a β10-like peptide is a peptide that is structurally complementary to an OgLuc polypeptide lacking a β10 chain, resulting in enhanced luminescence of the complex compared to an OgLuc polypeptide without a 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).

[0062] As used in this article, the term "β" 1-8 "β-like polypeptides" refer to polypeptides that possess the sequence of β chains 1-8 of the OgLuc polypeptide and have structural similarity to it, but lack β chains 9 and 10. Other "β-like polypeptides"... Y-Z "Like polypeptides" can be named similarly (e.g., β-peptides). 1-4 Like polypeptide, β2-8 Like polypeptide, β 5-10 (e.g., polypeptides).

[0063] As used in this article, the term "NANOLUC" refers to artificial luciferase or bioluminescent peptides commercially produced by Promega Corporation.

[0064] As used in this article, the term "LgBiT" refers to the term corresponding to β. 1-9 The polypeptide is a type of polypeptide that can be used, for example, for binary complementation to form a bioluminescent complex, and corresponds to SEQ ID NO: 3037.

[0065] As used in this article, the term "SmBiT" refers to the term corresponding to β. 10 The peptide is a type of peptide that can be used, for example, for binary complementation to form a bioluminescent complex, but has low affinity for LgBiT (e.g., requires promotion of complex formation) and corresponds to SEQ ID NO: 3039.

[0066] As used in this article, the term "HiBiT" refers to the term corresponding to β. 10 The peptide is a type of peptide that can be used, for example, for binary complementation to form a bioluminescent complex, but has the high affinity of LgBiT (e.g., no need to promote complex formation). An exemplary HiBiT peptide corresponds to SEQ ID NO: 3038.

[0067] As used in this article, the term "LgTrip" refers to the term corresponding to β. 1-8 A polypeptide of the β9-like peptide. An exemplary LgTrip corresponds to SEQ ID NO: 3045 and can be used, for example, with β9-like peptides and β... 10 In the ternary complementarity of peptides, a bioluminescent complex can be formed, or it can interact with β-peptides. 9-10 The two complementary peptides form a bioluminescent complex.

[0068] As used in this article, the term "SmTrip10" refers to the term corresponding to β. 10 The peptide is a peptide that can be used, for example, three-part complementarity to form a bioluminescent complex.

[0069] As used herein, the term “SmTrip9” refers to a peptide corresponding to a β9-like peptide, which can be used, for example, for triploidal complementation to form a bioluminescent complex.

[0070] 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. If the fragments of the sp polypeptide are structurally complementary and can form an active complex, they can restore the activity of the original polypeptide. Detailed Implementation

[0071] This article provides compositions comprising double-stranded RNA (dsRNA) binding domains linked to components of a complementary system. When a pair of dsRNA binding domains bind to dsRNA, a detectable complex of the complementary components is formed, and dsRNA can be detected / quantified.

[0072] Protein kinase R (PKR, Uniprot # P19525) is an intracellular dsRNA sensor containing a dsRNA-binding domain. Figure 1 (Blue and red). After PKR dimerizes on dsRNA, the kinase domain ( Figure 1 The PKR (green) is activated, thereby initiating downstream signaling pathways, which activate inflammatory pathways and shut down protein translation. The PKR binds to dsRNA in a sequence-independent manner and requires only that the dsRNA be at least 30 base pairs long (no upper limit). The PKR binds to the phosphate backbone of the dsRNA and does not interact with nucleotide bases (see, for example, Nanduri et al., EMBO J, 1998; incorporated in its entirety by reference).

[0073] This article provides a peptide construct that utilizes the dsRNA binding function of PKR and uses it to facilitate the formation of a detectable complex in the presence of dsRNA. In some embodiments, a pair 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 an associated signal. The presence and / or quantity of dsRNA in a sample (e.g., environment, organism, drug, etc.) can be detected / quantified based on the signal generated by the system described herein.

[0074] Experiments were conducted during the development of the embodiments described herein to test the exemplary systems within the scope of this document. First, commercially available LgBiT and SmBiT components of the NanoBiT system (Promega Corp, Madison, WI) were fused with the dsRNA-binding domain of PKR (see, for example...). Figure 2 ), and luminescence was detected in the presence of formalin substrate and gradually increasing concentrations of dsRNA. Figure 3 Second, splitting GFP (LgGFP and SmGFP) was fused with the dsRNA-binding domain of PKR, and fluorescence was detected in the presence of increased dsRNA concentration. Figure 4These experiments demonstrate that the dsRNA-binding domain of PKR can be used to promote structural complementarity between complementary peptides / peptides and to form detectable complexes and signals in a dsRNA concentration-dependent manner. This article provides systems for linking any suitable component of a detectable complex to the dsRNA-binding domain, and the uses of such systems for detecting dsRNA in samples.

[0075] I. PKR dsRNA binding domain

[0076] Interferon (IFN)-induced double-stranded RNA (dsRNA) activated protein kinase R (PKR) is an IFN-stimulated gene (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.; Pindel, A. and Sadler, A. (2011). J. Interferon Cytokine Res. 31, 59–70.; incorporated by reference in whole) and acts as a pathogen recognition receptor (Gilfoy, FD and Mason, PW (2007). J. Virol. 81, 11148–11158.; incorporated by reference in whole), which proceeds by recognizing dsRNA (a typical byproduct of viral infection) induced by IFN. 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; dsRNA binding induces PKR dimerization and allows exposure of the catalytic site, autophosphorylation, and kinase activation (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.; incorporated herein by reference). Activation of PKR catalyzes 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.; citation incorporated herein by reference) thereby blocking the initiation of mRNA translation, which leads to a complete halt in cellular and viral protein synthesis and can result in apoptosis in response to viral infection (Balachandran et al. (1998). EMBO J. 17, 6888–6902.; citation incorporated herein by reference).

[0077] In some embodiments, this document provides compositions of fusions comprising a dsRNA-binding domain and a component of a detectable complex. In some embodiments, the dsRNA-binding domain is capable of binding dsRNA in a sequence-independent manner. In some embodiments, the dsRNA-binding domain does not preferentially bind to the ends of dsRNA or specific RNA structures. In some embodiments, the dsRNA-binding domain binds dsRNA but not single-stranded RNA (ssRNA) or DNA (double-stranded or single-stranded). In some embodiments, the dsRNA-binding domain binds ssRNA and / or DNA at sufficiently low levels that any signal generated by such binding is within the background measured herein.

[0078] In some embodiments, the dsRNA-binding domain of the fusion compound or system described herein corresponds to the PKR dsRNA-binding domain. In some embodiments, the dsRNA-binding domain of the fusion compound described herein has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 3061 (PKR dsRNA-binding domain). In some embodiments, the dsRNA-binding domain of the fusion compound described herein has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity with SEQ ID NO: 3061 (PKR dsRNA-binding domain).

[0079] In some embodiments, the dsRNA-binding domain of the fusion compound or system described herein comprises one or more portions corresponding to a portion of the PKR dsRNA-binding domain. In some embodiments, the dsRNA-binding domain of the fusion compound or system described herein comprises a portion corresponding to a first dsRNA-binding motif of the PKR dsRNA-binding domain (SEQ ID NO: 3062). In some embodiments, all or a portion of the dsRNA-binding domain of the fusion compound described herein has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 3062 (PKR dsRNA-binding motif 1). In some embodiments, all or a portion of the dsRNA-binding domain of the fusion compound described herein has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity with SEQ ID NO: 3062 (PKR dsRNA-binding motif 1). In some embodiments, the dsRNA-binding domain of the fusion compound or system herein includes a portion of a second dsRNA-binding motif corresponding to the PKR dsRNA-binding domain (SEQ ID NO: 3063). In some embodiments, all or a portion of the dsRNA-binding domain of the fusion compound herein has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NO: 3063 (PKR dsRNA-binding motif 2). In some embodiments, all or a portion of the dsRNA-binding domain of the fusion compound herein has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence similarity with SEQ ID NO: 3063 (PKR dsRNA-binding motif 2). In some embodiments, the dsRNA binding domain of the fusion herein comprises a first segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range thereof) sequence identity with SEQ ID NO: 3062 (PKR dsRNA binding motif 1), and a second segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range thereof) sequence identity with SEQ ID NO: 3063 (PKR dsRNA binding motif 2).In some embodiments, the dsRNA-binding domain of the fusion comprises a first segment having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) 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 therebetween) 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 variants thereof) are fused by a linker of 1-100 amino acids in length. In some embodiments, the linker comprises a native linker present in the PKR dsRNA binding domain.

