Fusion proteins for purifying nucleic acids

Abstract

Provided herein are fusion proteins and methods for purifying nucleic acids from a composition. The disclosed compositions and methods allow for faster, more efficient purification of nucleic acids compared to traditional affinity chromatography.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Application No. 63 / 578,551, filed August 24, 2023, and U.S. Application No. 63 / 554,715, filed February 16, 2024, the contents of which are incorporated herein by reference in their entirety for all purposes. Reference to electronic sequence listing

[0002] The contents of the electronic sequence list (ISOL_011_02WO_SeqList_ST26.xml; size: 1,566,780 bytes; and creation date: August 21, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure generally relates to fusion proteins, compositions containing fusion proteins, and methods for using fusion proteins to purify nucleic acids. Background Technology

[0004] The use of nucleic acids as therapeutic agents is rapidly increasing, as highlighted by mRNA-based vaccines against SARS-CoV-2. Purification of nucleic acids, such as mRNA, is challenging because mRNA is a large molecule, much larger than proteins. Due to its large size, mRNA does not interact well with conventional affinity chromatography resins. Furthermore, the impurity profile of nucleic acid compositions is often complex, and it is difficult to find a purification platform that can remove all contaminants from the composition.

[0005] There is a need in the art for improved compositions and methods for rapidly and cost-effectively purifying nucleic acids, such as mRNA. Summary of the Invention

[0006] This disclosure provides fusion proteins containing nucleic acid-binding proteins and methods for purifying nucleic acids using these fusion proteins, as well as methods for using these fusion proteins.

[0007] This document provides compositions comprising a fusion protein. In embodiments, the fusion protein comprises a nucleic acid binding protein (NBP) and a polypeptide having phase behavior. In the embodiments, the NBP is selected from any of the following: RNA-specific adenosine deaminase 1 (ADAR1), ADAR1 double-stranded RNA-binding domain 3 (dsRBD3), Bacillus subtilis cold shock protein B (Bs-CspB), cold shock domain Y-box protein (CSD-Y box), eukaryotic translation initiation factor 4E (eIF4e), Fox-1 protein (FOX1), heterogeneous nucleoribonucleoprotein Q1 (hnRNPQ1), Homo sapiens zinc finger CCCH type 14 protein (HsZC3H14), poly-A binding protein (PABP), poly-A binding protein nucleus 1 (PABPN1), triangular pentapeptide repeat protein A (PPRpA), Pumilio-like repeat protein A (PUFpA), Staufen, 12-O-tetradecanoylphorbolol-13-acetate inducible sequence 11D (TIS11D), Z-DNA / RNA binding protein 1 (ZBP1), and zinc finger nuclease (ZNF). In the embodiments, the NBP has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 61-103. In the embodiments, the NBP is selected from any one of the following: ADAR1 double-stranded RNA-binding domain 3 (dsRBD3), heterogeneous nucleoribonucleoprotein Q1 (hnRNPQ1), Homo sapiens zinc finger CCCH type 14 protein (HsZC3H14), poly-A binding protein nucleus 1 (PABPN1), triangular pentapeptide repeat protein A (PPRpA), and Pumilio-like repeat protein A (PUFpA). In the embodiments, the NBP has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 89, 93, 95, or 97-99.In the embodiments, the polypeptide exhibiting phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 57 or 60. In the embodiments, the polypeptide exhibiting phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 57 or 60.

[0008] In an embodiment, the fusion protein comprises a linker. In an embodiment, the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 143-216.

[0009] In embodiments, the NBP binds to RNA, DNA, or both. In embodiments, the NBP binds to RNA selected from any of the following: double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), mRNA, precursor mRNA, polyadenosine (polyA) RNA, Z-conformation RNA (Z-RNA), or combinations thereof. In embodiments, the NBP binds to the 3' end of mRNA, the 3' end of mRNA, the 3' untranslated region (UTR) of mRNA, the polyA tail of mRNA, or AU-rich elements of mRNA, or combinations thereof. In embodiments, the NBP binds to precursor mRNA. In embodiments, the NBP binds to introns, exons, polyA tails, or combinations thereof of precursor mRNA. In embodiments, the NBP binds to DNA. In embodiments, the NBP binds to the following: single-stranded DNA, double-stranded DNA, polyadenosine (polyA) DNA, Z-conformation DNA (Z-DNA), or combinations thereof.

[0010] In this embodiment, the fusion protein is encoded by a nucleic acid. In this embodiment, the nucleic acid has a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acids of any one of SEQ ID NO:119-133. In this embodiment, the fusion protein is encoded by a vector. In an embodiment, the vector comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acids of any one of SEQ ID NOs: 104-118, 134, and 135. In another embodiment, the vector has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acids of any one of SEQ ID NOs: 119-133.

[0011] This document provides methods for purifying nucleic acids, including (i) contacting a composition comprising the nucleic acid and at least one contaminant with a fusion protein disclosed herein, wherein the fusion protein binds to the nucleic acid to form a complex; (ii) contacting the complex with a first environmental factor to increase the size of the complex; (iii) separating the complex from at least one contaminant; and (iv) separating the nucleic acid from the fusion protein by contacting the complex with a second environmental factor. In embodiments, the complex is separated from at least one contaminant based on size. In embodiments, size-based separation is performed using any of the methods selected from: tangential flow filtration, membrane chromatography, analytical ultracentrifugation, high-performance liquid chromatography, membrane chromatography, conventional flow filtration, acoustic separation, centrifugation, countercurrent centrifugation, and rapid protein liquid chromatography. In an embodiment, the first environmental factor includes one or more of the following: (a) changes in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of viral particles, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturants; or (c) the application of electromagnetic waves or sound waves. In an embodiment, the second environmental factor includes one or more of the following: (a) changes in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of viral particles, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturants; or (c) the application of electromagnetic waves or sound waves. In an embodiment, the at least one contaminant is selected from solvents, proteins, peptides, carbohydrates, nucleic acids, viruses, cells (e.g., bacterial, yeast, or mammalian cells), carbohydrates, lipids, or lipopolysaccharides.

[0012] This document provides a fusion protein comprising Bacillus subtilis cold shock protein B (Bs-CspB) and a phase-behaving polypeptide. In embodiments, the Bs-CspB has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 90. In embodiments, the phase-behaving polypeptide comprises P and G motifs, the P and G motifs comprising a plurality of proline residues and a plurality of glycine residues. In embodiments, the P and G motifs comprise at least about 10% proline residues and at least about 20% glycine residues. In an embodiment, the phase-behaving polypeptide comprises a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 217), or a random, disordered analogue thereof; wherein Xaa can be any amino acid other than proline. In an embodiment, n is an integer from 1 to 360, including endpoints.In the embodiments, the phase-behaving polypeptide comprises an amino acid sequence selected from the following: a. (GRGDSPY)n (SEQ ID NO: 1); b. (GRGDSPH)n (SEQ ID NO: 2); c. (GRGDSPV)n (SEQ ID NO: 3); d. (GRGDSPYG)n (SEQ ID NO: 4); e. (RPLGYDS)n (SEQ ID NO: 5); f. (RPAGYDS)n (SEQ ID NO: 6); g. (GRGDSYP)n (SEQ ID NO: 7); h. (GRGDSPYQ)n (SEQ ID NO: 8); i. (GRGNSPYG)n (SEQ ID NO: 9); j. (GVGVP)n (SEQ ID NO: 10); k. (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 10). 11); l.(GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 12); m.(GVGVPGWGVPGVGVPGWGVPGVGVP)m (SEQ ID NO: 13); n.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m (SEQ ID NO: 14); o.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m (SEQ ID NO: 15); and p.(GAGVPGVGVPGAGVPGVGVPGAGVP)m (SEQ ID NO: 16); or random, disordered analogues thereof; wherein: n is an integer in the range of 20-360, inclusive; and m is an integer in the range of 4-25, inclusive. In the embodiments, the polypeptide having phase behavior comprises an amino acid sequence selected from the following: a. (GVGVP)m (SEQ ID NO: 22); b. (ZZPXXXXGZ)m (SEQ ID NO: 23); c. (ZZPXGZ)m (SEQ ID NO: 24); d. (ZZPXXGZ)m (SEQ ID NO: 25); or e. (ZZPXXXGZ)m (SEQ ID NO: 26), wherein m is an integer between 10 and 160, including the endpoints, wherein X is any amino acid other than proline or glycine, and wherein Z is any amino acid, if present.

[0013] In an embodiment, the phase-behavior polypeptide comprises an amino acid sequence selected from the following: a. (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 17); or b. (GVGVPGVGVPGLGVPGVGVPGVGVP)m (SEQ ID NO: 18); wherein m is an integer between 2 and 32, including the endpoints. In an embodiment, the polypeptide having phase behavior comprises an amino acid sequence selected from the following: a. (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 19), where m is 8 or 16; b. (GVGVPGAGVP)m (SEQ ID NO: 20), where m is an integer between 5 and 80, including the endpoints; or c. (GXGVP)m (SEQ ID NO: 21), where m is an integer between 10 and 160, including the endpoints, and wherein each repeating X is independently selected from the group consisting of: glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine. In the embodiments, the polypeptide exhibiting the phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 56 or 262-264. In the embodiments, the polypeptide exhibiting the phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 262-264. In the embodiments, the polypeptide exhibiting phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 56.In the embodiments, the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 90, and at least 90% identity with the polypeptides of any one of SEQ ID NO: 56 and 262-264. In the embodiments, the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 90, and at least 90% identity with the polypeptide of SEQ ID NO: 56.

[0014] In an embodiment, the fusion protein comprises a linker. In an embodiment, the linker has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 261. In an embodiment, the linker has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 261. In the embodiments, the fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 225 or 226.

[0015] In the embodiments, the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 225 or 226. In the embodiments, the Bs-CspB binds to RNA, DNA, or both. In the embodiments, the Bs-CspB binds to RNA, DNA, or both. In the embodiments, the Bs-CspB binds to ssRNA. In the embodiments, the Bs-CspB binds to the 3' end, 5' end, coding region, non-coding region, or combination thereof of mRNA. In the embodiments, the Bs-CspB binds to precursor mRNA. In the embodiments, the Bs-CspB binds to introns, exons, 5' UTR, 3' UTR, or combinations thereof of precursor mRNA. In the embodiments, the Bs-CspB binds to DNA. In the embodiments, the Bs-CspB binds to single-stranded DNA, double-stranded DNA, polyadenosine (polyA) DNA, Z-conformation DNA (Z-DNA), or combinations thereof.

[0016] This document provides a nucleic acid encoding a fusion protein. In embodiments, the nucleic acid has a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the nucleic acids in SEQ ID NO:119-133.

[0017] This article provides a vector encoding a fusion protein. This article provides a vector containing nucleic acids. This article provides a vector having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid of any one of SEQ ID NO: 104-118, 134, and 135.

[0018] This document provides a method for purifying single-stranded nucleic acids, the method comprising: (i) contacting a composition comprising the single-stranded nucleic acid and at least one contaminant with a fusion protein as described in any one of claims 1-28, wherein the fusion protein binds to the single-stranded nucleic acid to form a complex; (ii) adding a first environmental factor to the composition comprising the complex, thereby increasing the size of the complex; (iii) separating the complex from at least one contaminant; and (iv) separating the single-stranded nucleic acid from the fusion protein by contacting the complex with a second environmental factor, thereby forming a product comprising the single-stranded nucleic acid. In an embodiment, the single-stranded nucleic acid comprises ssRNA. In an embodiment, the complex is separated from at least one contaminant based on size. In an embodiment, size-based separation is performed using a method selected from any of the following: tangential flow filtration, membrane chromatography, analytical ultracentrifugation, high-performance liquid chromatography, conventional flow filtration, acoustic separation, centrifugation, countercurrent centrifugation, and rapid protein liquid chromatography. In an embodiment, size-based separation is performed using a method including centrifugation. In an embodiment, the first environmental factor includes one or more of the following: (a) changes in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of viral particles, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturants; or (c) the application of electromagnetic waves or sound waves. In an embodiment, the second environmental factor includes one or more of the following: (a) changes in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of viral particles, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturants; or (c) the application of electromagnetic waves or sound waves. In an embodiment, the at least one contaminant is selected from solvents, proteins, peptides, carbohydrates, double-stranded nucleic acids, viruses, cells (e.g., bacterial, yeast, or mammalian cells), carbohydrates, lipids, or lipopolysaccharides. In an embodiment, the at least one contaminant is a double-stranded nucleic acid. In an embodiment, the double-stranded nucleic acid is dsRNA. In an embodiment, salt is added at a concentration ranging from about 0.5 M to about 3 M. In an embodiment, salt is added at a concentration of about 1.2 M to about 1.7 M. In an embodiment, based on the total nucleic acid content in the product of step (iv), the product contains about 70% to about 100% single-stranded nucleic acid. In an embodiment, the single-stranded nucleic acid contains ssRNA. In an embodiment, based on the total nucleic acid content in the product, the product contains about 10% or less of at least one contaminant. In an embodiment, the at least one contaminant contains dsRNA. In an embodiment, the purification method includes removing at least about 1-log of contaminant compared to the amount of contaminant in the composition of step (i).In an embodiment, the purification method includes removing 1 to 10-log of contaminants compared to the amount of contaminants in the composition of step (i). In an embodiment, in step (i), the fusion protein is present at a concentration of about 1 µM to about 200 µM. In an embodiment, in step (i), the fusion protein is present at a concentration of about 30 µM to about 60 µM. Attached Figure Description

[0019] The accompanying drawings, which are incorporated herein and form part of this specification, illustrate some, but not unique or exclusive, exemplary embodiments and / or features. The embodiments and drawings disclosed herein are intended to be illustrative rather than restrictive.

[0020] Figure 1 This study demonstrates a comparison of the nonspecific binding of various peptides exhibiting phase behavior to RNA templates and linearized plasmid DNA of varying lengths and chain strengths using agarose gel electrophoresis. Successful binding of the biopolymer to nucleic acid species is indicated by its retention within the well. Peptide 40L80 (SEQ ID NO: 264), exhibiting phase behavior, demonstrates nonspecific binding to a wide range of nucleic acid species.

[0021] Figure 2 The elution percentage of total RNA captured directly from an in vitro transcription (IVT) reaction transcribed from a range of mRNA template sizes using an ssRNA purification reagent comprising a fusion protein (SEQ ID NO: 225) containing Bs-CspB (SEQ ID NO: 90) and a phase-behaving polypeptide (referred to as 100V80, SEQ ID NO: 56). The elution percentage reflects the final yield after elution compared to the total RNA present in the initial IVT reaction.