[0080] II. Detectable complex

[0081] In some embodiments, the present system comprises a pair of fusion compounds: (1) a first fusion compound comprising a dsRNA-binding domain linked to a first component of the detectable complex, and (2) a second fusion compound comprising a dsRNA-binding domain linked to a second component of the detectable complex. In some embodiments, the components of the complex have sufficiently low affinity for each other to require promotion to form the complex and generate a detectable signal (or the signal generated after promotion is significantly greater than the unpromoted signal, such that the unpromoted signal is present in the background). In some embodiments, binding of the dsRNA-binding domain linked to a component of the detectable complex promotes the formation of the detectable complex and the generation of a detectable signal.

[0082] In some embodiments, the complex can generate any suitable signal that allows detection of the binding of the complementary fusion pair to dsRNA. For example, upon complementation, the detectable complex can generate any suitable signal, such as fluorescence, luminescence, enzyme activity, ligand binding, etc. In some embodiments, the component corresponds to a fragment (or a variant of such fragment) of an enzyme or other protein capable of generating a detectable signal. In such embodiments, the component forms an enzyme- or other protein-like complex facilitated by the binding of the dsRNA-binding domain to dsRNA, and the corresponding signal can be detected. In other embodiments, the component can form a detectable complex that does not correspond to an existing enzyme or complex.

[0083] In some embodiments, the detectable complex corresponds to a protein that has been split into two fragments capable of non-covalently interacting to form a complex exhibiting the functional activity of the protein (see, for example, Shekhawat and Ghosh. Curr Opin Chem Biol. Dec. 2011; 15(6): 789–797; incorporated herein by reference in its entirety). In some embodiments, a suitable protein is one with detectable activity that can be reversibly eliminated by fragmenting the protein into two separate components, wherein the activity is restored when the fragments are re-associated with dsRNA by binding to dsRNA rather than covalently with a dsRNA-binding domain fused to the respective fragment. Any protein that can be split into fragments can be used in the embodiments herein, the fragments being able to associate (e.g., with a promoting effect) to restore the activity of the parent protein.Examples of such proteins include β-lactamases (Galarneau et al., Nat. Biotech. 2002;20(6):619–622.; incorporated by reference), ubiquitin (Johnsson and Varshavsky A., Proc Natl Acad Sci US A. 1994;91:10340–13044.; incorporated by reference), dihydrofolate reductase (DHFR) (Pelletier et al., Proc Natl Acad Sci US A. 1998;95:12141–12146.; incorporated by reference), focal adhesion kinase (FAK), Gal4, GFP, and variants (Ghosh et al., J. Am. Chem. Soc. 2000;122:5658–5659.; Hu and Kerppola. Nat. Biotechnol. 2003; 21:539–1545; incorporated by reference in whole) (e.g., EGFP), horseradish peroxidase, infrared fluorescent protein, various luciferases (Remy and Michnick. Nat. Meth. 2006;3(12):977–979.; Paulmurugan and Gambhir. Ana. Chem. 2003;759(7):1584–1589; incorporated by reference in whole) (e.g., recombinase-enhanced bimolecular luciferase, Gaussia princeps luciferase, firefly (Luker et al. Proc Natl Acad Sci US A. 2004;101:12288–122893; incorporated by reference in whole) etc.), tobacco etching virus (TEV) protease (Wehr et al. Nat. Meth. 2006;3(12):985–993; incorporated by reference in whole), thymidine kinase (Massoud et al. Nat. Med. 2010;16(8):921–927; incorporated by reference in its entirety), branched acid mutases (Muller et al. Prot. Sci. 2010;19(5):1000–1010; incorporated by reference in its entirety), etc. 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 are capable of producing protein-like activity upon complex formation. Other examples of detectable complex components are described below.

[0084] A. HaloTag

[0085] In some embodiments, this document provides fusions of a dsRNA-binding domain with a complementary peptide / peptide pair capable of interacting with each other (e.g., facilitated by the binding of the dsRNA-binding domain to dsRNA) to form an active modified dehalogenase complex capable of covalently bonding with a haloalkane ligand. In some embodiments, a first fusion comprising a first complementary peptide or polypeptide fragment of a modified dehalogenase is provided, and a second fusion comprising a second complementary peptide or polypeptide fragment of a modified dehalogenase is provided, wherein, upon interaction (e.g., facilitated by the binding of the dsRNA-binding domain to dsRNA), the complementary peptide / peptide forms an active modified dehalogenase complex capable of covalently bonding with a haloalkane ligand. In some embodiments, the complementary peptide / peptide is a fragment of a cleavage mutant dehalogenase.

[0086] In some embodiments, the peptide / peptide component capable of forming the modified dehalogenase complex is a fragment of a cleavage mutant dehalogenase, such as a fragment of the commercially available HALOTAG protein (Promega) and / or a mutant dehalogenase disclosed in U.S. Publication 2006 / 0024808, the disclosure of which is incorporated herein by reference.

[0087] In some embodiments, a cleavage-modified dehalogenase is provided as a component of the compositions, systems, and methods described herein. 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 mutant dehalogenase fragment has a substitution that, if present in a full-length modified dehalogenase (or corresponding complex), forms a covalent bond with a haloalkane ligand. In some embodiments, the first and second fragments of the mutant dehalogenase are capable of interacting (e.g., facilitated by the binding of the linked dsRNA-binding domain to dsRNA) to form an active modified dehalogenase complex.

[0088] HALOTAG is a 297-residue self-labeled polypeptide (33 kDa) derived from a bacterial hydrolase (dehalogenase) that has been modified to covalently bind to its ligand haloalkane moiety. The HALOTAG ligand can be linked to solid surfaces (e.g., beads) or functional groups (e.g., fluorophores), and the HALOTAG polypeptide can be fused to a variety of proteins of interest, allowing for covalent attachment of the protein to a solid surface or functional group. The HALOTAG polypeptide is a modified dehalogenase with a genetically modified active site that specifically binds to the haloalkane ligand chloroalkane linker with an enhanced and increased ligand-binding rate (Pries et al., The Journal of Biological Chemistry. 270(18):10405–11; incorporated by reference in its entirety). The reaction that forms a bond between the protein tag and the chloroalkane linker is rapid and essentially irreversible under physiological conditions (Waugh DS (June 2005). Trends in Biotechnology. 23(6):316–20; incorporated by reference in its entirety). In native hydrolases, nucleophilic attack on 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 the amino acid residue within the wild-type hydrolase (Chen et al. (February 2005) Current Opinion in Biotechnology. 16(1):35–40; quotations incorporated herein by reference). This results in enzyme regeneration after the reaction. However, with the modified haloalkane dehalogenase HALOTAG, the reaction intermediate cannot undergo a second reaction because it cannot be hydrolyzed due to a mutation in the enzyme. This results in the intermediate persisting as a stable covalent adduct without the associated reverse reaction (Marks et al. (August 2006) Nature Methods. 3(8):591–6; quotations incorporated herein by reference). Various HALOTAG ligands, functional groups, fusions, assays, modifications, uses, etc., are described in U.S. Patent Nos. 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 US 2014 / 0322794. The entire contents of each patent are incorporated herein by reference.

[0089] In some embodiments, the modified dehalogenase fragments, complementary peptides, complementary polypeptides, etc., described herein are based on the HALOTAG complementation 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 comprising two or more peptide or polypeptide components described herein correspond to the HALOTAG protein and are capable of binding haloalkyl ligands in a similar manner.

[0090] In some embodiments, such as those described in U.S. Provisional Application No. 63 / 338,323 and PCT Application No. PCT / US23 / 20959, all of which are incorporated herein by reference in their entirety, extensive experiments have demonstrated the feasibility of generating HALOTAG fragments (and variants thereof) capable of interacting to form modified dehalogenase complexes capable of binding haloalkyl ligands, and the feasibility of optimizing variants of the HALOTAG fragments to obtain desired properties. As described herein, embodiments are not limited to the HALOTAG sequence. In some embodiments, this document provides dehalogenases with cleavage modifications that differ in sequence from HALOTAG (SEQ ID NO: 1) (e.g., as fusions with dsRNA-binding domains).

[0091] In some embodiments, compositions and systems are provided comprising components of a cleavage-modified dehalogenase, such as cleavage HALOTAG (“spHT”) or variants thereof (e.g., as a fusion with a dsRNA-binding domain). In some embodiments, the systems and compositions herein comprise spHT peptides and polypeptides (e.g., as part of the fusions described herein).

[0092] 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 a modified dehalogenase sequence derived from SEQ ID NO: 1 (HALOTAG).

[0093] MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETF QAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG.

[0094] In some embodiments, the spHT peptide and polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) have at least 70% sequence identity with a portion of SEQ ID NO: 1 (e.g., >70% sequence identity, >75% sequence identity, >80% sequence identity, >85% sequence identity, >90% sequence identity, >95% sequence identity, >96% sequence identity, >97% sequence identity, >98% sequence identity, >99% sequence identity). In some embodiments, the spHT peptide and polypeptide (e.g., as part of a fusion with a dsRNA-binding domain) have 100% sequence identity with all or a portion of SEQ ID NO: 1. In some embodiments, the spHT peptide and polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) have at least 70% sequence similarity to all or a portion of SEQ ID NO: 1 (e.g., >70% sequence similarity, >75% sequence similarity, >80% sequence similarity, >85% sequence similarity, >90% sequence similarity, >95% sequence similarity, >96% sequence similarity, >97% sequence similarity, >98% sequence similarity, >99% sequence similarity). In some embodiments, the spHT peptide and polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) have 100% sequence similarity to all or a portion of SEQ ID NO: 1.

[0095] In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA-binding domain) contains A at position 2 corresponding to SEQ ID NO: 1. In other embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA-binding domain) contains S at position 2 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA-binding domain) contains V at position 47 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA-binding domain) contains T at position 58 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA-binding domain) contains G at position 78 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains F at position 88 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains M at position 89 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains F at position 128 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with the dsRNA binding domain) contains T at position 155 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains K at position 160 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with a dsRNA-binding domain) contains V at position 167 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with a dsRNA-binding domain) herein contains T at position 172 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with a dsRNA-binding domain) contains M at position 175 corresponding to SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains G at position 176 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains N at position 195 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains E at position 224 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains D at position 227 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains K at position 257 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains A at position 264 corresponding to SEQ ID NO: 1. In some embodiments, the peptide or polypeptide herein contains N at position 272 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains L at position 273 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains S at position 291 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide herein (e.g., as part of a fusion with the dsRNA binding domain) contains T at position 292 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains E at position 294 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains I at position 295 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains S at position 296 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide (e.g., as part of a fusion with the dsRNA binding domain) contains G at position 297 corresponding to SEQ ID NO: 1.

[0096] In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an S at position 2 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an L at position 47 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an S at position 58 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a D at position 78 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a Y at position 88 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an L at position 89 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a C at position 128 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an A at position 155 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an E at position 160 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an A at position 167 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an A at position 172 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a K at position 175 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a C at position 176 corresponding to SEQ ID NO: 1.In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a K at position 195 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an A at position 224 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an N at position 227 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an E at position 257 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a T at position 264 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an H at position 272 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a Y at position 273 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have a P at position 291 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an A at position 292 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not have an amino acid at position 294 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at position 295 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at position 296 corresponding to SEQ ID NO: 1. In some embodiments, the spHT peptide or polypeptide described herein (e.g., as part of a fusion with a dsRNA-binding domain) does not contain an amino acid at position 297 corresponding to SEQ ID NO: 1.

[0097] 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 a portion of SEQ ID NO:1 (e.g., >70% sequence similarity or identity, >75% sequence similarity or identity, >80% sequence similarity or identity, >85% sequence similarity or identity, >90% sequence similarity or identity, >95% sequence similarity or identity, >96% sequence similarity or identity, >97% sequence similarity or identity, >98% sequence similarity or identity, >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., at least 70% sequence similarity or 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., at least 70% sequence similarity or identity with the second portion). In some embodiments, the sp dehalogenase (e.g., spHT) comprises two fragments that together have 100% sequence similarity or identity with all or a portion of SEQ ID NO: 1. For example, the first fragment of the sp polypeptide has 100% sequence similarity or identity with the first portion of SEQ ID NO: 1, and the second fragment of the sp polypeptide has 100% sequence similarity or identity with the second portion of SEQ ID NO: 1.

[0098] 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 location in the parental 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 polypeptide of theoretically 100 amino acids is separated by an sp site (referred to herein as the sp site of 57) between residues 57 and 58 of the parental polypeptide, the first component polypeptide would correspond to positions 1-57, and the second component polypeptide would correspond to positions 58-100. In some embodiments herein, the sp site within SEQ ID NO: 1 can appear at any position from position 5 to position 290 of SEQ ID NO: 1. In some embodiments, SEQ ID NO: 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 that collectively contain amino acids corresponding to each position in SEQ ID NO: 1. For example, a peptide having the sequence of SEQ ID NO: 26 and a peptide having the sequence of SEQ ID NO: 27 together comprise amino acids corresponding to each position in SEQ ID NO: 1. Any peptide and polypeptide pair (or two polypeptides) corresponding to two of SEQ ID NO: S2-577 and together comprising amino acids corresponding to each position in SEQ ID NO: 1 (with or without positional deletions or repetitions) can be used in the embodiments described herein. In some embodiments, the spHT dehalogenase comprises any of the following fragment pairs (e.g., fused with a dsRNA-binding domain): SEQ ID NO: NO: 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 47, 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, 6 8 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, 10 0 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 W 125, 126 W 127, 128 W 129, 130 W 131, 132 W 133, 134 W 135, 136 W 137, 138 W 139, 140 W 141, 142 W 143, 144 W 145, 146 W 147, 148 W 149, 150 W 151, 152 W 153, 154 W 1 55, 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, 19 6 W 197, 198 W 199, 200 W 201, 202 W 203, 204 W 205, 206 W 207, 208 W 209, 190 W 211, 212 W 213, 214 W 215, 216 W 217, 218 W 219, 220 W 221, 222 W 223, 224 W 225, 226 W 227 , 228 W 229, 300 W 301, 302 W 303, 304 W 305, 306 W 307, 308 W 309, 310 W 311, 312 W 313, 314 W 315, 316 W 317, 318 W 319, 320 W 321, 322 W 323, 324 W 325, 326 W 327, 328 W 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, 3 60 W 361, 362 W 363, 364 W 365, 366 W 367, 368 W 369, 370 W 371, 372 W 373, 374 W 375, 376 W 377, 378 W 379, 380 W 381, 382 W 383, 384 W 385, 386 W 387, 388 W 389, 390 W 39 1, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422 Wa 423, 424 Wa 425, 426 Wa 427, 428 Wa 429, 430 Wa 431, 432 Wa 433, 434 Wa 435, 436 Wa 437, 438 Wa 439, 440 Wa 441, 442 Wa 443, 444 Wa 445, 446 Wa 447, 448 Wa 449, 450 Wa 451, 452 Wa 453,454 and 455, 456 and 457, 458 and 459, 460 and 461, 462 and 463, 464 and 465, 466 and 467, 468 and 46 9, 470 and 471, 472 and 473, 474 and 475, 476 and 477, 478 and 479, 480 and 481, 482 and 483, 484 and 485, 486 and 487, 488 and 489, 490 and 491, 492 and 493, 494 and 495, 496 and 497, 498 and 499, 50 0 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 53 1, 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, 56 2 and 563, 564 and 565, 566 and 567, 568 and 569, 570 and 571, 572 and 573, 574 and 575, and 576 and 577. ,

[0099] In some embodiments, spHT comprises pairs of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NO:2-577, which together contain amino acids corresponding to each position in SEQ ID NO:1, but with 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) deleted at the C-terminus or N-terminus of one or both segments. For example, a pair corresponding to SEQ ID NO:7 and 28 together corresponds to the position in SEQ ID NO:1, but with 11 residues deleted. In some embodiments, any pair of SEQ ID NO:2-577 together corresponds to the sequence of SEQ ID NO:1, but with up to 40 amino acids deleted, all of which are within the range of spHT herein. In some embodiments, the deletion is adjacent to the cleavage site. In some embodiments, the deletion corresponds to the N-terminus or C-terminus of SEQ ID NO:1.