[0022] Figure 3A and Figure 3B The ssRNA purification reagent containing Bs-CspB and 100V80 showed high selectivity for ssRNA. Figure 3A The following diagram illustrates the logarithmic activity of dsRNA during RNA capture of purified RNA incorporating dsRNA at a given ssRNA purification reagent:RNA molar ratio. 10 Removal value (LRV). This reagent retains selectivity for ssRNA and is able to remove dsRNA greater than 4-log. Figure 3B The elution percentage of total dsRNA in samples with a range of dsRNA incorporation percentages is shown. Selectivity was still maintained in the capture of purified RNA despite dsRNA incorporation values ​​as high as 10% of total RNA.

[0023] Figure 4 Screening of ssRNA binding candidates for a broad range of ssRNA templates and dsRNA targets in independent capture reactions using agarose gel electrophoresis is demonstrated. The fusion protein containing Bs-CspB and 100V80 selectively captures a broad range of ssRNA templates. The presence of bands indicates uncaptured nucleic acids. Detailed Implementation definition

[0024] Unless otherwise expressly indicated by the context, the singular forms “a / an” and “the” used herein and in the appended claims include plural referents. Thus, for example, reference to “protein” may refer to a single protein or a mixture of such proteins, and reference to “method” includes reference to equivalent steps and / or methods known to those skilled in the art, and so on.

[0025] As used herein, the terms “about” or “approximately” when preceding a numerical value indicate a range of that value plus or minus 10%. For example, “about 100” covers 90 and 110.

[0026] Furthermore, as used herein, “and / or” means and covers any and all possible combinations of one or more of the related listed items, as well as the absence of combinations when interpreted in an alternative manner (“or”).

[0027] Unless the context otherwise requires, the various features described herein are specifically intended for use in any combination.

[0028] Furthermore, this disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For further illustration, if, for example, the specification indicates that a particular amino acid may be selected from A, G, I, L, and / or V, then this language also indicates that the amino acid may be selected from any subset of those amino acids, such as A, G, I, or L; A, G, I, or V; A or G; L only; etc., as if each such sub-combination were explicitly set forth herein. Furthermore, this language also indicates that one or more of these specified amino acids may be omitted. For example, in a particular embodiment, the amino acid is not A, G, or I; not A; not G or V; etc., as if each such possible omission were explicitly set forth herein.

[0029] As used herein, the term "environmental factor" is any factor that alters one or more properties of a composition when applied to a composition containing a fusion protein. Non-limiting examples of environmental factors include changes in one or more of temperature, pH, salt concentration, concentration of the fusion protein, concentration of the nucleic acid, or pressure; the addition of one or more surfactants, molecular crowding agents, denaturants, reducing agents, or oxidizing agents; or the application of electromagnetic or acoustic waves.

[0030] As used herein, the term "contaminant" can refer to any undesirable substance in the purified composition. In embodiments, a contaminant is any substance other than the nucleic acid to be purified. Non-limiting examples of contaminants include, but are not limited to, solvents, proteins, peptides, carbohydrates, nucleic acids, viruses, cells (e.g., bacterial, yeast, or mammalian cells), carbohydrates, lipids, or lipopolysaccharides. In embodiments, a contaminant is an endotoxin or a fungal toxin. In embodiments, the cell is an immune cell. In embodiments, the immune cell is a T cell, B cell, NK cell, peripheral blood mononuclear cell, monocyte, macrophage, or neutrophil. In embodiments, the cell is a T cell expressing a chimeric antigen receptor (CAR). In embodiments, the contaminant is a double-stranded nucleic acid.

[0031] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence. The term “peptide” can refer to short-chain amino acids, including, for example, native peptides, recombinant peptides, synthetic peptides, or combinations thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins.

[0032] A "polynucleotide" is a sequence of nucleotide bases and can be RNA, DNA, or a DNA-RNA hybrid sequence (including naturally occurring and / or non-naturally occurring nucleotides). In the examples, the polynucleotide is a single-stranded or double-stranded DNA sequence.

[0033] As used herein, “isolated” or “purified” (or grammatically equivalent) nucleic acid means that the nucleic acid is at least partially separated from at least some other components (e.g., cell lysates) in the starting material containing the nucleic acid. In the examples, the “isolated” or “purified” nucleic acid may be enriched by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more compared to the starting material.

[0034] As used herein, the term "peptide exhibiting phase behavior" refers to any peptide capable of undergoing a phase transition. In the embodiments, the peptide undergoes a phase transition due to the application of environmental factors. Exemplary peptides exhibiting phase behavior include elastin-like peptides (ELPs) and arthropod elastin-like peptides (RLPs).

[0035] As used herein, the term "fusion protein" refers to a polypeptide produced when two heterologous nucleotide sequences or fragments thereof encoding two (or more) different polypeptides not found to fuse together in nature are fused together in the correct translation reading frame.

[0036] As used herein, the term "nucleic acid binding protein" (also known as NBP) can refer to any amino acid sequence (protein, peptide, etc.) that binds to a target nucleic acid. In embodiments, an NBP may comprise a full-length, truncated, or modified form of a receptor for the target nucleic acid. In embodiments, an NBP may be an antigen-binding portion of a monoclonal antibody (e.g., Fab), a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand for the target nucleic acid; a peptide with sufficient affinity for the target nucleic acid; a single-domain conjugate, such as a camel conjugate; an artificial conjugate, such as Darpin; or a single-chain conjugate derived from a T-cell receptor.

[0037] As used herein, the term "fragment" includes a truncated form of a protein or polypeptide when it refers to a protein or polypeptide. For example, a fragment of an NBP may comprise about 50% to about 99.9% of the full-length NBP. In embodiments, fragments of NBP comprise about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% of the amino acids of the full-length NBP.

[0038] As used herein, the term "capture efficiency" in relation to the fusion protein described herein refers to the amount of nucleic acid captured by the fusion protein relative to the amount of nucleic acid present in the starting composition. The capture efficiency is determined using the following equation: 100 x (amount of nucleic acid captured by the fusion protein / amount of nucleic acid in the composition before purification).

[0039] In the context of two or more nucleic acid or polypeptide sequences, the term "percentage identity" refers to two or more sequences or subsequences that have a specified percentage of identical nucleotide or amino acid residues when compared. Unless otherwise stated, sequence identity is determined using the Basic Local Alignment Search Tool (BLAST®) from the National Center for Biotechnology Information (NCBI), available at blast.ncbi.nlm.nih.gov / Blast.cgi. In the examples, sequence identity is calculated over the entire length of the compared sequences. In the examples, sequence identity is calculated over fragments of approximately 20, 50, 75, 100, 250, 500, 750, or 1000 amino acids in each compared sequence. Fusion protein

[0040] This disclosure provides fusion proteins and methods for purifying nucleic acids using these fusion proteins. In embodiments, the fusion protein comprises an NBP (i.e., a nucleic acid-binding protein) that binds to a target nucleic acid and a polypeptide having phase behavior, wherein the NBP is coupled to the polypeptide having phase behavior. Nucleic acid binding protein

[0041] In embodiments, the fusion protein comprises a nucleic acid binding protein (NBP). In embodiments, the NBP binds to one or more nucleic acids. In embodiments, the NBP binds to single-stranded nucleic acids (e.g., single-stranded DNA or RNA). In embodiments, the NBP binds to double-stranded nucleic acids (e.g., double-stranded DNA or RNA). In embodiments, the NBP binds to DNA. In embodiments, the DNA is single-stranded or double-stranded. In embodiments, the NBP binds to RNA. In embodiments, the RNA is single-stranded or double-stranded. In embodiments, the NBP binds to both DNA and RNA, wherein the DNA and RNA are single-stranded, double-stranded, or a combination of both. In embodiments, the NBP binds to polyadenosine (polyA) DNA. In embodiments, the NBP binds to polyadenosine (polyA) RNA. In embodiments, the NBP binds to polythymidine (polyT) DNA. In embodiments, the NBP binds to polyuridine (polyU) RNA. In embodiments, the NBP binds to polycytidine (polyC) RNA. In embodiments, the NBP binds to polyguanidine (polyG) RNA. In one example, NBP binds to polycytidine (polyC) DNA. In another example, NBP binds to polyguanidine (polyG) DNA.

[0042] In the embodiments, NBP binds to the following: DNA, microRNA, capped RNA, DNA, double-stranded RNA, transfer RNA, ribosomal RNA, intranuclear small RNA, regulatory RNA, ribozymes, transfer RNA, or messenger RNA.

[0043] In this embodiment, NBP binds to AU-rich RNA elements (AREs). AREs are elements rich in adenosine monophosphate (AMAP) and uridine monophosphate (URP) in the 5′ or 3′ untranslated region of mRNA. AREs contain the core sequence AUUUA. AREs are determinants of RNA stability and are commonly found in the mRNA of proto-oncogenes, nuclear transcription factors, and cytokines. Proteins that bind to AREs are called ARE-binding proteins (ARE-BPs). In this embodiment, ARE-BPs stabilize mRNA. Non-limiting examples of ARE-BPs include human antigen R (huR, also known as “ELAV”), tritrityl nucleotide (tristetrapolin, TTP), AU-rich RNA-binding proteins (AUFs), and Fragile X intellectual disability syndrome-associated protein 1 (FXR1). The following articles describe ARE-BP and are incorporated herein by reference in their full text: Otsuka et al. Front. Genet. [Fronts in Genetics], May 2, 2019; Brennan and Steitz. Cell Mol Life Sci. [Cell Molecular Life Sciences], Feb. 2001; 58(2):266-77; Carballo et al. 1998. Science, 281, 1001-1005; Mazan-Mamczarz et al. Oncogene [Oncogenes], Vol. 27, pp. 6151–6163 (2008); Vasudevan and Steitz. Cell. [Cell], Mar 23, 2007; 128(6):1105-18; Curr Cancer Drug Targets. [Current Cancer Drug Targets] 2019; 19(5):382-399; J Biol Chem. [Journal of Biochemistry] April 28, 2017; 292(17):6869-6881. doi: 10.1074 / jbc.M116.772947. Electronic publication on March 16, 2017; Wiley Interdiscip Rev RNA. [Wiley Interdiscip Rev RNA] July-August 2014; 5(4):549-64. doi: 10.1002 / wrna.1230; and Elife. August 2, 2017; 6:e26129. doi: 10.7554 / eLife.26129; Mazan-Mamczarz et al. 2008. NucleicAcids Research, 37, 204-214. In the examples, NBP binds to ARE. In the embodiments, the NBP associated with ARE is incorporated with a bonding element of huR, TTP, AUF, or FXR1.ARE discloses in its references, which are incorporated herein by reference in their entirety: Barreau C et al. AU-rich elements and associated factors: arethere unifying principles? [AU-rich elements and associated factors: are there unifying principles?] NucleicAcids Res. 2006 Jan 3; 33(22):7138-50. In the examples, NBP binds to Z-conformation DNA (Z-DNA).

[0044] In the embodiments, NBP binds to Z-conformation RNA (Z-RNA). Typically, Z-DNA and R-DNA are the left-handed structures of DNA and RNA, respectively, as described in the reference, which is incorporated herein by reference in its entirety: Barreau C et al. AU-richelements and associated factors: are there unifying principles? [AU-rich elements and associated factors: are there unifying principles?] Nucleic Acids Res. [Nucleic Acids Research] 3 Jan 2006; 33(22):7138-50.

[0045] In this embodiment, NBP binds to microRNA.

[0046] In one embodiment, NBP binds to small nuclear RNA (snRNA). In another embodiment, NBP binds to small nucleolar RNA (snoRNA).

[0047] In this embodiment, NBP binds to regulatory RNA.

[0048] In the examples, NBP is bound to ribozymes.

[0049] In one embodiment, NBP binds to transfer RNA (tRNA). In another embodiment, NBP binds to long non-coding RNA (lncRNA).

[0050] In the embodiments, NBP binds to mRNA. In the embodiments, NBP binds to precursor mRNA. In the embodiments, NBP binds to precursor mRNA introns. In the embodiments, NBP binds to precursor mRNA exons. In the embodiments, NBP binds to the 3′ end of mRNA. In the embodiments, NBP binds to the 5′ end of mRNA. In the embodiments, NBP binds to the 5′ cap of mRNA. In the embodiments, NBP binds to positively charged intrinsically disordered regions (IDRs) or nucleic acids. In the embodiments, NBP binds to nucleic acid sequences. In the embodiments, NBP binds to nucleic acid structures. In the embodiments, NBP binds to nucleic acid secondary structures. In the embodiments, NBP binds to nucleic acid tertiary structures. In the embodiments, NBP binds to naturally occurring nucleic acids. In the embodiments, NBP binds to synthetically prepared nucleic acids. In the embodiments, the nucleic acids are naturally occurring but have been modified by synthetic means.

[0051] In this embodiment, NBP binds to a nucleic acid sequence, wherein the length of the nucleic acid sequence is from about 30 nucleotides to about 10,000 nucleotides. In this embodiment, the length of the nucleic acid is at least about 30 nucleotides. In the embodiments, the length of the nucleic acid is at least about 35 nucleotides, such as at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or at least about 10,000 nucleotides. In some embodiments, the nucleic acid is at least about 100 nucleotides long. In some embodiments, the nucleic acid is at least about 250 nucleotides long. In some embodiments, the nucleic acid is at least about 500 nucleotides long. In some embodiments, the nucleic acid is at least about 1,000 nucleotides long. In some embodiments, the nucleic acid is at least about 1,500 nucleotides long. In some embodiments, the nucleic acid is at least about 2,000 nucleotides long. In some embodiments, the nucleic acid is at least about 2,500 nucleotides long. In some embodiments, the nucleic acid is at least about 3,000 nucleotides long. In some embodiments, the nucleic acid is at least about 4,000 nucleotides long. In some embodiments, the nucleic acid is at least about 5,000 nucleotides long. In some embodiments, the nucleic acid is at least about 6,000 nucleotides long. In some embodiments, the nucleic acid is at least about 7,000 nucleotides long. In some embodiments, the nucleic acid is at least about 8,000 nucleotides long. In some embodiments, the nucleic acid is at least about 9,000 nucleotides long. In some embodiments, the nucleic acid is at least about 10,000 nucleotides long.

[0052] In this embodiment, the nucleic acid has a diameter or length of about 0.001 µm to about 500 µm. In this embodiment, the nucleic acid has a diameter between 1 nm and 100 µm (including the endpoints). In this embodiment, the nucleic acid has a diameter between 1 nm and 100 nm (including the endpoints). In this embodiment, the nucleic acid has a diameter between 100 nm and 1 µm (including the endpoints). In this embodiment, the nucleic acid has a diameter between 1 µm and 50 µm (including the endpoints). In this embodiment, the nucleic acid has a diameter between 50 µm and 100 µm (including the endpoints).