[0100] In some embodiments, spHT comprises pairs of peptides and polypeptides (or two polypeptides) corresponding to two of SEQ ID NO:2-577, which together contain amino acids corresponding to each position in SEQ ID NO:1, but with up to 40 amino acid repeats (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or in between) at the C-terminus or N-terminus of one or both segments. For example, a pair corresponding to SEQ ID NO:6 and 29 together corresponds to the position in SEQ ID NO:1, but has 11 residue repeats. In some embodiments, any pair of SEQ ID NO:2-577 together corresponds to the sequence of SEQ ID NO:1, but has up to 40 amino acid repeats, all of which are within the scope of spHT herein. In some embodiments, the repeats are adjacent to the splitting site. In some embodiments, the repeats correspond to the N-terminus or C-terminus of SEQ ID NO:1.

[0101] Using any sp site fragment, for example, the position corresponding to position 5 and position 290 of SEQ ID NO: 1, is readily conceived and is within the scope of this document.

[0102] In some implementations, a value corresponding to SEQ ID NO is provided. spHT at the following sp sites of 1: 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, 1 47, 148, 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, 179, 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, 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, 265, 266, 267, 268269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290.

[0103] In some embodiments, spHT is provided having sp sites at positions corresponding to positions 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 SEQ ID NO: 1.

[0104] In some embodiments, the spHT peptides and polypeptides described herein (e.g., fused with a dsRNA binding domain) contain one or more (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) substitutions or deletions relative to one of SEQ ID NO: 2-557. In some embodiments, sp peptides and polypeptides (e.g., fused with a dsRNA-binding domain) are provided that have 70%-100% sequence identity with one of SEQ ID NO: 2-557 (e.g., >70% sequence identity, >75% sequence identity, >80% sequence identity, >85% sequence identity, >90% sequence identity, >95% sequence identity, >96% sequence identity, >97% sequence identity, >98% sequence identity, >99% sequence identity). In some embodiments, sp peptides and polypeptides (e.g., fused with a dsRNA-binding domain) are provided that have 70%-100% sequence similarity (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%) with one of SEQ ID NO: 2-557.

[0105] In some embodiments, a pair of sp peptides and / or polypeptides (e.g., fused with a dsRNA-binding domain) capable of forming an active sp dehalogenase complex (active spHT complex) is provided. In some embodiments, such pairs have at least 70% sequence identity or similarity to the two in SEQ ID NO:2-557 and together contain residues corresponding to 100% of the positions in SEQ ID NO:1, thereby allowing up to 40 deletions or repeats at the C-terminus or N-terminus of the peptide / polypeptide.

[0106] In some implementations, the first fragment of the spHT complement pair (e.g., fused with the dsRNA binding domain) corresponds to SEQ ID NO: Positions 1 to 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, 14 6, 147, 148, 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, 179, 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, 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, 264265, 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.

[0107] In some implementations, the second fragment of the spHT complement pair (e.g., fused with the dsRNA binding domain) corresponds to SEQ ID NO: Position 1: 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, 14 8, 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, 179, 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, 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, 265, 266267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290 to position 294.

[0108] In some implementations, the repeating portion of the spHT complementary pair has a length of 1-40 amino acids (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 therein).

[0109] In some implementations, the length of the missing portion of the spHT complement pair is 1-40 amino acids (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 therein).

[0110] The exemplary spHT fragment sequences of SEQ ID NO: 2-577 have 100% sequence identity with the portion of SEQ ID NO: 1; those sequences that were not aligned do not have a portion with 100% sequence identity with SEQ ID NO: 1. However, as described herein, spHT peptides and polypeptides may have less than 100% sequence identity with SEQ ID NO: 1 (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99% but less than 100% sequence identity). Therefore, this document provides peptides and polypeptides having less than 100% sequence identity with one of SEQ ID NO: 2-577 (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99% but less than 100% sequence identity) and which may be used in complementary pairs and complexes herein.

[0111] In some embodiments, the spHT complementary pair herein comprises a peptide corresponding to SEQ ID NO: 578 and a polypeptide corresponding to SEQ ID NO: 1188. SEQ NO: 578 and 1188 are fragments of SEQ ID NO: 1 and have 100% sequence identity with the 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; this peptide is referred to herein as “SmHT”. In some embodiments, the spHT complementary pair comprises a polypeptide having 100% sequence identity with SEQ ID NO: 1188; this polypeptide is referred to herein as “LgHT”. Numerous experiments have been performed to analyze variants of SmHT and LgHT. SEQ ID NO: 579-1187 correspond to peptide variants of SEQ ID NO: 588 up to all positions being substituted. Peptides of each of SEQ ID NO: 578-1187 were synthesized, and various properties, including the ability to form active complexes with complementary LgHT variant polypeptides, were tested. SEQ ID NO: 1189-3033 correspond to polypeptide variants having one or more substitutions relative to SEQ ID NO: 1188. Peptides of each of SEQ ID NO: 1188-3033 were synthesized, and various properties were tested, including the ability to form active complexes with complementary SmHT variant peptides.

[0112] In some embodiments, this document provides an SmHT peptide or a SmHT variant peptide (e.g., fused with a dsRNA-binding domain) having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range therebetween) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NO: 578-1187. In some embodiments, the peptide (e.g., fused with a dsRNA-binding domain) corresponds to SmHT (SEQ ID NO: 578), but is substituted with one or more of one or more of SEQ ID NO: 588-1187 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a range therebetween) relative to SEQ ID NO: 578. In some implementations, the SmHT variant (e.g., fused with the dsRNA binding domain) has 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or a range thereof) nonconserved substitutions relative to one of SEQ ID NO: 578-1187.

[0113] In some embodiments, this document provides a SmHT peptide or a SmHT variant peptide (e.g., fused with a dsRNA-binding domain) comprising:

[0114] 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 (SEQ ID NO: 3034); and / or

[0115] 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 (SEQ ID NO: 3035);

[0116] Each X is any amino acid (e.g., proteogenic amino acid).

[0117] In some embodiments, this document provides an LgHT polypeptide or an LgHT variant polypeptide (e.g., fused with a dsRNA-binding domain) having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or a range therebetween) sequence similarity (e.g., conserved or semi-conserved similarity) to one of SEQ ID NO: 1188-3033. In some embodiments, the polypeptide (e.g., within the fusion herein or as a standalone reporter molecule or tag, etc.) corresponds to LgHT (SEQ ID NO: 1188), but is substituted with one or more of SEQ ID NO: 1189-3033 (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 a range therebetween) relative to SEQ ID NO: 1188. In some implementations, the LgHT variant (e.g., fused with a dsRNA binding domain) has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100% or in between) sequence identity with one of SEQ ID NO: 1188-3033.

[0118] B. NanoLuc

[0119] In some embodiments, this document provides fusions of dsRNA-binding domains with complementary peptide / peptide pairs, said peptide / peptide pairs being capable of interacting with each other (e.g., facilitated by the binding of the dsRNA-binding domain to dsRNA) to form a luminescent complex capable of interacting with a luminescent substrate to produce luminescence. In some embodiments, the luminescent complex is capable of producing significantly enhanced luminescence upon interaction with the luminescent substrate compared to a single complementary peptide / peptide or in the presence of a luminescent substrate without a complementary pair.

[0120] In some embodiments, a first fusion comprising a first complementary peptide or polypeptide fragment of luciferase is provided, and a second fusion comprising a second complementary peptide or polypeptide fragment of luciferase is provided, wherein, upon interaction (e.g., facilitated by binding of a dsRNA-binding domain to dsRNA), the complementary peptide / polypeptide formation is capable of producing a luminescent, active luciferase complex in the presence (contact) of a suitable substrate of luciferase. In some embodiments, the complementary peptide / polypeptide is a fragment that cleaves luciferase.

[0121] In some embodiments, the peptide / peptide component capable of forming the luciferase complex is a fragment of luciferase, such as those derived from commercially available NANOLUC luciferase (Promega), including but not limited to NANOBIT (Promega), SMBIT peptide (Promega), and LGBIT peptide (Promega), which are capable of promoting the formation of the luminescent complex. In some embodiments, the peptide / peptide component capable of forming the luciferase complex is any peptide and peptide component described in U.S. Patent No. 9,797,889 and / or U.S. Application Serial No. 16 / 439,565 (incorporated herein by reference in its entirety).

[0122] In some embodiments, this document provides compositions (e.g., fusion peptides) and systems (e.g., multiple complementary fusion peptides, substrates, etc.) comprising peptide / peptide fragments capable of interacting (e.g., facilitated by binding of dsRNA to dsRNA via a dsRNA-binding domain fused thereto) to form an active luminescent complex capable of generating luminescence using a suitable substrate.

[0123] In some embodiments, this document provides fusion peptides and systems thereof (e.g., multiple complementary fusion peptides, substrates, etc.) comprising a dsRNA-binding domain fused to a complementary peptide / peptide fragment, the complementary peptide / peptide fragment being capable of interacting (e.g., facilitated by the binding of the dsRNA-binding domain fused thereto to dsRNA) to form an active bioluminescent complex capable of producing luminescence upon interaction with a suitable luminescent substrate. In some embodiments, a first fusion comprising a peptide / peptide fragment of a luminescent protein is provided, and a second fusion comprising a complementary peptide / peptide fragment of a luminescent protein is provided, wherein, upon interaction (e.g., facilitated by the binding of the dsRNA-binding domain fused thereto to dsRNA), the complementary peptide / peptide fragment forms an active bioluminescent complex capable of producing luminescence upon interaction with a suitable luminescent substrate. In some embodiments, the complementary peptide / peptide is a fragment of a cleaved luminescent protein (e.g., luciferase).

[0124] In some embodiments, this document provides fusion pairs of a dsRNA-binding domain and a component of a binary complementary system capable of forming a luminescent complex. In other embodiments, a tertiary or multi-level complementary system (e.g., three or more components) may be used in fusions with the dsRNA-binding domain and system described herein. For example, fusions of a dsRNA-binding domain and three or more components of a system may be provided. Alternatively, the system may comprise two fusions of a dsRNA-binding domain and a luminescent complex component, and one or more additional components of the luminescent complex as separate components.

[0125] Natural deep-sea shrimp luciferase (OgLuc) and commercially available NANOLUC luciferase (Promega Corporation) each contain polypeptides (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10) comprising 10 β (beta) chains. U.S. Patent No. 9,797,889 (incorporated herein by reference in its entirety) describes the development and use of complementary systems comprising β1-9-like polypeptides and β10-like peptides (some of the OgLuc / NANOLUC-based polypeptide and peptide sequences in U.S. Patent No. 9,797,889 differ in polypeptide and peptide sequence from the corresponding sequences of NANOLUC and wild-type natural OgLuc). Similarly, U.S. Patent Application Serial No. 16 / 439,565 (incorporated herein by reference in its entirety) describes the development and use of complementary systems comprising two or more OgLuc / NANOLUC peptides and / or polypeptides (some of the OgLuc / NANOLUC-based polypeptides and peptide sequences in U.S. Patent No. 16 / 439,565 differ from the corresponding sequences in NANOLUC and wild-type natural OgLuc).

[0126] In some embodiments, a peptide component of a binary bioluminescent complex having greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) sequence identity with SEQ ID NO: 3036 is provided in a fusion with the dsRNA-binding domain herein, wherein when the peptide component of the binary bioluminescent complex contacts the polypeptide consisting of SEQ ID NO: 3037 (e.g., facilitated by binding of the dsRNA-binding domain to dsRNA) in the presence of a substrate of the bioluminescent complex (e.g., greater luminescence of the component of the complex in the presence of a substrate), a detectable bioluminescent signal is generated. 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, a detectable bioluminescent signal is generated when the peptide component of the binary bioluminescent complex contacts (e.g., promoted by binding of the dsRNA binding domain to dsRNA) a polypeptide component of the binary bioluminescent complex having a sequence identity greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) with SEQ ID NO: 3037. In some embodiments, a detectable bioluminescent signal is generated or significantly increased when the peptide associates with a polypeptide comprising SEQ ID NO: 3037 or composed of SEQ ID NO: 3037. In a preferred embodiment, the peptide exhibits one or more alterations (e.g., enhancements) in one or more properties compared to the peptide of SEQ ID NO: 3038 or 3039, wherein said properties are selected from: affinity for the peptide composed of SEQ ID NO: 3037, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the peptide composed of SEQ ID NO: 3037 (e.g., in the context of dsRNA-binding domain fusions herein).

[0127] Exemplary sequences of peptide components of binary bioluminescent complexes that can be used in embodiments herein (e.g., as part of a fusion with a dsRNA-binding domain) are described, for example, in U.S. Patent No. 9,797,889 (incorporated entirely by reference). While peptide components of binary bioluminescent complexes (e.g., within fusions herein) are not limited to these sequences, in some embodiments, peptide components of binary bioluminescent complexes herein may be selected from the amino acid sequences of SEQ ID NOs: 3-438 and 2162-2365 of U.S. Patent No. 9,797,889 (incorporated entirely by reference).

[0128] In some embodiments, this document provides a peptide component (e.g., as a fusion with a dsRNA-binding domain) of a binary bioluminescent complex having greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) sequence identity with SEQ ID NO: 3038, wherein, when a substrate of the bioluminescent complex is present (e.g., greater luminescence of the component of the complex in the presence of a substrate), the peptide component of the binary bioluminescent complex contacts (e.g., facilitated by binding of the dsRNA to the dsRNA-binding domain fused thereto) a polypeptide (e.g., fused with a dsRNA-binding domain) composed of SEQ ID NO: 3037, generating a detectable bioluminescent signal. In some embodiments, the peptide has less than 100% sequence identity with SEQ ID NO: 3036. In some embodiments, a detectable bioluminescent signal is generated when a peptide component of a binary bioluminescent complex (e.g., fused with a dsRNA-binding domain) contacts a polypeptide component of a binary bioluminescent complex having greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) sequence identity with SEQ ID NO: 3037. In some embodiments, a detectable bioluminescent signal is generated or significantly increased when the peptide associates with a polypeptide comprising SEQ ID NO: 3037 or composed of SEQ ID NO: 3037.

[0129] Exemplary sequences of peptide components of binary bioluminescent complexes that can be used in the embodiments herein (e.g., as fusions with dsRNA-binding domains) are described, for example, in U.S. Patent No. 9,797,889 (incorporated entirely by reference). Although the peptide components of the binary bioluminescent complexes herein are not limited to these sequences, in some embodiments, the peptide components of the binary bioluminescent complexes herein may be selected from SEQ ID NO: 441-2156 of U.S. Patent No. 9,797,889 (incorporated entirely by reference).

[0130] In some embodiments, this document provides a fusion of a dsRNA-binding domain and a polypeptide component of a binary bioluminescent complex having greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%, 100%) sequence identity with SEQ ID NO: 3037, wherein when the polypeptide contacts the peptide composed of SEQ ID NO: 3036 or 3038 (e.g., within the fusion herein) in the presence of a substrate of the bioluminescent complex (e.g., greater luminescence of the component of the complex in the presence of a substrate), a detectable bioluminescent signal is generated.

[0131] In some embodiments, this document provides fusion pairs, each fusion containing a dsRNA binding domain, and a first fusion containing a first component of a bioluminescent complex having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or a range thereof) sequence identity with the first fragment of SEQ ID NO: 3041, and a second fusion containing a second component of a bioluminescent complex having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or a range thereof) sequence identity with the complementary portion of SEQ ID NO: 3041. In some embodiments, the first component has a sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3042, and the complementary component has a sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3050. In some embodiments, the first component has a sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3043, and the complementary component has a sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3051. In some embodiments, the first component has a sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3044, and the complementary component has a sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3052.In some embodiments, the first component has 40% or greater sequence identity with SEQ ID NO: 3045 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or a range thereof), and the complementary component has 40% or greater sequence identity with SEQ ID NO: 3053 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or a range thereof). In some embodiments, when the first component associates with the complementary component, the bioluminescent signal is significantly increased (e.g., promoted by the binding of the fused dsRNA-binding domain to dsRNA).

[0132] Exemplary sequences of peptide and polypeptide components of binary or multi-component bioluminescent complexes that can be used in embodiments herein for fusion with dsRNA binding domains are described, for example, in U.S. Patent Application Serial No. 16 / 439,565 (incorporated in its entirety by reference). Although the peptide and polypeptide components of binary or multi-component bioluminescent complexes herein are not limited to these sequences, in some embodiments, the peptide or polypeptide components of binary or multi-component bioluminescent complexes herein may be selected from SEQ ID NO: 1-804 of U.S. Patent Application Serial No. 16 / 439,565 (incorporated in its entirety by reference).