[0053] In the examples, the size (i.e., diameter or length) of the nucleic acid was approximately 0.001 µm, approximately 0.002 µm, approximately 0.003 µm, approximately 0.004 µm, approximately 0.005 µm, approximately 0.006 µm, approximately 0.007 µm, approximately 0.008 µm, approximately 0.009 µm, approximately 0.010 µm, approximately 0.020 µm, approximately 0.030 µm, approximately 0.040 µm, approximately 0.050 µm, approximately 0.060 µm, approximately 0.070 µm, approximately 0.080 µm, approximately 0.090 µm, approximately 0.1 µm, approximately 0.2 µm, approximately 0.3 µm, approximately 0.4 µm, approximately 0.5 µm, approximately 0.6 µm, approximately 0.7 µm, approximately 0.8 µm, approximately 0.9 µm, approximately 1 µm, approximately 2 µm, approximately 2 µm, approximately 2 µm, approximately 2 µm, approximately 0.001 µm, approximately 0.002 µm, approximately 0.003 µm, approximately 0.004 µm, approximately 0.005 µm, approximately 0.006 µm, approximately 0.007 µm, approximately 0.008 µm, approximately 0.009 ... µm, approximately 3 µm, approximately 4 µm, approximately 5 µm, approximately 6 µm, approximately 7 µm, approximately 8 µm, approximately 9 µm, approximately 10 µm, approximately 11 µm, approximately 12 µm, approximately 13 µm, approximately 14 µm, approximately 15 µm, approximately 16 µm, approximately 17 µm, approximately 18 µm, approximately 19 µm, approximately 20 µm, approximately 21 µm, approximately 22 µm, approximately 22 µm, approximately 23 µm, approximately 24 µm, approximately 25 µm, approximately 26 µm, approximately 27 µm, approximately 28 µm, approximately 29 µm, approximately 30 µm, approximately 31 µm, approximately 32 µm, approximately 33 µm, approximately 34 µm, approximately 35 µm, approximately 36 µm, approximately 37 µm, approximately 38 µm, approximately 39 µm, approximately 40 µm, approximately 41 µm, approximately 42 µm, approximately 43 µm Approximately 44 µm, approximately 45 µm, approximately 46 µm, approximately 47 µm, approximately 48 µm, approximately 49 µm, approximately 50 µm, approximately 55 µm, approximately 60 µm, approximately 65 µm, approximately 70 µm, approximately 75 µm, approximately 80 µm, approximately 85 µm, approximately 90 µm, approximately 95 µm, approximately 100 µm, approximately 150 µm, approximately 200 µm, approximately 250 µm, approximately 300 µm, approximately 350 µm, approximately 400 µm, approximately 450 µm, or approximately 500 µm or greater, including all values ​​and ranges between them. In some examples, the nucleic acid has a size greater than or equal to 10 µm. In some examples, the nucleic acid has a size greater than or equal to 25 µm. In some examples, the nucleic acid has a size greater than or equal to 50 µm. In some examples, the nucleic acid has a size greater than or equal to 100 µm.

[0054] In some embodiments, the nucleic acid has a size (i.e., molar mass) of about 2 kDa to about 1000 MDa. In the examples, the molar mass of the nucleic acid was approximately 2 kDa, approximately 5 kDa, approximately 15 kDa, approximately 20 kDa, approximately 20 kDa, approximately 25 kDa, approximately 30 kDa, approximately 35 kDa, approximately 40 kDa, approximately 45 kDa, approximately 50 kDa, approximately 55 kDa, approximately 60 kDa, approximately 65 kDa, approximately 70 kDa, approximately 75 kDa, approximately 80 kDa, approximately 85 kDa, approximately 90 kDa, approximately 95 kDa, approximately 100 kDa, approximately 150 kDa, approximately 200 kDa, approximately 250 kDa, approximately 300 kDa, approximately 350 kDa, approximately 400 kDa, approximately 450 kDa, approximately 500 kDa, approximately 550 kDa, approximately 600 kDa, approximately 650 kDa, approximately 700 kDa, approximately 750 kDa, approximately 800 kDa, approximately 850 kDa, approximately 900 kDa. kDa, approximately 950 kDa, approximately 1000 kDa, approximately 1 MDa, approximately 5 MDa, approximately 10 MDa, approximately 15 MDa, approximately 20 MDa, approximately 25 MDa, approximately 50 MDa, approximately 75 MDa, approximately 100 MDa, approximately 125 MDa, approximately 150 MDa, approximately 175 MDa, approximately 200 MDa, approximately 225 MDa, approximately 250 MDa, approximately 275 MDa, approximately 300 MDa, approximately 325 MDa, approximately 350 MDa, approximately 400 MDa, approximately 425 MDa, approximately 450 MDa, approximately 500 MDa, approximately 550 MDa, approximately 600 MDa, approximately 650 MDa, approximately 700 MDa, approximately 750 MDa, approximately 800 MDa, approximately 850 MDa, approximately 900 MDa, approximately 950 MDa, or approximately 1000 MDa, including all values ​​and ranges within it.

[0055] In some embodiments, the fusion protein comprises 1 to about 100 NBPs, 1 to about 75 NBPs, 1 to about 50 NBPs, 1 to about 40 NBPs, 1 to about 30 NBPs, 1 to about 20 NBPs, 1 to about 15 NBPs, 1 to about 10 NBPs, or 1 to about 5 NBPs. In embodiments, the fusion protein comprises about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 NBPs. In embodiments, a single polypeptide having phase behavior may be coupled to multiple NBPs, such as about 1 to 100 NBPs.

[0056] In the embodiments, the fusion protein may have two or more NBPs, each of which binds to different nucleic acids or to the same nucleic acid.

[0057] In this embodiment, the affinity of NBP for nucleic acids is modulated to facilitate the separation of nucleic acids from fusion proteins.

[0058] In the embodiments, NBP is selected from any of the following: RNA-specific adenosine deaminase 1 (ADAR1), ADAR1 double-stranded RNA-binding domain 3 (dsRBD3), Bacillus subtilis cold shock protein B (Bs-CspB), cold shock domain Y-box protein (CSD-Y-box), eukaryotic translation initiation factor 4E (eIF4e), Fox-1 protein (FOX1), heterogeneous nucleoribonucleoprotein Q1 (hnRNPQ1), human zinc finger CCCH type 14 protein (HsZC3H14), polya-binding protein (PABP), polya-binding protein nucleus 1 (PABPN1), triangular pentapeptide repeat protein A (PPRpA), Pumilio homology domain (PUM-HD), Pumilio-like repeat protein A (PUFpA), Staufen, 12-O-tetradecanoylphorbolol-13-acetate inducible sequence 11 D (TIS11D), Z-DNA / RNA binding protein 1 (ZBP1), and zinc finger nuclease (ZNF). In the examples, NBP is ADAR1. In the examples, NBP is ADAR1 dsRBD3. In the examples, NBP is Bs-CspB. In the examples, NBP is CSD-Y box. In the examples, NBP is eIF4e. In the examples, NBP is FOX1. In the examples, NBP is hnRNPQ1. In the examples, NBP is HsZC3H14. In the examples, NBP is PABP. In the examples, NBP is PABPN1. In the examples, NBP is PPRpA. In the examples, NBP is PUFpA. In the examples, NBP is PUM-HD. In the examples, NBP is Staufen. In the examples, NBP is TIS11D. In the examples, NBP is ZBP. In the examples, NBP is ZNF.

[0059] In this embodiment, NBP is RNA-specific adenosine deaminase 1 (ADAR1) or a fragment, subunit, or domain thereof. ADAR1 is a polypeptide that catalyzes the posttranscriptional deamination of adenosine, thereby converting them into inosine. ADAR1 binds to and catalyzes double-stranded RNA. ADAR1 is described in the following reference, which is incorporated herein by reference in its entirety: Song B et al. The role of RNA editing enzyme ADAR1 in human disease. [RNA editing enzyme ADAR1 in human disease] Wiley Interdiscip Rev RNA. [Wiley Interdiscip Rev RNA] Jan 2022; 13(1):e1665. ADAR1 comprises one or more Z-DNA binding domains, one or more dsRNA binding domains, and a deaminase domain. In this embodiment, NBP is ADAR1 double-stranded RNA binding domain 3 (dsRBD3).

[0060] In the embodiments, ADAR1 dsRBD3 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 89.

[0061] In this embodiment, the NBP comprises a cold shock domain (CSD). The CSD comprises five antiparallel β-strands, forming a β-barrel structure called an oligosaccharide / oligonucleotide binding fold. The CSD binds to single-stranded RNA and single-stranded DNA. In this embodiment, the CSD consists of 60 to 80 amino acids, such as about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 amino acids. In this embodiment, the CSD contains about 70 amino acids. In this embodiment, the CSD is a bacterial CSD. In this embodiment, the bacterial CSD shows a preference for ssDNA up to 10-fold over ssRNA.

[0062] In the embodiments, NBP is Bacillus subtilis cold shock protein B (Bs-CspB) or a fragment, subunit, or domain thereof. Bs-CspB is also referred to as CspB from Bacillus subtilis (Bscscp). Bs-CspB binds to single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). Bs-CspB is a polypeptide that acts as an RNA chaperone protein and a transcription antiterminant. Bs-CspB is described in the following reference, which is incorporated herein by reference in its entirety: Sachs R et al. RNA singlestrands bind to a conserved surface of the major cold shock protein in crystals and solution. [RNA single strand binds to a conserved surface of the major cold shock protein in crystals and solution] RNA. Jan 2012; 18(1):65-76. Bs-CspB contains one or more nucleic acid binding motifs.

[0063] In the embodiments, the fusion protein comprises Bs-CspB. In the embodiments, Bs-CspB binds to RNA, DNA, or both. In the embodiments, Bs-CspB binds to RNA selected from any of the following: double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), mRNA, precursor mRNA, polyadenosine (polyA) RNA, Z-conformation RNA (Z-RNA), or combinations thereof. In the embodiments, Bs-CspB binds to ssRNA. In the embodiments, Bs-CspB binds to the following: the 3' end of mRNA, the 3' end of mRNA, the 3' untranslated region (UTR) of mRNA, the polyA tail of mRNA, or AU-rich elements of mRNA, or combinations thereof. In the embodiments, Bs-CspB binds to introns, exons, polyA tails, or combinations thereof of precursor mRNA. In the embodiments, Bs-CspB binds to DNA. In the embodiments, Bs-CspB binds to single-stranded DNA, double-stranded DNA, polyadenosine (polyA) DNA, Z-conformation DNA (Z-DNA), or combinations thereof.

[0064] In the embodiments, Bs-CspB has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 90.

[0065] In the embodiments, NBP is a cold-shock domain Y-box protein (CSD-Y box) or a fragment, subunit, or domain thereof. The CSD-Y box is also referred to as the Y-box protein 1 (YB1) cold-shock domain (CSD) (YB1-CSD). The CSD-Y box binds to single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). The CSD-Y box is a polypeptide that regulates nucleic acid metabolism, for example, through DNA repair, precursor mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. The CSD-Y box is described in the following reference, which is incorporated herein by reference in its entirety: Heinemann U. and Roske Y. Cold-Shock Domains-Abundance, Structure, Properties, and Nucleic-Acid Binding. [Cold-Shock Domains-Abundance, Structure, Properties, and Nucleic-Acid Binding] Cancers [Cancer] (Basel) Jan 7, 2021; 13(2):190. The CSD-Y box contains one or more nucleic acid binding motifs.

[0066] In the embodiments, the CSD-Y box has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 91.

[0067] In this embodiment, NBP is eukaryotic translation initiation factor 4E (eIF4e) or a fragment, subunit, or domain thereof. eIF4e binds to the 5′ cap of mRNA. eIF4e is a polypeptide that works in conjunction with other proteins to bind to mRNA and allow recruitment of ribosomes for translation initiation. eIF4e is described in the following reference, which is incorporated herein by reference in its entirety: Davis, MR, et al. Nuclear eIF4E Stimulates 3′-end Cleavage of Target RNAs. Cell Reports, 27, 1397-1408. eIF4e contains a cap-binding pocket.

[0068] In the embodiments, eIF4e has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 86, 87, 88, and 96.

[0069] In the embodiments, eIF4e has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 86 and 87.

[0070] In the embodiments, NBP is the Fox-1 protein (FOX1) or a fragment, subunit, or domain thereof. FOX1 binds to introns and exons of precursor mRNA. FOX1 is a polypeptide that promotes or inhibits exon expression by regulating alternative splicing. FOX1 is described in the following reference, which is incorporated herein by reference in its entirety: Kuryanagi H. Fox-1 family of RNA-binding proteins. Cell Mol Life Sci. Dec. 2009; 66(24):3895-907. FOX1 contains an RNA recognition motif (RRM).

[0071] In the embodiments, FOX1 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 92.

[0072] In this embodiment, NBP is heteronuclear ribonucleoprotein Q1 (hnRNPQ1) or a fragment, subunit, or domain thereof. hnRNPQ1 binds to mRNA. hnRNPQ1 is a polypeptide that regulates mRNA processing events, such as precursor mRNA splicing, mRNA transport, and translation regulation. hnRNPQ1 is described in the following reference, which is incorporated herein by reference in its entirety: Xing, L. et al. Negative regulation of RhoA translation and signaling by hnRNP-Q1 affects cellular morphogenesis. [Negative regulation of RhoA translation and signaling by hnRNP-Q1 affects cellular morphogenesis] Molecular Biology of the Cell 2012 23:8, 1500-1509. hnRNPQ1 contains one or more RNA recognition motifs (RRMs), an acidic domain, and an Arg-Gly-Gly (RGG) box domain.

[0073] In the embodiments, hnRNPQ1 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 93.

[0074] In the examples, NBP is a Homo sapiens zinc finger CCCH type 14 protein (HsZC3H14) or a fragment, subunit, or domain thereof. HsZC3H14 binds to poly(A) RNA. HsZC3H14 is a polypeptide that regulates the length of the 3′-poly(A) tail. HsZC3H14 is described in the following reference, which is incorporated herein by reference in its entirety: Rha J et al. The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice. [RNA-binding protein ZC3H14 is required for proper poly(A) tail length control, synaptic protein expression, and brain function in mice] HumMol Genet. [Human Molecular Genetics] 2017 Oct 1; 26(19):3663-3681. HsZC3H14 contains an N-terminal proline-tryptophan-isoleucine (PWI)-like domain and a C-terminal tandem CysCysCysHis(CCCH) zinc finger (ZF) domain.

[0075] In the embodiments, HsZC3H14 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 94 and 95.

[0076] In the embodiments, NBP is a poly(A)-binding protein (PABP) or a fragment, subunit, or domain thereof. PABP binds to poly(A) RNA. PABP is a polypeptide that mediates mRNA cyclization. PABP is described in the following reference, which is incorporated herein by reference in its entirety: Mangus DA et al., Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. [Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression] Genome Biol [Genomics Biology] 4, 223 (2003). PABP contains one or more RNA recognition motifs (RRMs).

[0077] In the embodiments, PABP has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 74-85.

[0078] In the embodiments, PABP has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 77.