[0133] In some embodiments, this document provides β6-7-like peptides comprising SEQ ID NO: 3054 and 3055 (e.g., within the fusions described herein). In some embodiments, this document provides β6-7-like peptides having sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) with SEQ ID NO: 3054 and 3055.

[0134] In some embodiments, this document provides β7-8-like peptides comprising SEQ ID NO: 3055 and 3056 (e.g., within the fusions described herein). In some embodiments, this document provides β7-8-like peptides having sequence identity of SEQ ID NO: 3055 and 3056 having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof).

[0135] In some embodiments, this document provides a β8-9-like peptide comprising SEQ ID NO: 3056 / 3059 or 3056 / 3060 (e.g., within the fusions described herein). In some embodiments, this document provides a β8-9-like peptide having sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or within the range thereof) to NO: 3056 / 3059 or 3056 / 3060.

[0136] In some embodiments, this document provides a β9-10-like peptide comprising SEQ ID NO: 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058 (e.g., within the fusions described herein). In some embodiments, this document provides a β8-9-like peptide having sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater (e.g., 100%) or within the range thereof) to NO: SEQ ID NOS: 3059 / 3057, 3059 / 3058, 3060 / 3057, or 3060 / 3058 (e.g., 100%) or within the range thereof).

[0137] In some embodiments, this document provides β6-8-like peptides or polypeptides comprising SEQ ID NO: 3054-3056 (e.g., within the fusions described herein). In some embodiments, this document provides β6-8-like peptides or polypeptides having sequence identity of NO: SEQ ID NO: 3054-3056 having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or within the range thereof).

[0138] In some embodiments, this document provides a β7-9-like peptide or polypeptide comprising SEQ ID NO: 3055 / 3056 / 3059 or 3055 / 3056 / 3060 (e.g., within the fusions described herein). In some embodiments, this document provides a β7-9-like peptide or polypeptide having sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or within the range thereof) to NO: SEQ ID NO: 3055 / 3056 / 3059 or 3055 / 3056 / 3060.

[0139] In some embodiments, this document provides a β8-10-like peptide or polypeptide comprising SEQ ID NO: 3056 / 3059 / 3057, 3056 / 3059 / 3058, 3056 / 3060 / 3057, or 3056 / 3060 / 3058 (e.g., in the fusions described herein). In some embodiments, this document provides a β7-9-like peptide or polypeptide having sequence identity of 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater (e.g., 100%) or within the range thereof) to NO: SEQ ID NO: 3056 / 3059 / 3057, 3056 / 3059 / 3058 (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater (e.g., 100%) or within the range thereof).

[0140] C. Fluorescent proteins

[0141] In some embodiments, this document provides fusions of dsRNA-binding domains with complementary peptide / peptide pairs, said peptide / peptide pairs being capable of interacting with each other (e.g., facilitated by the binding of the dsRNA-binding domain to dsRNA) to form a fluorescent complex capable of emitting fluorescence (emission spectrum) within a detectable range when excited with an appropriate wavelength (excitation spectrum). In some embodiments, the fluorescent complex is capable of producing fluorescence that is significantly enhanced than either of the individual complementary peptides / peptides.

[0142] In some embodiments, a first fusion comprising a first complementary peptide or polypeptide fragment of a fluorescent protein is provided, and a second fusion comprising a second complementary peptide or polypeptide fragment of a fluorescent protein is provided, wherein, upon interaction (e.g., facilitated by binding of a dsRNA-binding domain to dsRNA), the complementary peptide / polypeptide forms an active fluorescent complex capable of emitting fluorescence within a detectable range (emission spectrum) when excited with an appropriate wavelength (excitation spectrum). In some embodiments, the complementary peptide / polypeptide is a fragment of a fluorescent protein.

[0143] In some embodiments, fragments of fluorescent proteins are provided for use in the embodiments described 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, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, cyanin, dansyl chloride, phycocyanin, and phycoerythrin. Table 1 provides examples of existing splitting fluorescent proteins that can be used in the embodiments described herein (e.g., for use with two components fused to a dsRNA binding domain).

[0144] Table 1. Exemplary Splitting Fluorescent Proteins

[0145] EBFP2 EBFP2(1-10) and Capri(1-10) were used with GFP(11). Multiplexed labeling of cellular proteins with split fluorescentprotein tags. TamuraR, Jiang F, Xie J,Kamiyama D. CommunBiol. 2021 Cerulean Cerulean(1-10) used with GFP(11) Multiplexed labeling of cellular proteins with split fluorescentprotein tags. TamuraR, Jiang F, Xie J,Kamiyama D. CommunBiol. 2021 ECFP ECFP pairs C155 with N173 derived from Cerulean or Venus. Identification of newfluorescent proteinfragments for bimolecularfluorescencecomplementationanalysis underphysiologicalconditions. Shyu YJ,Liu H, Deng X, Hu CD.Biotechniques. 2006 sfGFP Split the superfolder GFP, our most desired GFP(1-10) and GFP(11). Versatile proteintagging in cells withsplit fluorescentprotein. Kamiyama etal. Nat Commun. 2016 spGFP Splitting superposition GFP Split-superpositiveGFP reassembly is afast, efficient, androbust method for detecting protein-protein interactions in vivo. Blakeley BD,Chapman AM, McNaughtonBR. Mol Biosyst. 2012 mNeonGreen2 mNG2(1-10) and mNG2(11) Improved splitfluorescent proteins for endogenous proteinlabeling. Feng et al. NatCommun. 2017 mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) are used together with mNG2(11) Improved yellow-greensplit fluorescentproteins for proteinlabeling and signalamplification. Zhou S,Feng S, Brown D, HuangB. PLoS One. 2020 Venus pBiFC-VN173, pBiFC-VC155 Identification of newfluorescent proteinfragments for bimolecularfluorescencecomplementationanalysis underphysiologicalconditions. Shyu YJ,Liu H, Deng X, Hu CD.Biotechniques. 2006 Venus Improved N-terminalfragment VN155(I152L),for use with VC155 An improved bimolecular fluorescencecomplementation assaywith a high signal-to-noise ratio. Kodama Y,Hu CD. Biotechniques.2010 mVenus pET-BiFC contains two fragments of split mVenus (aa 155), including the I152L fragment in the N-terminal segment. An enhanced recombinant amino-terminal acetylationsystem and novel invivo high-throughputscreen for molecules affecting alpha-synucleinoligomerisation. Eastwood T, Baker K, Brooker H, Frank S, Mulvihill DP. FEBSLett. 2017 mVenus Split mVenus (aa 155), including I152L in the N-terminal segment. Constant rate of p53tetramerization inresponse to DNA damagecontrols the p53response. Gaglia G,Lahav G. Mol SystBiol. 2014 EYFP Gateway vector containing EYFP split at position 175 Arabidopsis homolog of the yeast TREX-2 mRNAexport complex:components andanchoring nucleoporin.Lu et al. Plant J. 2010 mScarlet Split wrmScarlet variant of mScarlet Split-wrmScarlet andsplit-sfGFP: tools for faster, easier fluorescent labelingof endogenous proteins in Caenorhabditiselegans. Goudeau et al. Genetics. 2021 mRuby4 mRuby4(1-10) is paired with sfCherry2(11). Multiplexed labeling of cellular proteins with split fluorescentprotein tags. TamuraR, Jiang F, Xie J,Kamiyama D. CommunBiol. 2021 sfCherry2 sfCherry2(1-10) and sfCherry2(11) are also photoactivated variants of PAsfCherry2(1-10). Improved splitfluorescent proteins for endogenous proteinlabeling. Feng et al. NatCommun. 2017 sfCherry3 Improved sfCherry3C(1-10) used with sfCherry2(11) Bright split redfluorescent proteins for the visualization of endogenous proteins and synapses. Feng Set al. Commun Biol.2019 iRFP iSplit contains the PAS and GAF ​​domains of iRFP713. It requires the presence of heme oxygenase to form chromophores (rich in eukaryotic cells). A Near-Infrared BiFCReporter for In VivoImaging of Protein-Protein Interactions. Filonov GS, VerkhushaVV. Chem Biol. 2013 iRFP The GAF domain, derived from miRFP709 and miRFP670, pairs with the common PAS domain. Heme oxygenase is required to form the chromophore (rich in eukaryotic cells). Bright monomeric near-infrared fluorescentproteins as tags and biosensors for multiscale imaging. Shcherbakova et al. NatCommun. 2016 FAST Fluorescent activation and absorption conversion tag (FAST), used in conjunction with yellow-green or orange-red fluorophores. A split fluorescent reporter with rapidand reversiblecomplementation. TeboAG, Gautier A. NatCommun. 2019

[0146] As an exemplary implementation, the components of splitting GFP (the peptide of SEQ ID NO: 3070 and the polypeptide of SEQ ID NO: 3068) are fused into the dsRNA binding domain (SEQ ID NO: 3061) via the linker of SEQ ID NO: 3064. In some embodiments, this document provides fusion pairs, each fusion containing a dsRNA binding domain, and a first fusion containing a first component of a luciferin complex having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) sequence identity with the first fragment of SEQ ID NO: 3068, and a second fusion containing a second component of a bioluminescent complex having 40% or greater (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater (e.g., 100%) or in the range thereof) sequence identity with the complementary portion of SEQ ID NO: 3070.

[0147] III. Fusion

[0148] In some embodiments, the fusions described herein comprise a dsRNA-binding domain directly linked to a component of the detectable complex. In some embodiments, the dsRNA-binding domain comprises two directly linked dsRNA-binding motifs. However, in other embodiments, the dsRNA-binding domain and a component of the detectable complex and / or two dsRNA-binding motifs in the dsRNA-binding domain are fused via a linker. Such linkers can be any suitable sequence and are up to 100 amino acids in length (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 amino acids or in the range thereof). In some embodiments, the fusions described herein comprise a linker with an optimized geometry that promotes complementarity, ligand / substrate binding, dsRNA binding, etc.

[0149] In one embodiment, the components of the fusion herein (e.g., dsRNA binding domains and / or motifs, detectable complex components, adapters, etc.) can be arranged in any suitable orientation to allow dsRNA binding, formation of structural complementarity in the detectable complex, and generation of a 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 an adapter). 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 an adapter). 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 an adapter), 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 an adapter).

[0150] IV. Substrate, ligand, and cofactor

[0151] In some embodiments, the detectable complexes used in the embodiments described herein utilize substrates, ligands, cofactors, etc., to generate a detectable signal. In such embodiments, systems and methods are provided that comprise suitable substrates, cofactors, and / or ligands of the detectable complex and its components.

[0152] In some embodiments, the ligands / substrates of the present invention are permeable to the cell membrane (i.e., capable of entering the cell from the outside (e.g., eukaryotes, prokaryotes) without causing chemical, enzymatic, or mechanical damage to the cell membrane).

[0153] In some embodiments, the ligands herein comprise cleavable connectors, such as those described in U.S. Patent No. 10,618,907, which is incorporated herein by reference in its entirety.

[0154] For the systems and methods described herein that utilize various cleavage enzymes as detectable complexes, appropriate substrates and / or cofactors are provided for such systems. For example, dihydrofolate, ATP, tetramethylbenzidine, chorismite, etc., can be provided to generate detectable signals with the assembled detectable complexes.

[0155] A. Halogenated alkyl ligands

[0156] In embodiments where the detectable complex comprises a modified dehalogenase complex (e.g., cleavage of HALOTAG), systems and methods comprising a haloalkane ligand are provided. The modified dehalogenase complex (e.g., structural complementarity formation facilitated by the binding of the fusion compound to dsRNA as described herein) binds the haloalkane ligand, and the functional group linked to the haloalkane allows for detection. In some embodiments, the haloalkane ligand has the formula (I): R-linker-AX, where R is the detectable functional group, where the linker is a multi-atom straight or branched chain comprising C, N, S, or O, or a group comprising one or more rings, such as saturated or unsaturated rings, such as one or more aryl rings, heteroaryl rings, or any combination thereof, where AX is a ligand of the modified dehalogenase (e.g., HALOTAG) (e.g., where A is (CH2)). 4-20 And X is a halogen (e.g., Cl or Br). Suitable ligands are described, for example, in U.S. Patent Nos. 11,072,812; 11,028,424; 10,618,907; and 10,101,332; all of which are incorporated herein by reference in their entirety. In some embodiments, X of formula (I) is a methanesulfonamide or trifluoromethanesulfonamide, rather than a halide; this embodiment yields an exchangeable ligand that reversibly binds 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; all of which are incorporated herein by reference in their entirety.

[0157] In some embodiments, R is one or more functional groups (e.g., fluorophores, biotin, luminescent materials, or fluorescent or luminescent molecules). Exemplary functional groups used in this invention include, but are not limited to, amino acids, proteins (e.g., enzymes, antibodies, or other immunogenic proteins), radionuclides, nucleic acid molecules, drugs, lipids, biotin, avidin, streptoavidin, magnetic beads, solid supports, electronically opaque molecules, chromophores, MRI contrast agents, dyes (e.g., thallium dyes, calcium-sensitive dyes (e.g., 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-thallyl)-phenoxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (Fluo-3)), sodium-sensitive dyes (e.g., 1,3-benzenediacarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy- [6,2-benzofurandiyl]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.

[0158] In some implementations, the ligand contains a fluorescent functional group (R). Suitable fluorescent functional groups include, but are not limited to, stilbazolium derivatives (Marquesa et al., Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling. ChemRxiv. Cambridge: Cambridge OpenEngage;). 2021; incorporated by reference), xanthones (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), anthocyanin derivatives (e.g., anthocyanin, indole-carbon anthocyanin, oxycarbon anthocyanin, sulfur-carbon anthocyanin, naphthalene derivatives (e.g., dansyl sulfonyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., waterfall blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresol violet, oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethyl derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA FLOUR (Invitrogen), DYLIGHT FLUOR (ThermoScientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU and SQUARE dye (SETABioMedicals), QUASAR and CAL FLUOR dye (Biosearch Technologies), SURELIGHT dye (APC, RPE, PerCP, phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc.

[0159] In some embodiments, the ligand comprises a fluorescent functional group (R). A fluorescent functional group is a functional group that generates and enhances a fluorescence signal upon binding of the ligand to a target (e.g., the binding of a haloalkane to a modified dehalogenase). The problem of background signal 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 target binding. Exemplary fluorescent dyes used in the embodiments herein include the JANELIA FLUOR family of fluorophores, such as:

[0160] JANELIA FLUOR 549, SE:

[0161] ,

[0162] JANELIA FLUOR 646, SE:

[0163] ,

[0164] JANELIA FLUOR 585, SE:

[0165] ,

[0166] JANELIA FLUOR 635, SE:

[0167] as well as

[0168] JANELIA FLUOR 669, SE:

[0169]

[0170] (See, for example, U.S. Patent Nos. 9,933,417; 10,018,624; 10,161,932; and 10,495,632; each of which is incorporated herein by reference in its entirety.) In some embodiments, exemplary conjugates of JANELIA FLUOR 549 and JANELIA FLUOR 646 with haloalkane ligands (e.g., HALOTAG) for the modified dehalogenase are commercially available (Promega Corp.). The use and design of fluorescent functional groups, dyes, probes, and ligands are described, for example, in Grimm et al., Nat Methods. 3117 Oct;14(10):987-994.; Wang et al., Nat Chem. 3120 Feb;12(2):165-172; all of which are incorporated herein by reference in their entirety.

[0171] B. Bioluminescent substrates

[0172] In some embodiments, the systems and methods described herein include using a luminescent material to generate a luminescent complex that can detect a luminescent signal. In some embodiments, a suitable luminescent material is selected and paired with the luminescent complex.

[0173] In some embodiments, the systems and methods described herein (comprising bioluminescent complexes and / or components thereof) utilize imidazopyrazine luminescent substrates to generate bioluminescence. Such embodiments include those utilizing luminescent complexes based on NANOLUC, NANOBIT, and NANOTRIP. In some embodiments, the substrate is coelenterate.

[0174] .