[0079] In the embodiments, NBP is poly(A)-binding protein core 1 (PABPN1) or a fragment, subunit, or domain thereof. PABPN1 binds to the poly(A) RNA tail. PABPN1 is a polypeptide that regulates RNA processing, such as preventing the nuclear export of unspliced ​​RNA and regulating the length of the poly(A) tail. PABPN1 is described in the following reference, which is incorporated herein by reference in its entirety: Mangus DA et al., Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol [Genomics Biology] 4, 223 (2003). PABPN1 is an RNA recognition motif (RRM).

[0080] In the embodiments, PABPN1 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 97.

[0081] In the embodiments, NBP is a trigonal pentapeptide repeat (PPR) or a fragment, subunit, or domain thereof. The PPR binds to the 3′ or 5′ end of the RNA transcript. The PPR is a polypeptide that regulates RNA stability and translation activation. In the embodiments, NBP is PPR protein a (PPRpA). A PPR contains 20-50 amino acids, for example, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 amino acids. In the embodiments, the PPR contains about 35 amino acids. In the embodiments, NBP comprises a PPR repeated about 2-30 times within the NBP sequence. In an embodiment, the NBP contains a PPR repeated approximately 10-30 times within the NBP sequence. In an embodiment, the PPR may be repeated approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times within the NBP. In an embodiment, the PPR is repeated at least 10 times. The PPR repeats may be continuous or separated by one or more amino acids. The PPR repeats form two antiparallel α-helices. In an embodiment, the NBP containing the PPR forms a solenoid structure. In an embodiment, the NBP containing the PPR binds to single-stranded RNA, single-stranded DNA, or mRNA. In an embodiment, the NBP containing the PPR binds to the 5' cap of mRNA. In the embodiments, the NBP containing the PPR binds to the 3' poly-A tail of the mRNA. The PPR is described in the following reference, which is incorporated herein by reference in its entirety: Manna, S. An overview of pentatricopeptide repeat proteins and their applications, Biochimie, 113, 2015, 93-99. The PPR contains one or more PPR motifs and one or more helical-turn-helical motifs.

[0082] In the embodiments, PPRpA has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 98.

[0083] In the embodiments, NBP is a Pumilio-like repeat (PUF) or a fragment, subunit, or domain thereof. PUFs bind to the 3′-UTR of mRNA in the cytosol and to the precursor of rRNA in the nucleolus. PUFs are polypeptides that act as post-transcriptional and translational regulators. In the embodiments, NBP is PUF protein A (PUFpA). The PUF domain contains eight α-helical repeats of a conserved 36-amino acid sequence, forming a concave RNA-binding surface. In the embodiments, NBP contains 1-8 α-helical repeats of the PUF domain, for example, 1, 2, 3, 4, 5, 6, 7, or 8 α-helical repeats. In the embodiments, NBP containing PUF binds to a poly-A tail. In the embodiments, NBP containing PUF binds to mRNA. PUF is described in the following reference, which is incorporated herein by reference in its entirety: Wang M et al., The PUF Protein Family: Overview on PUF RNA Targets, Biological Functions, and Post Transcriptional Regulation. Int J Mol Sci. 2018 Jan 30; 19(2):410. PUF contains one or more Pumilio homology domains (PUM-HD).

[0084] In the embodiments, PUFpA has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 99.

[0085] In the embodiments, NBP is the Pumilio homology domain (PUM-HD) or a fragment, subunit, or domain thereof. PUM-HD binds to the 3′-UTR of mRNA and represses their translation. PUM-HD is a polypeptide that acts as a post-transcriptional and translational regulator. PUF is described in the following reference, which is incorporated herein by reference in its entirety: Wang M et al. The PUF Protein Family: Overview on PUF RNA Targets, Biological Functions, and Post Transcriptional Regulation. Int J Mol Sci. 2018 Jan 30; 19(2):410. PUM-HD contains RNA recognition motifs.

[0086] In the embodiments, PUM-HD has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 102 and 103.

[0087] In the embodiments, NBP is the staufen protein or a fragment, subunit, or domain thereof. Staufen binds to double-stranded RNA (dsRNA). Staufen is a polypeptide that plays a role in RNA transport, decay, and translational repression. Staufen is described in the following reference, which is incorporated herein by reference in its entirety: Visentin S et al. A multipronged approach to understanding the form and function of hStaufen protein. RNA. 2020 Mar; 26(3):265-277. Staufen comprises one or more dsRNA-binding domains (RBDs) and one or more tubulin-binding domains (TBDs).

[0088] In the embodiments, Staufen has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 100.

[0089] In the embodiments, NBP is 12-O-tetradecanoylphorbol-13-acetate (TPA) inducible sequence 11D (TIS11D) or a fragment, subunit, or domain thereof. TIS11D binds to AU-rich elements in mRNA. TIS11D is a polypeptide that functions in RNA metabolism and secondary hematopoiesis. TIS11D is described in the following reference, which is incorporated herein by reference in its entirety: Morgan, BR et al., Probing the Structural and Dynamical Effects of the Charged Residues of the TZF Domain of TIS11d, 2015 Biophysical Journal, 108:6, 1503-1515. TIS11D contains one or more CCCH-type tandem zinc finger domains and one or more (R / K)YKTEL motifs.

[0090] In the embodiments, TIS11D has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 61-66.

[0091] In the embodiments, TIS11D has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 65.

[0092] In the embodiments, NBP is Z-DNA / RNA binding protein 1 (ZBP1) or a fragment, subunit, or domain thereof. ZBP1 binds to Z-conformation DNA and RNA. ZBP1 is a polypeptide that functions in the innate immune response by binding to exogenous nucleic acids and inducing the production of type I interferon. ZBP1 is described in the following reference, which is incorporated herein by reference in its entirety: Maelfait J et al. Sensing of viral and endogenous RNA by ZBP1 / DAI induces necroptosis. EMBO J. [Journal of the European Society for Molecular Biology] Sep 1, 2017; 36(17):2529-2543. ZBP1 contains one or more Z-binding domains (ZBD).

[0093] In the embodiments, ZBP1 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 67-73.

[0094] In the embodiments, ZBP1 has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 65.

[0095] In the examples, NBP is a zinc finger nuclease (ZNF) or a fragment, subunit, or domain thereof. ZNF binds to double-stranded DNA and RNA. ZNF is a polypeptide that regulates gene expression at the transcriptional and translational levels. ZNF is described in the following reference, which is incorporated herein by reference in its entirety: Chaves-Arquero B et al. The distinct RNA-interaction modes of a small ZnF domain underlay TUT4(7) diverse action in miRNA regulation. [The distinct RNA-interaction modes of a small ZnF domain underlay TUT4(7) diverse action in miRNA regulation] RNA Biol. [RNA Biology] 2021 Nov 12; 18(Supplement 2):770-781. ZNF contains one or more C2H2 domains, one or more CCHC domains, and a catalytic domain.

[0096] In the embodiments, ZNF has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 101.

[0097] This article describes an NBP comprising Bs-CspB. In the examples, the NBP binds to double-stranded RNA. In the examples, the NBP comprises a dsRNA-binding protein (dsRBD) or a fragment thereof. The dsRBD is described in the following reference, which is incorporated herein by reference in its entirety: Banerjee et al. RNA Biol. [RNA Biology] Oct 2014; 11(10):1226-1232.

[0098] In this embodiment, the NBP binds to capped mRNA. In this embodiment, the NBP binding to the capped mRNA comprises eukaryotic translation initiation factor 4E (eIF4E), eukaryotic translation initiation factor 3 subunit D (eIF3D), or a combination thereof.

[0099] In the embodiments, NBP binds to the groove of DNA or RNA. Non-limiting examples of nucleic acid-binding proteins that bind to the groove of DNA or RNA include the transactivator (Tat) of the transcript of human immunodeficiency virus 1 (HIV-1), the HIV-1 REV protein, and the RSG-1.2 peptide. The RSG-1.2 peptide is a synthetic peptide that binds to the Rev response element present in the env gene of the HIV-1 genome. The RSG-1.2 peptide is described in the following article, which is incorporated herein by reference in its entirety: Kumar et al. PLoS One. [PLoS ONE] 2011; 6(8):e23300.

[0100] In this embodiment, the NBP binds to mRNA. In this embodiment, the NBP binding to mRNA is a ribosomal protein. In this embodiment, the ribosomal protein is a 70S ribosome or an 80S ribosome. In this embodiment, the ribosomal protein is derived from the 40S or 60S subunit of the 80S ribosome. In this embodiment, the ribosomal protein is derived from the 30S or 50S subunit of the 70S ribosome. In this embodiment, the ribosomal protein is selected from the group consisting of: L3 ribosomal protein, L4 ribosomal protein, L13 ribosomal protein, L20 ribosomal protein, L22 ribosomal protein, L24 ribosomal protein, L24e ribosomal protein, S12 ribosomal protein, S14 ribosomal protein, and eukaryotic initiation factor 4E binding protein 1 (4EBP1).

[0101] In this embodiment, the NBP that binds to mRNA is part of the spliceosome. In this embodiment, the NBP that is part of the spliceosome is a splicing factor. In this embodiment, the splicing factor is selected from ASF / SF2 splicing factor, serine-rich / arginine-rich splicing factor 4 (SRp75), and serine-rich and arginine-rich splicing factor 1 (SRSF1).

[0102] In the examples, the NBP that binds to mRNA is a protein localized to the p-particle. In the examples, the protein localized to the p-particle is selected from the group consisting of LAF-1, MEG-1, and MEG-3. LAF-1, MEG-1, and MEG-3 are described in the following references, which are incorporated herein by reference in their entirety: Leacock et al. Genetics, Vol. 178, No. 1, January 1, 2008, pp. 295–306; Wu et al. Mol Biol Cell. 2019 Feb. 1; 30(3): 333–345; Elbaum-Garfinkle et al. Proc Natl Acad SciUSA. 2015JJ 9; 112(23):7189-94.

[0103] In the embodiments, the NBP that binds to mRNA is a protein that removes or promotes the removal of the 5' cap from mRNA, referred to herein as a "decapping protein". In the embodiments, the protein that removes or promotes the removal of the 5' cap from mRNA is Dcp1, Dcp2, or a combination thereof. Dcp1 and Dcp2 are described in the following reference, which is incorporated herein by reference in its entirety: Valkov et al., Nature Structural & Molecular Biology, Vol. 23, pp. 574–579 (2016).

[0104] In the examples, the NBP bound to mRNA is a component of the processing body (p-body). In the examples, the components of the p-body are Edc3, DHX9, or Xrn1. The components of the p-body are described in the following reference, which is incorporated herein by reference in its entirety: Luo et al., Biochemistry [Biochemistry] 2018, 57, 17, 2424–2431.

[0105] In this embodiment, the NBP that binds to mRNA is a stem-loop binding protein (SLBP). SLBP binds to the stem-loop structure of the histone 3' untranslated region (UTR) in replication-dependent histone mRNA. In this embodiment, the NBP that binds to mRNA is a heterologous nucleoribonucleoprotein (hnRNP). hnRNP is described in the following reference, which is incorporated herein by reference in its entirety: Geuens et al., Hum Genet. [Human Genetics] 2016; 135: 851–867.

[0106] In this embodiment, the NBP that binds to the mRNA is GroEL.

[0107] In the embodiments, NBP is a protein involved in in vitro transcription. Non-limiting examples of NBPs involved in in vitro transcription include T7 RNA polymerase, RNase inhibitors, 2'-O-methyltransferase, inorganic pyrophosphatase, poly(A) polymerase, DNase I, calf intestinal phosphatase, Antarctic phosphatase, the D1 subunit of vaccinia virus mRNA capping enzyme, guanine-7-methyltransferase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), guanylate transferase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), RNA triphosphatase (present in the D1 subunit of vaccinia virus mRNA capping enzyme), and the D12 subunit of vaccinia virus mRNA capping enzyme. The following references describe the proteins mentioned above and are incorporated herein by reference in their full text: Dickson et al. Prog Nucleic Acid Res Mol Biol. [Advances in Molecular Biology of Nucleic Acid Research] 2005; 80: 349–374; Shuman et al. J Biol Chem. [Journal of Biochemistry] 10 Dec 1980; 255(23):11588–11598; Luo et al. J Virol. [Journal of Virology] 10 June 1995; 69(6): 3852–3856; Kobori et al. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 1 November 1984 81 (21) 6691-6695.

[0108] In the embodiments, NBP is selected from the group consisting of: poly(A)-binding protein (PABP), eukaryotic translation initiation factor 4E (eIF4E), eukaryotic translation initiation factor 3 subunit D (eIF3D), heterogeneous nucleoribonucleoprotein (hnRNP), RNA-specific adenosine deaminase 1 (ADAR1), RNA-specific adenosine deaminase 2 (ADAR2), CspB from Bacillus subtilis (Bscscp), Y-box protein 1 cold shock domain (YB1-CSD), Fox-1 protein (FOX1), poly(A)-binding protein (PABP), Staufen protein, TIS11d, zinc finger protein (ZNF), and Z-DNA binding protein 1 (ZBP1). The protein contains retinoic acid-induced gene-I (RIG-I)-like protein, toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR3), toll-like receptor 8 (TLR8), retinoic acid-induced gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), interferon-inducible protein 1 (IFIT1) with tetrapeptide repeats, protein kinase R (PKR), 2'-5'-oligoadenylate synthase, oligoadenylate synthase-like (OASL) protein (e.g., OAS1, OAS2, OAS3, or OASL), ribonuclease E (RNase E), gamma-interferon-inducible protein Ifi-16 (IF116), and cyclic GMP-AMP synthase (cGAS). The following references describe the selected proteins and are incorporated herein by reference in their full text: Kuryanagi. Cell Mol Life Sci. 2009; 66(24): 3895–3907; Baou et al. J Biomed Biotechnol. 2009; 2009: 634520; and Brisse and Ly. Front. Immunol. 2019, July 17; 10: 1586; Rehwinkel et al. Nature Reviews Immunology, Vol. 20, pp. 537–551 (2020); and Brisse et al. Front. Immunol. 2019; 10: 1586; Luo et al. Cell. 2011, October 14; 147(2): 409–422.

[0109] In an embodiment, the NBP comprises one or more RNA-binding domains (RBDs) and one or more intrinsically disordered regions (IDRs). In an embodiment, the IDR comprises RG[G] repeats, rich RS / RG domains, K / R sheets, molecular recognition features, low-complexity sequences, triangular pentapeptide domains, or combinations thereof.