[0175] In some embodiments, the substrate is a coelentin derivative, such as formalan, formalan analogs (e.g., fluoroformazan), coelentin-n, coelentin-f, coelentin-h, coelentin-hcp, coelentin-cp, coelentin-c, coelentin-e, coelentin-fcp, dideoxycoelentin (“coelentin-hh”), coelentin-i, coelentin-icp, coelentin-v, and 2-methylcoelentin, as disclosed in WO 2003 / 040100; U.S. Application Serial No. 12 / 056,073 (paragraph

[0086] ); U.S. Patent No. 8,669,103; and those disclosed in U.S. Provisional Application No. 63 / 379,573; the disclosures of the aforementioned patents are incorporated herein by reference in their entirety.

[0176] In some implementations, the substrate is formazan:

[0177] .

[0178] In some implementations, the substrate is flufomarazine:

[0179] .

[0180] Suitable luminescent organisms for use in the bioluminescent complexes of this paper will be understood. For example, firefly luciferin with the following structure:

[0181] Luciferin is a luciferin found in many species of the Lampyridae family and is a substrate for beetle luciferase. Latina luciferin has the following structure:

[0182] It is a bacterial luciferin derived from the freshwater snail *Latia neritoides*, possessing the following structure:

[0183] It can be used as a substrate for many bacterial luciferases. Coelenterin has the following structure:

[0184] It is found in radiolarians, comb jellies, cnidarians, squid, burrowing stars, copepods, chaetognaths, fish, and shrimp, and is a luminescent substrate for luciferases in these organisms. Variants and derivatives of coelenterate, such as formazan and fluformazan, can be used in the embodiments described herein (e.g., with Oplophorus-derived bioluminescent complexes). Other luminescent substrates include those from the following: *Dystrophariae*.

[0185] ,

[0186] Sea Firefly Fluorescein (Sea Firefly Fluorescein):

[0187] as well as

[0188] N. nambi:

[0189] .

[0190] The pairing of appropriate bioluminescent proteins or complexes with luminescent organisms is understood in the art.

[0191] V. Method

[0192] In some embodiments, this document provides a method for detecting the presence and / or quantifying the amount of dsRNA in a sample, 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 a detectable complex) and any necessary substrates, ligands, cofactors, etc.; and (b) detecting and / or quantifying the signal generated by the detectable complex. In some embodiments, the amount (e.g., intensity) of the signal is correlated with the amount of dsRNA in the sample. In some embodiments, the signal is compared with a signal from a control sample having a known concentration of dsRNA. In some embodiments, the signal is compared with a determined value corresponding to a known concentration of dsRNA in the sample.

[0193] In some embodiments, the sample is a biological sample, an environmental sample, a drug sample (e.g., a therapeutic agent containing RNA (e.g., ssRNA)), or any suitable sample type that may contain dsRNA (e.g., as a contaminant). In some embodiments, the sample contains therapeutic RNA, and the method described herein is performed as a quality control test to ensure that the amount of dsRNA in the sample is sufficiently low.

[0194] experiment

[0195] Example 1

[0196] Splitting bioluminescent protein

[0197] Fusions of PKR dsRNA binding domains with LgBiT and SmBiT (PKR-LgBiT and PKR-SmBiT) were expressed in *E. coli* and purified using a His tag. Protein expression and purification were satisfactory. Experiments conducted during the development of the embodiments described herein demonstrated that when the PKR-LgBiT and PKR-SmBiT components (25 ng / ml each) were added to a sample containing Poly(I:C) (synthetic dsRNA analogs) and formazan, sensitive (limit of detection < 0.1 ng / ml) and rapid (1 hour assay time) dsRNA quantification was achieved in an easy-to-use (add-mix-read) assay format. Figure 3 The optimal incubation time was determined to be 1 hour. The exemplary assays are quantitative and have a linear range of approximately 2 logarithms.

[0198] Example 2

[0199] Split fluorescent protein

[0200] 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 demonstrated that when the PKR-LgBiT and PKR-SmBiT components were added to a sample containing dsRNA and exposed to the excitation wavelength of GFP, they enabled sensitive and rapid dsRNA quantification. Figure 4 ).

[0201] References

[0202] The following references are incorporated into this paper in their entirety by way of citation.

[0203] Cheng et al. Visualizing double-stranded RNA distribution anddynamics in living cells by dsRNA bidding-dependent fluorescencecomplementation. Virology. 2015 (PMID: 26351203).

[0204] Monsion et al. Efficient detection of long dsRNA in vitro and in vivousing the dsRNA biding domain from FHV B2 protein. Front Plant Sci. 2018.(PMID: 29449856)

Claims

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

2. The system of 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 of claim 2, wherein the first and second components of the detectable complex exhibit low affinity for each other in the absence of a promoting effect of binding to the dsRNA via the first and second dsRNA binding domains.

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

5. The system of claim 1, wherein the first dsRNA binding domain and the second dsRNA binding domain comprise the same amino acid sequence.

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

8. The system of claim 7, wherein the dsRNA binding domain comprises the dsRNA binding motif of SEQ ID NO: 3062.

9. The system of claim 7, wherein the dsRNA binding domain comprises the dsRNA binding motif of SEQ ID NO: 3063.

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

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

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

13. The system of claim 1, wherein the dsRNA binding domain has at least 70% sequence similarity to SEQ ID NO: 3061.

14. The system of claim 13, wherein the dsRNA binding domain has at least 70% sequence identity with SEQ ID NO: 3061.

15. The system of claim 14, wherein the dsRNA binding domain comprises SEQ ID NO: 3061.

16. The system of claim 1, wherein the detectable complex is capable of generating a detectable signal.

17. The system of claim 16, wherein the amount of signal generated by the detectable complex is correlated with the amount of dsRNA in the sample via the system.

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

19. The system of claim 1, wherein the first and second components of the detectable complex are fragments of proteins capable of generating a detectable signal, and wherein the detectable complex is capable of generating the detectable signal when the first and second components of the detectable complex associate.

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

21. The system of 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 of 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, dichlorotriazineamine fluorescein, anthocyanin, dansyl chloride, phycocyanin, and phycoerythrin.

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

24. The system of claim 23, wherein the first component has at least 70% sequence identity with SEQ ID NO: 3068, and the second component has at least 70% sequence identity with SEQ ID NO: 3070.

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

26. The system of 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 of claim 26, wherein the enzyme is selected from β-lactamase, dihydrofolate reductase (DHFR), focal adhesion kinase (FAK), Gal4, and horseradish peroxidase.

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

29. The system of 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 of claim 29, wherein the luciferase is selected from... genus *Spinylobacter* Luciferase, firefly luciferase, click beetle luciferase Seaweed Luciferase, sea firefly luciferase, Jellyfish essence Luminescent proteins and oberin luminescent proteins.

31. The system of claim 28, wherein the first and second components of the detectable complex together have at least 70% sequence identity with SEQ ID NO: 3041.

32. The system of claim 31, wherein the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3042, and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3050.

33. The system of claim 31, wherein the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3043, and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3051.

34. The system of claim 31, wherein the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3044, and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3052.

35. The system of claim 31, wherein the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3045, and the first component of the detectable complex has at least 70% sequence identity with SEQ ID NO:3053.

36. The system of claim 38, further comprising the substrate.

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

38. The system of claim 38, wherein the detectable complex is a modified dehalogenase complex, and wherein 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 of claim 38, wherein the modified dehalogenase comprises SEQ ID NO:

1.

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

41. The system of claim 40, wherein the haloalkyl ligand comprises an R-connector-AX, wherein R is a detectable moiety, X is a halogen, and AX is a substrate for a dehalogenase.

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

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

44. The system of claim 43, further comprising the substrate of the luminescent complex.

45. The system of claim 44, wherein the substrate of the luminescent complex is an imidazopyrazine luminescent material.

46. ​​The system of claim 44, wherein the imidazopyrazine luminescent agent is coelenterin or formazan.

47. The system of claim 43, further comprising dsRNA.

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

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

50. The system of claim 48, wherein the dsRNA binding domain has at least 70% sequence similarity to SEQ ID NO: 3061.

51. The system of claim 50, wherein the dsRNA binding domain has at least 70% sequence identity with SEQ ID NO: 3061.

52. The system of claim 50, wherein the dsRNA binding domain comprises SEQ ID NO: 3061.

53. The system of 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 of 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 of 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, the method comprising contacting the sample with a system as described in any one of claims 1-55 and detecting a signal from the detectable complex, wherein the amount of signal detected is correlated with the amount of dsRNA in the sample.

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

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