[0110] In embodiments, NBP includes one or more of the following domains: short linear motifs (SLiM), RG repeats, RGG repeats, RS / RG rich domains, K / R basic patches, molecular recognition features, low-complexity sequences, RNA recognition motifs, double-stranded RNA binding domains, K homology domains, and zinc finger domains (e.g., CCHH ZF domain, CCCC (Ran-BP2) domain, CCCH...). ZF domain, RGG domain, Pumillo family domain, trigonal pentapeptide domain, cold shock domain, helicase domain, La motif, Piwi-Argonaute-Zwille (PAZ) domain, P element-induced useless testis, pseudouridine synthase and archaeopurin transglycosylation (PUA), Pumillo-like repeat (PUM), ribosome S1-like (S1), Sm and Sm-like (Sm / Lsm) repeats, thiouridine synthase and RNA methyltransferase and pseudouridine synthase (THUMP), and domains with YT521-B homology. The following references describe many of these fields and are incorporated in full by reference: Balcerak et al. Open Biol. 2019 Jun; 9(6) 190096; Jarvelin et al. Cell Communications Signal, 2016: 14, 9; Corley et al. Mol. Cell. 2020 Apr; 78(1): 9-29; De Franco et al. Sci Rep. 2019: 9, 2484; Shotwell et al. 2020. Wiley Interdiscip Rev RNA, 11, e1573; Simon et al. 2019. Molecular Cell, 75, 66-75.e5; Varadi et al. 2015, PLoSOne, 10, e0139731; Zeke et al., 2020, WIREs RNA, n / a, e1714.

[0111] In this embodiment, the NBP comprises a short linear motif (SLiM). An SLiM consists of up to ten amino acid residues predominantly located outside the protein's structural domains. The SLiM binds to RNA nonspecifically with low affinity. The SLiM is typically repeated multiple times throughout the protein.

[0112] In the embodiments, the NBP contains a pseudouridine synthase and a archaeopurin transglycosylation (PUA) domain. The PUA domain is 67–94 amino acids in length, with a β1α1β2β3β4β5α2β6 structure forming a pseudobarrel surrounded by two α-helices. In the embodiments, the NBP containing the PUA binds to double-stranded RNA.

[0113] In this embodiment, the NBP includes an S1 RNA-binding domain. In this embodiment, the NBP including the S1 RNA-binding domain interacts with single-stranded RNA, double-stranded RNA, or mRNA. In this embodiment, the S1 RNA-binding domain contains about 60 to about 80 amino acids, such as about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, or about 80 amino acids. In this embodiment, the S1 RNA-binding domain contains about 70 amino acids.

[0114] In this embodiment, the NBP contains an Sm RNA-binding motif. Sm RNA-binding motifs are present in Sm and sm-like (Lsm) proteins of eukaryotes and archaea, as well as in Hfq proteins of prokaryotes. An Sm motif consists of approximately 70 residues with an α1β1β2β3β4β5 topology, forming a curved antiparallel β-sheet. Sm-containing proteins readily polymerize through interactions between chains β4 and β5 in the two Sm motifs. In this embodiment, the NBP contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Sm motifs. In this embodiment, the NBP contains two Sm motifs. In this embodiment, the Sm binding motif binds to RNA through hydrogen bonding and base stacking interactions.

[0115] In this embodiment, the NBP contains thiouridine synthase and RNA methyltransferase and pseudouridine synthase (THUMP) domains. The THUMP domain is present in many tRNA-modifying enzymes. The THUMP domain is located near the RNA-modifying domain and sometimes near the N-terminal ferrugin-like domain. The THUMP domain exhibits an α1α2β1α3β2β2 topology, forming parallel α-helices flanking the β-sheet. In this embodiment, the NBP containing the THUMP domain binds to tRNA.

[0116] In this embodiment, the NBP comprises a YT521-B homology domain. In this embodiment, the YT521-B homology domain comprises 100-150 amino acids, for example, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 1... 22, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, or about 150 amino acids. In the examples, the NBP containing the YT521-B homologous domain binds to methylated adenosine.

[0117] In this embodiment, the NBP contains a Piwi-Argonautre-Zwille (PAZ) domain. In this embodiment, the PAZ domain facilitates the binding of small interfering mRNAs and / or microRNA guides to mRNA targets. In this embodiment, the PAZ domain is derived from the Dicer or Argonaute protein. The PAZ domain displays a six-stranded β-barrel with two α-helices at the top and unique appendages on opposite sides containing β-hairpins and short α-helices.

[0118] In this embodiment, the NBP includes a P-element-induced useless testis (PIWI) domain. In this embodiment, the PIWI domain facilitates the binding of small interfering mRNAs and / or microRNA guides to mRNA targets. In this embodiment, the PIWI domain is present on the Argonaute protein. The PIWI domain tertiary structure forms an RNase H-like fold consisting of a five-stranded β-sheet with α-helices on both sides.

[0119] In this embodiment, the NBP includes a PAZ domain and a PIWI domain.

[0120] In this embodiment, the NBP contains an RS / RG-rich domain. The RS / RG-rich domain contains repeats of arginine-serine (RS), arginine-glycine (RG), or combinations thereof. The RS / RG-rich domain mediates specific or non-specific interactions with RNA. Examples of proteins containing RS / RG-rich domains include SR proteins and SR-like proteins, such as serine / arginine-rich splicing factor 1 (SRSF1) and the RNA helicase DDX23.

[0121] In this embodiment, the NBP contains a helicase domain. Helicases consist of six superfamilies (SF), including SF1, SF2, SF3, SF4, SF5, and SF6. In this embodiment, the helicase domain is a eukaryotic RNA and DNA helicase from the SF1 or SF2 superfamily. Non-limiting examples of families within the SF1 and SF2 superfamilies include the Upf1-like family, DEAD-box, DEAH, RIG-I-like, Ski2-like, and NS3 family. In this embodiment, the helicase domain is a bacterial or viral helicase from the SF3, SF4, SF5, or SF6 superfamily. ATP binding to the helicase promotes a higher affinity of the helicase domain for RNA. ATP hydrolysis promotes a conformational change, causing the helicase to unwind its substrate and / or translocate a nucleotide.

[0122] In this embodiment, the NBP contains a La motif. The La motif consists of five α-helices and three β-chains, forming a small antiparallel β-sheet that folds against a modified “winged helix.” In this embodiment, the La motif binds to the 3' terminal UUU-OH element on the small RNA transcribed by polymerase III. The La motif contains 80 to 100 amino acids, such as about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 amino acids. In this embodiment, the La motif contains about 90 amino acids.

[0123] In an embodiment, the NBP comprises an RG[G] repeat. RG[G] repeat sequences are known to have extensive degenerate binding. An RG[G] repeat is an arginine- and glycine-rich motif consisting of at least three RG / RGG repeats (e.g., 3-500) separated by 10 amino acid residues. RG / RGG motifs comprise RGG and / or RG repeats of varying lengths with spacers of different amino acids. In an embodiment, the NBP comprises a di-RGG motif. A di-RGG motif contains two repeating RGG sequences separated by 0-4 amino acids. In an embodiment, the NBP comprises a di-RG motif. A di-RG motif contains two repeating RG sequences separated by 0-4 amino acids. In an embodiment, the NBP comprises a tri-RGG motif. A tri-RGG motif contains three repeating RG sequences separated by 0-4 amino acids. In an embodiment, the NBP comprises a tri-RG motif. A tri-RG motif contains three repeating RG sequences separated by 0-4 amino acids. These motifs are described in the following article, which is incorporated herein by reference in its entirety: Thandapani et al. (2013). Molecular Cell, 50, 613-623.

[0124] In embodiments, the amino acid sequence of the NBP comprises one or more RG, RGG, RGGR, RGGGR, or combinations thereof. In embodiments, the NBP comprising RG, RGG, RGGR, or RGGGR, or combinations thereof, is mediated by hydrogen bonding and base stacking with DNA and RNA via an arginine moiety. In embodiments, the NBP comprising RG, RGG, RGGR, RGGGR, or combinations thereof binds to the DNA G-quadruplex. An exemplary protein containing repeats of RGG, RGGR, or RGGGR is the RNA-binding protein FUS. In embodiments, the NBP comprises FUS. In embodiments, the NBP sequence comprises consecutive repeats of RGG, RGGR, RGGGR, or combinations thereof. An exemplary NBP containing combinations of repeats of RGG, RGGR, or RGGGR may comprise the sequence RGGRGGRGGRRGGRRGGRRGGGRRGG. In embodiments, the NBP may comprise one or more RGG, RGGR, or RGGGR scattered throughout its sequence. In embodiments, the NBP contains 1 to 100 RG, RGG, RGGR, or RGGGR sequences. RGG, RGGR, and RGGGR can be distributed throughout the sequence (separated by one or more amino acids) or continuously. In the examples, NBP comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 5 3. Approximately 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, approximately 99 or approximately 100 RG, RGG, RGGR, or RGGGR repeats. RG, RGG, RGGR, and RGGGR repeats may be continuous or scattered throughout the sequence. The following article describes an exemplary RGG sequence and is incorporated herein by reference in its entirety: Simon et al., Molecular Cell (2019), 75, 66-75.e5.

[0125] In some embodiments, the NBP includes an RG domain. The RG domain contains approximately 2 to approximately 500 RG (arginine-glycine) repeats. In some embodiments, the NBP includes an RGG domain. The RGG domain contains approximately 2 to approximately 500 RGG (arginine-glycine-glycine) repeats. In some embodiments, the NBP includes an RGGR domain. The RGGR domain contains approximately 2 to approximately 500 RGGR (arginine-glycine-glycine-arginine) repeats. In some embodiments, the NBP includes an RGGGR domain. The RGGGR domain contains approximately 2 to approximately 500 RGG (arginine-glycine-glycine-arginine) repeats. In some embodiments, the NBP includes a mixed RG domain. The mixed RG domain contains 2 to 500 simultaneous repeats of RG, RGG, RGGR, and / or RGGGR. For example, the mixed RG domain may contain RGG, followed by RG, followed by RGGR, followed by RG, followed by RGGGR.

[0126] In this embodiment, the NBP comprises a K / R basic sheet. The K / R basic sheet contains 4-8 consecutive lysine, arginine, or combinations thereof. The K / R basic sheet forms a highly positively charged and exposed interface for binding to RNA. K / R basic sheets are often contained within multiple clusters of the same protein.

[0127] In this embodiment, the NBP includes a molecular recognition feature (MoRF). In this embodiment, the MoRF is up to 25, 50, or more amino acids long, or 25 to 50 amino acids long. The MoRF undergoes a dynamic transition from disorder to order upon ligand binding.

[0128] In the embodiments, NBP comprises a low complexity (LC) sequence. In the embodiments, the LC sequence contains up to 100 amino acids and consists of numerous repetitions of the same amino acid or several amino acids. LC sequences can aggregate into amyloid filaments and undergo a reversible phase transition to a hydrogel-like state. Examples of proteins containing LC sequences are FUS and hnRNPA2.

[0129] In an embodiment, the NBP contains an RNA recognition motif (RRM). The RRM binds to RNA. Typically, the binding is sequence-specific. In an embodiment, the RRM contains approximately 75 to approximately 125 amino acids, for example, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 105, approximately 110, approximately 115, approximately 120, or approximately 125 amino acids in length. In an embodiment, the RRM contains approximately 85 amino acids. The RRM typically employs a β1α1β2β3α2β4 topology, forming two α-helices abutting antiparallel β-sheets, wherein conserved RNA-binding RNP1 and RNP2 motifs are housed in the central β1 and β3 strands.

[0130] In the embodiments, the NBP comprises a double-stranded RNA-binding domain (dsRBD). In the embodiments, the dsRBD contains approximately 55 to approximately 80 amino acids, or approximately 65 to approximately 70 amino acids. In the embodiments, the dsRBD contains 68 amino acids. The dsRBD typically adopts an αβββα conformation. In the embodiments, the dsRBD appears as a tandem repeat or in combination with other RNA-binding domains. There are two subclasses of dsRBDs: type B and type A. Type A binds better to dsRNA compared to type B. dsRBDs typically bind in a shape-dependent manner and are not sequence-specific. However, ADAR2 is a rare example of a dsRBD exhibiting sequence-specific binding.

[0131] In the embodiments, the NBP contains a K-homology domain. In the embodiments, the K-homology domain contains 60 to 80 amino acids. In the embodiments, the K-homology domain contains 70 amino acids. There are two types of K-homology domains: type I or reverse type II. Type I K-homology domains employ a β1α1α2β2β'α' topology. Reverse type II K-homology domains employ an α'β'β1α1α2β2 topology. K-homology domains do not use aromatic amino acids for binding; instead, they use hydrogen bonds. NBPs containing K-homology domains are difficult to design due to their strict sequence specificity.

[0132] In an embodiment, the NBP comprises one or more zinc finger (ZF) domains. In an embodiment, the NBP comprises 1-100 ZF domains, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49. Approximately 50, approximately 51, approximately 52, approximately 53, approximately 54, approximately 55, approximately 56, approximately 57, approximately 58, approximately 59, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, approximately 84, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99 or approximately 100 ZF repetitions. In the embodiments, the zinc finger domain is selected from one of the following subtypes: CCHC (zinc digit), CCCH, CCCC (RanBP2), and CCHH. C and H refer to scattered cysteine ​​and histidine residues coordinated to zinc atoms. In the embodiments, the zinc finger domain contains about 20 to about 40 amino acids, such as about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, or about 40 amino acids. The CCHH ZF domain contains two conserved cysteine ​​and two conserved histidine residues. The CCHH ZF domain recognizes both structure-specific and sequence-specific elements. To date, there are no engineered versions of the CCHH ZF domain. The CCHH ZF domain binds to single-stranded and double-stranded DNA and RNA. CCCC ZF may not require a specific RNA conformation for binding. Typically, CCCC ZF recognizes short trinucleotide repeats. The engineering modification of CCCC ZF is described in the following reference, which is incorporated herein by reference in its entirety: De Franco et al., Sci Rep [Scientific Reports]: 2019: 9, 2484.

[0133] In the embodiments, the nucleic acid binding protein (NBP) is a polypeptide from Table 1, or a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the polypeptides in Table 1. In the embodiments, the NBP has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 90. Table 1. Exemplary amino acid sequences of NBP. Peptides exhibiting phase behavior

[0134] In the embodiments, the fusion protein described herein comprises one or more polypeptides having phase behavior. In the embodiments, the fusion protein described herein comprises 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 polypeptides having phase behavior. In the embodiments, the fusion protein comprises 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 polypeptides having phase behavior.

[0135] In the embodiments, the phase-behaving polypeptide is an arthropod elastin-like polypeptide (RLP). An arthropod elastin-like polypeptide is an elastomeric polypeptide with mechanical properties including desired resilience, compressive modulus, tensile modulus, shear modulus, elongation at break, maximum tensile strength, stiffness, springback, and compressive deformation. In the embodiments, the arthropod elastin-like polypeptide described herein is a polymer comprising one or more repeating polymers. In the embodiments, the polymeric repeats may have an amino acid sequence selected from any one of SEQ ID NO: 1-9.

[0136] In the embodiments, the arthropod elastin-like polypeptide comprises more than one type of repeat, such as the repeat of SEQ ID NO: 1 and the repeat of SEQ ID NO: 3.

[0137] In the embodiments, the arthropod elastin-like polypeptide described herein comprises up to 500 repetitions occurring within a given RLP. In the embodiments, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, approximately 250, approximately 260, approximately 270, approximately 280, approximately 290, approximately 300, approximately 310, approximately 320, approximately 330, approximately 340, approximately 350, approximately 360, approximately 370, approximately 380, approximately 390, approximately 400, approximately 450, or approximately 500 times are repeated.

[0138] In an embodiment, the RLP comprises one or more partial repeats. In an embodiment, the length of the partial repeat is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In an embodiment, the RLP contains one or more additional amino acids at its N-terminus or C-terminus that are not part of the repeating portion.

[0139] In the embodiments, one or more RLP repeats are out of order, i.e., they contain different amino acid sequences but retain the same amino acid composition. For example, repeats may have amino acid sequences different from SEQ ID NO: 8, but retain the same amino acid composition. In the embodiments, the polypeptide exhibiting phase behavior is an elastin-like polypeptide. Elastin-like polypeptides (ELPs) are biopolymers derived from elastin. In the embodiments, the polypeptide exhibiting phase behavior comprises P and G motifs containing a plurality of P residues and a plurality of G residues. In the embodiments, the P and G motifs contain at least about 10% proline and at least about 20% glycine. In the embodiments, the elastin-like polypeptide described herein comprises the sequence (Val-Pro-Gly-Xaa-Gly).n (SEQ ID NO: 217) is a polymer of repeating pentapeptides. In the examples, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 48, 149, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500, including all values ​​and ranges therein. In embodiments, n is an integer from 1 to 360, including endpoints. In embodiments, the polypeptide having phase behavior comprises an amino acid sequence selected from: a.(GRGDSPY) n (SEQ ID NO: 1) b.(GRGDSPH) n (SEQ ID NO: 2) c.(GRGDSPV) n (SEQ ID NO: 3) d.(GRGDSPYG) n (SEQ ID NO: 4) e.(RPLGYDS) n (SEQ ID NO: 5) f. (RPAGYDS) n (SEQ ID NO: 6) g.(GRGDSYP) n (SEQ ID NO: 7) h.(GRGDSPYQ) n (SEQ ID NO: 8) i.(GRGNSPYG) n (SEQ ID NO: 9) j.(GVGVP) n (SEQ ID NO: 10); k.(GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO: 11); l.(GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 12); m.(GVGVPGWGVPGVGVPGWGVPGVGVP) m (SEQ ID NO: 13); n.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP) m (SEQ ID NO: 14); o.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) m (SEQ ID NO: 15); and p.(GAGVPGVGVPGAGVPGVGVPGAGVP) m (SEQ ID NO: 16); Or a random, disordered equivalent thereof. In an embodiment, n is an integer in the range of 1-500 (1, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500, including any value or range therewith). In an embodiment, n is an integer in the range of 20-360, including the endpoints. In an embodiment, m is an integer in the range of 1-100 (1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, including any value within that range). In an embodiment, m is an integer in the range of 4-25, including the endpoints.

[0140] In the embodiments, the phase-behavior polypeptide comprises an amino acid sequence selected from the following: (GVGVP) m (SEQ IDNO: 22); (ZZPXXXXGZ) m (SEQ ID NO: 23); (ZZPXGZ) m (SEQ ID NO: 24); (ZZPXXGZ) m (SEQ IDNO: 25); or (ZZPXXXGZ) m (SEQ ID NO: 26), where m is an integer between 10 and 160, including the endpoints, where X (if present) is any amino acid other than proline or glycine, and where Z (if present) is any amino acid. In the embodiments, the polypeptide having phase behavior comprises an amino acid sequence selected from the following: (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 17); or (GVGVPGVGVPGLGVPGVGVPGVGVP) m (SEQ ID NO: 18); where m is an integer between 2 and 32, including the endpoints. This phase-behaving polypeptide comprises an amino acid sequence selected from the following: (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 19), where m is 8 or 16; (GVGVPGAGVP) m (SEQ ID NO: 20), where m is an integer between 5 and 80, inclusive; or (GXGVP) m (SEQ ID NO: 21), where m is an integer between 10 and 160, inclusive, and where each repeating X is independently selected from the group consisting of: glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine. In the embodiments, m is an integer in the range of 1-100 (1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, inclusive).

[0141] In the embodiments, the pentapeptide repeats are out of order, for example, they contain different amino acid sequences but maintain the same amino acid composition. For example, ELP may contain amino acid sequences different from SEQ ID NO: 217, but maintain the same amino acid composition, for example, 40% of the sequence is glycine, 20% of the sequence is Xaa, 20% of the sequence is proline, and 20% of the sequence is valine.

[0142] In an embodiment, the ELP comprises one or more partial repeats. In an embodiment, the length of the partial repeat is 1, 2, 3, or 4 amino acids. In an embodiment, the ELP contains one or more additional amino acids at its N-terminus or C-terminus that are not part of the repeating portion.

[0143] ELPs and RLPs undergo phase transitions due to environmental factors. When conjugated with one or more peptides, such as one or more NBPs, or expressed as fusion proteins with one or more other peptides, such as one or more NBPs, ELPs and RLPs retain their ability to undergo phase transitions. Polymers such as ELPs and RLPs have a transition temperature (Tt), also known as a cloud point temperature (Tc). In some embodiments, ELPs and RLPs undergo a reversible phase transition from a soluble phase to an insoluble phase at Tt. ELPs that transition from a soluble phase to an insoluble phase by heating or increasing salt concentration have a Tt known as the lower critical dissolution temperature (LCST). RLPs that transition from a soluble phase to an insoluble phase by cooling or decreasing salt concentration have a Tt known as the lower critical dissolution temperature (UCST). In embodiments, the phase transition is caused by a change in the secondary structure of the ELP and / or RLP. For example, the phase transition of ELP is caused by a change in the secondary structure from random coil (below Tt) to a type II β-turn. In this embodiment, the change in secondary structure is characterized by a method selected from: circular dichroism spectrophotometry, small-angle X-ray scattering, cryo-electron microscopy, ultraviolet-visible spectrophotometry, static light scattering, dynamic light scattering, nuclear magnetic resonance spectroscopy, solid-state nuclear magnetic resonance spectroscopy, infrared spectroscopy, Fourier transform infrared spectroscopy (FTIR), microscopy, and small-angle neutron scattering. In this embodiment, the phase transition of the ELP is not caused by a change in secondary structure.

[0144] In embodiments, the RLP and ELP described herein have transition temperatures between about 0°C and about 100°C. In embodiments, the RLP and ELP described herein have transition temperatures between about 10°C and about 50°C. In some embodiments, the transition temperature is about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, or about 24°C. Approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, approximately 40°C, approximately 41°C, approximately 42°C, approximately 43°C, approximately 44°C, approximately 45°C, approximately 46°C, approximately 47°C, approximately 48°C, approximately 49°C, approximately 5 0°C, approximately 51°C, approximately 52°C, approximately 53°C, approximately 54°C, approximately 55°C, approximately 56°C, approximately 57°C, approximately 58°C, approximately 59°C, approximately 60°C, approximately 61°C, approximately 62°C, approximately 63°C, approximately 64°C, approximately 65°C, approximately 66°C, approximately 67°C, approximately 68°C, approximately 69°C, approximately 70°C, approximately 71°C, approximately 72°C, approximately 73°C, approximately 74°C, approximately 75°C C, approximately 76°C, approximately 77°C, approximately 78°C, approximately 79°C, approximately 80°C, approximately 81°C, approximately 82°C, approximately 83°C, approximately 84°C, approximately 85°C, approximately 86°C, approximately 87°C, approximately 88°C, approximately 89°C, approximately 90°C, approximately 91°C, approximately 92°C, approximately 93°C, approximately 94°C, approximately 95°C, approximately 96°C, approximately 97°C, approximately 98°C, approximately 99°C, or approximately 100°C. In embodiments, the RLP described herein has a transition temperature from approximately 10°C to approximately 100°C.

[0145] In the embodiments, the Tt of the RLP and ELP described herein is regulated by manipulating the primary structure (e.g., amino acid sequence) of the RLP and ELP. In the embodiments, the hydrophobicity of the ELP or RLP is modulated. In the embodiments, the hydrophobicity of the ELP is modified by changing the identity of the guest residue Xaa. In the embodiments, an increase in the hydrophobicity of the ELP or RLP leads to a decrease in Tt. In the embodiments, a decrease in the hydrophobicity of the ELP or RLP leads to an increase in Tt. In the embodiments, the polarity of the ELP or RLP is regulated. In the embodiments, the polarity of the ELP is modulated by changing the identity of the guest residue Xaa. In the embodiments, an increase in the polarity of the ELP or RLP leads to an increase in Tt. In the embodiments, a decrease in the polarity of the ELP or RLP leads to a decrease in Tt.

[0146] In this embodiment, the number (n) of the ELP pentapeptide repeats is modulated to change Tt. In this embodiment, n of the pentapeptide repeat (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 217) is an integer from 1 to 500, including the endpoints. In this embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 48, 149, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500, including all values ​​and ranges in between.

[0147] In the embodiments, Xaa, also referred to herein as a “guest residue,” is any amino acid that does not eliminate the phase behavior of the ELP. In the embodiments, Xaa is any amino acid other than proline. In the embodiments, Xaa is selected independently for each repeat. For example, a given ELP may contain guest residues alanine, glycine, and valine in a ratio of 8:7:1. In some embodiments, Xaa is selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In the embodiments, Xaa is a non-classical amino acid selected from the group consisting of: 2,4-diaminobutyric acid, α-amino-isobutyric acid, alloleucine, 4-aminobutyric acid, 2-aminobutyric acid (Abu), α-Ahx, 6-aminohexanoic acid, 2-aminoisobutyric acid (Aib), 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designed amino acids such as β-methyl amino acids, Cα-methyl amino acids, Na-methyl amino acids, and general amino acid analogs. In the embodiments, Xaa is a D-isomer of a natural or non-classical amino acid.

[0148] In embodiments, the temperature t (Tt) of the RLP and ELP described herein is modulated by introducing one or more environmental factors into the composition containing RLP and / or ELP. In embodiments, the Tt of the ELP and / or RLP is modulated by adjusting the ionic strength of the solvent. In embodiments, the ionic strength of the solvent is adjusted by adding a salt. In embodiments, the ELP and / or RLP have lower Tt in solvents containing anions classified as kosmotropes. Kosmotrope anions are highly hydrated and affect the water shielding on the ELP and / or RLP. In embodiments, the Tt of the ELP and / or RLP can be modulated by adding anion as a chaotrope. At low concentrations, adding a chaotrope increases the Tt of the ELP and / or RLP. At high concentrations, adding a chaotrope decreases the Tt of the ELP and / or RLP. In embodiments, the Tt of the ELP and / or RLP can be modulated by introducing one or more reagents that break hydrogen bonds. Non-limiting examples of reagents that break hydrogen bonds include sodium dodecyl sulfate (SDS) and urea. In the examples, reagents that enhance hydrogen bond formation are used to modulate Tt. In the examples, reagents that enhance hydrophobic interactions are used to modulate Tt. Trifluoroethanol is a reagent that enhances hydrophobic interactions and hydrogen bond formation, resulting in a decrease in Tt.

[0149] In embodiments, the concentrations of ELP and / or RLP can be adjusted to regulate Tt. In embodiments, higher ELP and / or RLP concentrations result in lower Tt. In embodiments, lower ELP and / or RLP concentrations result in higher Tt.

[0150] In addition, the regulation of pH, light and ion concentration can also be used to regulate Tt.

[0151] In the embodiments, the amount (e.g., addition or removal) and identity (e.g., positive or negative) of charged amino acids (e.g., histidine, lysine, arginine, glutamic acid, aspartic acid, ornithine, or other non-naturally charged amino acids) are adjusted by pH regulation to achieve Tt adjustment.

[0152] In the embodiments, the ELP and / or RLP described herein are block copolymers. A block copolymer comprises two or more sequence domains or blocks, wherein the two or more blocks have different properties. Non-limiting examples of tunable properties include hydrophilicity, hydrophobicity, polarity, and secondary structure. In the embodiments, the block copolymer is amphiphilic, for example, it comprises at least one hydrophobic block and at least one hydrophilic block.

[0153] In the embodiments, the ELPs and / or RLPs described herein are assembled into various morphologies. Non-limiting examples of morphologies include spherical aggregates, micelles, vesicles, fibrils, nanofibrils, nanotubes, and hydrogels. In the embodiments, the RLPs and / or ELPs described herein are assembled into various morphologies upon the addition of environmental factors. In the embodiments, the RLPs and / or ELPs described herein are changed from one morphology to another upon the addition of environmental factors. In the embodiments, the RLPs and / or ELPs described herein are changed from one morphology to another upon the addition of nucleic acids.

[0154] In the embodiments, the addition of environmental factors causes the RLP and / or ELP to undergo a phase transition. In the embodiments, during the phase transition of the RLP and / or ELP, the RLP and / or ELP transform from one form to another.

[0155] In the embodiments, the phase transition of RLP and / or ELP causes the formation of dense liquid droplets.

[0156] In the embodiments, the polypeptide exhibiting phase behavior comprises an amino acid sequence selected from Table 2. In the embodiments, the polypeptide exhibiting phase behavior has at least 90% identity with the polypeptide of any one of SEQ ID NO: 56 and 262-264. In the embodiments, the polypeptide exhibiting phase behavior has at least 90% identity with the polypeptide of SEQ ID NO: 56. In the embodiments, the polypeptide exhibiting phase behavior comprises any one of SEQ ID NO: 56 and 262-264. In the embodiments, the polypeptide exhibiting phase behavior comprises SEQ ID NO: 56. Table 2. Exemplary amino acid sequences of peptides exhibiting phase behavior.

[0157] In the embodiments, the fusion protein comprises 1 to 500, 1 to 450, 1 to 400, 1 to 350, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 The fusion protein comprises 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, or 8 to 10 different polypeptides exhibiting phase behavior. In embodiments, the fusion protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different polypeptides exhibiting phase behavior. For example, the fusion protein may comprise a first polypeptide exhibiting phase behavior and a second polypeptide exhibiting phase behavior. In embodiments, the fusion protein comprises a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth polypeptide exhibiting phase behavior.

[0158] In embodiments, the fusion protein comprising an amino acid sequence selected from any one of SEQ ID NO: 1-60 and 217 further comprises up to 10, up to 15, up to 20, or up to 25 additional N-terminal and / or C-terminal amino acids. In embodiments, the fusion protein comprising an amino acid sequence selected from any one of SEQ ID NO: 1-60 and 217 further comprises an additional N-terminal methionine. In embodiments, the fusion protein comprising an amino acid sequence selected from any one of SEQ ID NO: 1-60 and 217 further comprises an additional C-terminal glycine. In embodiments, the fusion protein comprising an amino acid sequence selected from any one of SEQ ID NO: 56 and 262-264 further comprises up to 10, up to 15, up to 20, or up to 25 additional N-terminal and / or C-terminal amino acids. In embodiments, the fusion protein comprising an amino acid sequence selected from any one of SEQ ID NO: 56 and 262-264 further comprises an additional N-terminal methionine. In embodiments, the fusion protein comprising any one of the amino acid sequences of SEQ ID NO: 56 and 262-264 further comprises an additional C-terminal glycine. In embodiments, the fusion protein comprising the amino acid sequence selected from SEQ ID NO: 56 further comprises up to 10, up to 15, up to 20, or up to 25 additional N-terminal and / or C-terminal amino acids. In embodiments, the fusion protein comprising the amino acid sequence of SEQ ID NO: 56 further comprises an additional N-terminal methionine. In embodiments, the fusion protein comprising the amino acid sequence of SEQ ID NO: 56 further comprises an additional C-terminal glycine.

[0159] In the embodiments, the fusion protein contains polypeptide repeating units. In the embodiments, there are 5-500 polypeptide repeating units, including all ranges and values ​​therein. In the embodiments, the numbers 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 are present. 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 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, 200, 201, 202, 203, 204, 205 206, 207, 208, 209, 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, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 3 24, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383 384, 385, 386, 387, 388, 389, 390, 391, 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, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 4 43, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 polypeptide repeating units.

[0160] In the embodiments, the fusion protein has the same amino acid composition as ELP and / or RLP, but without repetitions. In the embodiments, the fusion protein comprises an amino acid sequence having approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with ELP and / or RLP. In the embodiments, the fusion protein comprises an amino acid composition having approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with ELP and / or RLP. In the embodiments, the fusion protein comprises a hydrophobic amino acid composition having approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identity with ELP and / or RLP.

[0161] In the embodiments, the polypeptide exhibiting the phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 56 and 262-264. In the embodiments, the polypeptide exhibiting the phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 262-264. In the embodiments, the polypeptide exhibiting phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 56.

[0162] In the embodiments, the peptides exhibiting phase behavior comprise non-repeating unstructured peptides. In the embodiments, the non-repeating unstructured peptides have an amino acid sequence containing at least 50 amino acids. In the embodiments, the non-repeating unstructured peptides have an amino acid sequence containing at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. In the embodiments, the peptides exhibiting phase behavior comprise P and G motifs containing a plurality of P residues and a plurality of G residues. In the embodiments, the P and G motifs comprise at least about 10% proline and at least about 20% glycine. In the embodiments, the sequence of the non-repeating unstructured peptide is at least about 10% proline (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%) and at least 20% glycine (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). In the embodiments, the non-repeating unstructured polypeptide has the following sequence, which contains at least about 40% of amino acids selected from the group consisting of: valine, alanine, leucine, lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

[0163] In the embodiments, the non-repeating unstructured polypeptide comprises a sequence that does not contain three consecutive identical amino acids, wherein any subsequence having 5-10 amino acids does not appear more than once in the non-repeating unstructured polypeptide, and wherein the non-repeating unstructured polypeptide comprises a subsequence that begins and ends with proline, and wherein the subsequence further comprises at least one glycine.

[0164] In the embodiments, the ELP and / or RLP described herein are expressed as components of a phase-behaving polypeptide. In the embodiments, the phase-behaving polypeptide is expressed in bacterial or mammalian cells. In the embodiments, the phase-behaving polypeptide is expressed in *E. coli*. In the embodiments, the phase-behaving polypeptide is expressed in insect cells (e.g., Sf9 cells). In the embodiments, the sequence of the non-repeating unstructured polypeptide is at least about 10% proline (e.g., at least 10%, 20%, 30%, 40%) and at least 20% glycine (e.g., at least 20%, 30%, 40%, or 50%) and at least 40% (e.g., at least 40%, 50%, 60%, or 70%) amino acids selected from the group consisting of valine, alanine, leucine, lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

[0165] In the embodiments, the ELP and / or RLP described herein are expressed as components of the fusion protein. In the embodiments, the fusion protein is expressed in bacterial or mammalian cells. In the embodiments, the fusion protein is expressed in *E. coli*. In the embodiments, the fusion protein is expressed in insect cells (e.g., Sf9 cells).

[0166] In the embodiments, the non-repeating unstructured polypeptide does not contain three consecutive identical amino acids. In the embodiments, the non-repeating unstructured polypeptide comprises a subsequence that appears only once in the non-repeating unstructured polypeptide sequence (e.g., a fragment of the non-repeating unstructured polypeptide). In the embodiments, the non-repeating unstructured polypeptide comprises a subsequence that begins and ends with proline. In the embodiments, the non-repeating unstructured polypeptide comprises a subsequence containing at least one glycine.

[0167] In the embodiments, the polypeptide exhibiting phase behavior comprises a signal peptide. In the embodiments, the signal peptide comprises an amino acid sequence selected from any one of SEQ ID NO: 218-220. In the embodiments, the signal peptide comprises an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100% identity with any one of SEQ ID NO: 218-220. In the embodiments, the signal peptide is a polypeptide from Table 3. Table 3. Exemplary amino acid sequences of signal peptides

[0168] In an embodiment, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 33), where m is 16. In an embodiment, the polypeptide exhibiting phase behavior comprises the amino acid sequence of SEQ ID NO: 34. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 38. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 43. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 47. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 60. Linkage between phase-behavioral peptides and NBP

[0169] In one embodiment, the fusion protein includes a linker. In another embodiment, the NBP is coupled to a polypeptide with phase behavior via the linker. In another embodiment, any linker that does not interfere with the function of the fusion protein may be used. In another embodiment, the fusion comprises one or more NBPs, one or more linkers, and one or more polypeptides with phase behavior from the C-terminus to the N-terminus. In yet another embodiment, the fusion comprises one or more NBPs, one or more linkers, and one or more polypeptides with phase behavior from the N-terminus to the C-terminus.

[0170] In this embodiment, the linker connects the NBP to a peptide exhibiting phase behavior. In this embodiment, the linker enables cooperative interactions between the phase-behaving peptide and the NBP. In this embodiment, the linker is a peptide. In this embodiment, the linker preserves the phase behavior of the phase-behaving peptide. In this embodiment, the linker preserves the T-phase of the phase-behaving peptide. t In the embodiments, the linker retains the structure of NBP. In the embodiments, the linker contains 1 to 50 amino acids. In the embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0171] In one embodiment, the stiffness of the connector is increased by including proline in the connector amino acid sequence.

[0172] In this embodiment, the flexibility of the connector is increased by including low-polarity amino acids, including threonine, serine, and glycine.

[0173] In embodiments, the connector can employ various secondary structures, including but not limited to α-helices, β-chains, and random coils. In one embodiment, the connector employs an α-helix and includes (EAAAK). n (SEQ ID NO: 143) amino acid repeats, where n is the number of repeats, which is an integer in the range of 1 to 20, including the endpoints.

[0174] In this embodiment, the connector is made of (G4S) n (SEQ ID NO: 144) is composed of n, where n can be an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In an embodiment, the peptide linker has repeats of (SGGG)n (SEQ ID NO: 145), where n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In an embodiment, the peptide linker has (GGGS). n (SEQ ID NO: 146) repeats, where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0175] In one embodiment, the linker has the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 147). In another embodiment, the linker has the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 148). In yet another embodiment, the linker contains only glycine.

[0176] In this embodiment, the peptide linker includes a protease cleavage site. In this embodiment, the protease cleavage site is a furin protease cleavage site.

[0177] In this embodiment, the polypeptide linker is poly(Gly). nA connector, where n is an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) (SEQ ID NO: 149). In other embodiments, the connector is selected from the group consisting of dipeptides, tripeptides, and tetrapeptides. In an embodiment, the connector is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0178] In the embodiments, the connector is selected from GKSSGSGSESKS (SEQ ID NO: 150), GSTGSGSGKSSEGKG (SEQ ID NO: 151), GSTGSGSGKSSEGSGSTKG (SEQ ID NO: 152), GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 153), EGKSSGSGSESKEF (SEQ ID NO: 154), SRSSG (SEQ ID NO: 155), and SGSSC (SEQ ID NO: 156).

[0179] In this embodiment, the linker is a self-cleaving peptide. In this embodiment, the self-cleaving peptide is a 2A peptide. 2A peptides are a class of peptides 18-22 amino acids long that induce ribosome jumping during protein translation in cells. In this embodiment, the 2A peptide is a T2A peptide having the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 157), a P2A peptide having the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 158), an E2A peptide having the amino acid sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 159), or an F2A peptide having the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 160). In this embodiment, the 2A peptide has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with any one of SEQ ID NOs 157-160. In the embodiments, the 2A peptide further comprises GSG (SEQ ID NO:161-164) at its N-terminus.

[0180] In an embodiment, the adapter comprises the amino acid sequence of any one of SEQ ID NO: 143-216. In an embodiment, the adapter has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence selected from any one of SEQ ID NO: 143-216. In an embodiment, the adapter has an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 261. In an embodiment, the adapter has an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 261.

[0181] In this embodiment, the connector is a polypeptide from Table 4. Table 4. Exemplary amino acid sequences of the linker sequence

[0182] In this embodiment, the connector is a chemical connector. In this embodiment, the chemical connector is selected from the group consisting of carbohydrate connectors, lipid connectors, fatty acid connectors, and polyether connectors.

[0183] In embodiments, the linker is a direct covalent link between amino acid residues of a phase-behaving polypeptide and an NBP. In embodiments, the fusion protein comprises a phase-behaving polypeptide and an NBP. In embodiments, amino acid residues of a phase-behaving polypeptide are covalently linked to amino acids in Bs-CspB. In embodiments, the fusion protein further comprises one or more linkers as described herein. In embodiments, the fusion protein comprises a phase-behaving polypeptide, a linker, and an NBP from its N-terminus to its C-terminus. In embodiments, the fusion protein comprises an NBP, a linker, and a phase-behaving polypeptide from its N-terminus to its C-terminus. In embodiments, the fusion protein comprises a phase-behaving polypeptide, a linker, and an NBP from its C-terminus to its N-terminus. In embodiments, the fusion protein comprises an NBP, a linker, and a phase-behaving polypeptide from its C-terminus to its N-terminus. In embodiments, the fusion protein comprises one or more phase-behaving polypeptides, one or more linkers, and one or more NBPs from its N-terminus to its C-terminus. In embodiments, the fusion protein comprises one or more NBPs, one or more linkers, and one or more phase-behaving polypeptides from its N-terminus to its C-terminus. A fusion protein construct containing Bs-CspB and a peptide exhibiting phase behavior.

[0184] In some embodiments, the fusion protein described herein comprises Bs-CspB and a peptide exhibiting phase behavior. In embodiments, the fusion protein comprises a peptide exhibiting phase behavior, a linker, and Bs-CspB from its N-terminus to its C-terminus. In embodiments, the fusion protein comprises Bs-CspB, a linker, and a peptide exhibiting phase behavior from its N-terminus to its C-terminus. In embodiments, the fusion protein comprises a peptide exhibiting phase behavior, a linker, and Bs-CspB from its C-terminus to its N-terminus. In embodiments, the fusion protein comprises Bs-CspB, a linker, and a peptide exhibiting phase behavior from its C-terminus to its N-terminus.

[0185] In the embodiments, the Bs-CspB has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 90. In the embodiments, the polypeptide exhibiting the same behavior has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 56 and 262-264. In the embodiments, the polypeptide exhibiting phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 56.

[0186] In the embodiments, the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 90, and at least 90% identity with the polypeptide of any one of SEQ ID NO: 56 and 262-264. In the embodiments, the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 90, and at least 90% identity with the polypeptide of SEQ ID NO: 56. In the embodiments, the fusion protein comprises SEQ ID NO: 90 and any one of SEQ ID NO: 56 and 262-264. In the embodiments, the fusion protein comprises SEQ ID NO: 90 and SEQ ID NO: 56.

[0187] In an embodiment, the fusion protein further comprises a linker. In an embodiment, the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO:261.

[0188] In an embodiment, the fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 225 or 226. In an embodiment, the fusion protein is at least 90% identical to the polypeptide of SEQ ID NO: 225. In an embodiment, the fusion protein is at least 90% identical to the polypeptide of SEQ ID NO: 225. In an embodiment, the fusion protein comprises SEQ ID NO: 225 or 226. In an embodiment, the fusion protein comprises SEQ ID NO: 225. In an embodiment, the fusion protein comprises a start codon. In an embodiment, the start codon comprises an N-terminal methionine. In an embodiment, the fusion protein comprises an N-terminal methionine. In the embodiments, the fusion protein containing an N-terminal methionine has the amino acid sequence of SEQ ID NO: 225. In the embodiments, the fusion protein not containing an N-terminal methionine has the amino acid sequence of SEQ ID NO: 226.

[0189] Exemplary sequences of the fusion proteins described above are shown in Table 5. Table 5. Amino acid sequences of fusion proteins containing Bs-CspB and a polypeptide exhibiting phase behavior. Methods using fusion proteins

[0190] This disclosure provides fusion proteins and methods of using them. In embodiments, the fusion protein comprises a nucleic acid-binding protein (NBP) that binds to nucleic acids and a polypeptide having phase behavior. In embodiments, the NBP comprises Bs-CspB. In embodiments, the nucleic acid-containing composition is free from one or more contaminants.

[0191] In an embodiment, the method for purifying nucleic acids includes contacting the nucleic acids with a fusion protein; wherein the nucleic acids bind to the fusion protein to form a complex; wherein the size of the complex is increased by a first environmental factor; wherein the complex is separated from at least one contaminant based on its size; and wherein the nucleic acids are separated from the fusion protein by a second environmental factor.

[0192] In an embodiment, a method for purifying single-stranded nucleic acids includes contacting a composition comprising single-stranded nucleic acids and at least one contaminant with a fusion protein, wherein the fusion protein binds to the single-stranded nucleic acid to form a complex; adding a first environmental factor to the composition comprising the complex to increase the size of the complex; separating the complex from the at least one contaminant; and separating the single-stranded nucleic acid from the fusion protein by contacting the complex with a second environmental factor to form a product comprising single-stranded nucleic acids. In an embodiment, the single-stranded nucleic acid comprises ssRNA, ssDNA, or both. In an embodiment, the single-stranded nucleic acid comprises ssRNA. In an embodiment, the at least one contaminant comprises double-stranded RNA.

[0193] In one embodiment, the method includes separating the complex from at least one contaminant based on size.

[0194] In an embodiment, the method for purifying nucleic acids includes contacting the nucleic acid with a fusion protein; wherein the nucleic acid binds to the fusion protein to form a complex; wherein the size of the complex increases; wherein the complex is separated from at least one contaminant based on its size; and wherein the nucleic acid is separated from the fusion protein by an environmental factor, thereby forming a product containing nucleic acids. In an embodiment, the nucleic acid comprises a single-stranded nucleic acid. In an embodiment, the nucleic acid comprises ssRNA. In an embodiment, the contaminant comprises a double-stranded nucleic acid. In an embodiment, the contaminant comprises dsRNA.

[0195] In an embodiment, a method for removing a contaminant from a composition containing nucleic acids includes contacting the contaminant with a fusion protein; wherein the contaminant binds to the fusion protein to form a complex; wherein the size of the complex is increased by a first environmental factor; wherein the complex is separated from the nucleic acid based on its size; and wherein the contaminant is separated from the fusion protein by a second environmental factor, thereby forming a product containing nucleic acids. In an embodiment, the nucleic acid comprises a single-stranded nucleic acid. In an embodiment, the nucleic acid comprises ssRNA. In an embodiment, the contaminant comprises a double-stranded nucleic acid. In an embodiment, the contaminant comprises dsRNA.

[0196] In some embodiments, the method of separating the first nucleic acid and the second nucleic acid includes contacting the first nucleic acid with a first fusion protein and contacting the second nucleic acid with a second fusion protein; wherein the first nucleic acid binds to the first fusion protein to form a first complex; wherein the second nucleic acid binds to the second fusion protein to form a second complex; and separating the first nucleic acid and the second nucleic acid by applying an environmental factor. In some embodiments, the first nucleic acid comprises a single-stranded nucleic acid. In some embodiments, the second nucleic acid comprises a double-stranded nucleic acid.

[0197] This document also provides a method for bringing a nucleic acid into proximity with another nucleic acid. In an embodiment, the method for bringing a first nucleic acid and a second nucleic acid into proximity includes contacting the first nucleic acid with a first fusion protein and contacting the second nucleic acid with a second fusion protein; wherein the first nucleic acid binds to the first fusion protein to form a first complex; wherein the second nucleic acid binds to the second fusion protein to form a second complex; and wherein environmental factors bring the first complex and the second complex into proximity with each other. In an embodiment, the method described herein brings the first nucleic acid and the second nucleic acid within a distance of about 10 µm, about 5 µm, about 1 µm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, about 300 nm, about 200 nm, about 100 nm, about 10 nm, about 1 nm, about 0.5 nm, or about 0.1 nm. In an embodiment, the first nucleic acid comprises a single-stranded nucleic acid. In an embodiment, the second nucleic acid comprises a double-stranded nucleic acid.

[0198] In some embodiments, the methods described herein utilize a fusion protein comprising NBP and a polypeptide exhibiting phase behavior. In some embodiments, the methods described herein utilize a fusion protein comprising Bs-CspB and a polypeptide exhibiting phase behavior. In some embodiments, the methods described herein utilize two or more different fusion proteins.

[0199] In embodiments, the methods described herein involve forming a complex. In embodiments, the methods described herein involve forming one or more complexes. In embodiments, the methods described herein involve forming one, two, three, four, five, or more complexes. A complex may be referred to as a "first complex" or a "second complex," etc.

[0200] In some embodiments, the complex comprises a fusion protein and nucleic acid. In some embodiments, the complex comprises a fusion protein and a contaminant. In some embodiments, the complex comprises a fusion protein and a second protein, such as an enzyme substrate, metabolite, or ligand (e.g., a ligand that binds to a cell receptor).

[0201] In embodiments, the components of the complex (e.g., the fusion protein and nucleic acid) bind to each other. In embodiments, the binding is reversible. Reversible binding means that the complex can dissociate, for example, separate into individual components. For example, if a complex is reversibly formed between the fusion protein and the nucleic acid, the fusion protein and the nucleic acid can subsequently dissociate. In embodiments, dissociation is triggered by environmental factors. In embodiments, reversible binding allows the separation of the nucleic acid from the fusion protein. In embodiments, reversible binding allows the separation of a contaminant from the fusion protein. In embodiments, reversible binding allows the separation of another molecule from the fusion protein. In embodiments, the nucleic acid comprises a single-stranded nucleic acid. In embodiments, the nucleic acid comprises ssRNA. In embodiments, the contaminant comprises a double-stranded nucleic acid. In embodiments, the contaminant comprises dsRNA.

[0202] In the embodiments, the reversible binding is non-covalent, meaning no covalent bonds are formed between the interacting components of the complex (such as between the fusion protein and the nucleic acid). In the embodiments, the non-covalent interaction results in the fusion protein and the nucleic acid. Non-limiting examples of non-covalent interactions include dipole-dipole forces, van der Waals forces, London dispersion forces, hydrogen bonds, hydrophobic interactions, and electrostatic interactions. In the embodiments, the non-covalent binding is disrupted by adding environmental factors. In the embodiments, the nucleic acid comprises a single-stranded nucleic acid. In the embodiments, the nucleic acid comprises ssRNA.

[0203] In this embodiment, the binding between the fusion protein and the target molecule (e.g., nucleic acid) is covalent. In this embodiment, the covalent bond between the fusion protein and the nucleic acid can be cleaved using, for example, a nuclease and / or a protease. In this embodiment, the nucleic acid comprises a single-stranded nucleic acid. In this embodiment, the nucleic acid comprises ssRNA.

[0204] In the embodiments, the size of the complex described herein increases after the application of an environmental factor. In the embodiments, the size of the complex formed between the fusion protein and the nucleic acid increases. In the embodiments, the size of the initial complex increases due to the aggregation of multiple complexes. In the embodiments, multiple complexes aggregate due to the self-assembly of the fusion protein. In the embodiments, multiple complexes aggregate due to the application of an environmental factor. In the embodiments, the size increase is stabilized by non-covalent interactions between multiple fusion proteins. In the embodiments, the size increase is stabilized by non-covalent interactions between peptides having phase behavior. In the embodiments, the non-covalent interactions are dipole-dipole forces, van der Waals forces, London dispersion forces, hydrogen bonds, hydrophobic interactions, and / or electrostatic interactions. In the embodiments, the nucleic acid comprises a single-stranded nucleic acid. In the embodiments, the nucleic acid comprises ssRNA.

[0205] In some embodiments, the method disclosed herein provides for forming multiple complexes in a mixture. In some embodiments, the size of all complexes increases. In some embodiments, the size of some complexes increases, while the size of others remains constant. In some embodiments, the size of one complex increases, while the size of another complex remains constant.

[0206] In the embodiments, the size of the initial complex increases by at least about 2 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times, at least about 100 times, or more. In the embodiments, the size of the initial complex increases by at least about 2 times. In the embodiments, the size of the initial complex increases by at least about 5 times. In the embodiments, the size of the initial complex increases by at least about 10 times. In the embodiments, the size of the initial complex increases by at least about 25 times.

[0207] As used herein, the phrase "size increase" can refer to an increase in the diameter of the composite or an increase in the mass of the composite. In the embodiments, the size increase is an increase in the molar mass of the composite. In the embodiments, the size increase is an increase in the hydrodynamic radius of the composite.

[0208] In the embodiments, the increase in the size of the complex can be visually observed with the naked eye. For example, the increase in the size of the complex can cause changes in the color, transparency, viscosity of the composition containing the complex, and / or can cause changes in the solubility of the complex (e.g., precipitation from solution), wherein such changes can be observed by a person without the use of any special equipment.

[0209] In the embodiments, those skilled in the art can measure the increase in the size of the complex according to methods known in the art. In the embodiments, the increase in the size of the complex can be measured using techniques selected from the group consisting of: X-ray scattering, small-angle X-ray scattering, wide-angle X-ray scattering, dynamic light scattering, analytical ultracentrifugation, size exclusion chromatography, and photon correlation spectroscopy.

[0210] In an embodiment, the size-increased complex is separated from the contaminant. In an embodiment, the size-increased complex containing nucleic acid and fusion protein is separated from the contaminant. In an embodiment, the size-increased complex containing contaminant and fusion protein is separated from the composition containing nucleic acid. In an embodiment, the size-increased first complex containing a first nucleic acid and a first fusion protein is separated from a second complex containing a second nucleic acid and a second fusion protein. In an embodiment, the size-increased complex containing contaminant and fusion protein is separated from the composition containing nucleic acid. In an embodiment, the size-increased first complex containing a first nucleic acid and a first fusion protein is separated from a second complex containing a second nucleic acid and a second fusion protein. In an embodiment, the first nucleic acid comprises a single-stranded nucleic acid. In an embodiment, the second nucleic acid comprises a single-stranded nucleic acid. In an embodiment, the single-stranded nucleic acid comprises ssRNA. In an embodiment, the contaminant comprises a double-stranded nucleic acid. In an embodiment, the contaminant comprises dsRNA.

[0211] In the embodiments, the separation of the complex from the contaminant can be observed visually with the naked eye.

[0212] In the embodiments, the separation of the complex from the contaminant is size-based. In the embodiments, size-based separation is performed using techniques selected from the group consisting of: tangential flow filtration (TFF), analytical ultracentrifugation, membrane chromatography, high-performance liquid chromatography, size exclusion chromatography, conventional flow filtration, acoustic separation, centrifugation, countercurrent centrifugation, and rapid protein liquid chromatography. In the embodiments, tangential flow filtration is used to separate the complex from at least one impurity based on size. In the embodiments, centrifugation is used to separate the complex from at least one impurity based on size. In the embodiments, about 100 relative centrifugal force (RCF) to about 16,000 RCF, for example, about 500 to about 16,000 RCF, or about 1,000 RCF to 16,000 RCF, is applied to separate the complex from at least one impurity. In an embodiment, a relative centrifugal force (RCF) of at least 500 is applied to separate the complex from at least one impurity, for example, at least about 500 RCF, at least about 600 RCF, at least about 700 RCF, at least about 800 RCF, at least about 900 RCF, at least about 1000 RCF, at least about 2000 RCF, at least about 3000 RCF, at least about 3500 RCF, at least about 4000 RCF, at least about 5000 RCF, at least about 6000 RCF, at least about 7000 RCF, at least about 8000 RCF, at least about 9000 RCF, at least about 10,000 RCF, at least about 11,000 RCF, at least about 12,000 RCF, at least about 13,000 RCF, at least about 14,000 RCF, at least about 15,000 RCF, at least about 16,000 RCF. RCF, at least about 17,000 RCF, at least about 18,000 RCF, at least about 19,000 RCF or at least about 20,000 RCF.

[0213] In this embodiment, TFF is used to separate the complex from contaminants based on size. In this embodiment, TFF can be used to separate the complex from at least one impurity based on size; this process is also referred to herein as "percolation". Percolation includes washing and elution steps. Washing removes impurities contained in the composition containing the complex. Elution separates the purified nucleic acid from the fusion protein. In this embodiment, TFF is used to concentrate the complex. In this embodiment, TFF can be used to increase the concentration of the complex within the composition; this process is also referred to herein as "concentration".

[0214] Tangential flow filtration uses microfiltration and ultrafiltration membranes to separate molecules. Microfiltration membranes typically have pore sizes between 0.1 µm and 10 µm. Ultrafiltration membranes typically have smaller pore sizes than microfiltration membranes, between 0.001 µm and 0.1 µm. In embodiments, membranes with pore sizes between about 0.001 µm and about 10 µm are used in the methods disclosed herein. In the embodiments, the membrane pore size is about 0.001 µm, about 0.01 µm, about 0.05 µm, about 0.1 µm, about 0.2 µm, about 0.3 µm, about 0.4 µm, about 0.5 µm, about 0.6 µm, about 0.7 µm, about 0.8 µm, about 0.9 µm, about 1.0 µm, about 2 µm, about 3 µm, about 4 µm, about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, or about 10 µm, including all values ​​and ranges therein. In the embodiments, the membrane pore size is about 0.1 µm. In the embodiments, the membrane pore size is about 0.2 µm.

[0215] In the embodiments, the membrane is made of hydrophilic poly(vinylidene fluoride) (PVDF), polyethersulfone (PES), cellulose phosphate, diethylaminoethyl cellulose, polysulfone, regenerated cellulose, nylon, nitrocellulose, cellulose acetate, polyethylene glycol-modified PES, modified polyethersulfone, and sulfonated PES or modified derivatives of the above materials.

[0216] In a transmembrane flow membrane (TFF), the membrane is positioned tangentially to the flow of the fluid mixture, such that the fluid mixture flows tangentially along a first side of the membrane. Simultaneously, the fluid medium is positioned to contact a second surface of the membrane. Transmembrane pressure is the force that drives the fluid through the membrane and carries permeable molecules.

[0217] In the embodiments, TFF is used for the separation of size-based complexes from contaminants at transmembrane pressures between about 0.1 bar and about 3 bar. In the embodiments, transmembrane pressures are about 0.1 bar, about 0.2 bar, about 0.3 bar, about 0.4 bar, about 0.5 bar, about 0.6 bar, about 0.7 bar, about 0.8 bar, about 0.9 bar, about 1.0 bar, about 1.1 bar, about 1.2 bar, about 1.3 bar, about 1.4 bar, about 1.5 bar, about 1.6 bar, about 1.7 bar, about 1.8 bar, about 1.9 bar, about 2.0 bar, about 2.1 bar, about 2.2 bar, about 2.3 bar, about 2.4 bar, about 2.5 bar, about 2.6 bar, about 2.7 bar, about 2.8 bar, about 2.9 bar, or about 3.0 bar, including all values ​​and ranges between them. In the embodiments, the transmembrane pressure is about 1.5 bar.

[0218] In this embodiment, the cross-flow rate is adjusted to improve the separation of the complex and contaminants described herein. The cross-flow rate is the rate at which the solution flows through the feed channel and across the membrane. It provides the force to remove molecules that restrict the flow of the filtrate. In this embodiment, the cross-flow rate is approximately 500 L / m. 2 / h and approximately 2000 L / m 2 Between / h. In the embodiment, the crossflow rate is approximately 500 L / m. 2 / h, approximately 600 L / m 2 / h, approximately 700 L / m 2 / h, approximately 800 L / m 2 / h, approximately 900 L / m 2 / h, approximately 1000 L / m 2 / h, approximately 1100L / m 2 / h, approximately 1200 L / m 2 / h, approximately 1300 L / m 2 / h, approximately 1400 L / m 2 / h, approximately 1500 L / m 2 / h, approximately 1600 L / m 2 / h, approximately 1700 L / m 2 / h, approximately 1800 L / m 2 / h, approximately 1900 L / m 2 / h, or approximately 2000 L / m 2 The values ​​and ranges are within the range of / h. In this embodiment, the crossflow rate is approximately 960 L / m. 2 / h. In an embodiment, TFF separation is performed using a membrane that allows contaminants to pass through while retaining a complex containing the fusion protein and nucleic acid. In an embodiment, a membrane that allows nucleic acids to pass through while retaining a complex containing the fusion protein and contaminants is used. In an embodiment, a membrane that allows nucleic acids to pass through while retaining a complex containing the fusion protein and contaminants is used. In an embodiment, a membrane that allows a complex containing a second fusion protein and a second nucleic acid to pass through while retaining a first complex containing the fusion protein and a first nucleic acid is used. In an embodiment, the first and second nucleic acids comprise single-stranded nucleic acids. In an embodiment, the first and second nucleic acids comprise ssRNA. In an embodiment, the contaminant comprises double-stranded nucleic acids. In an embodiment, the contaminant comprises dsRNA.

[0219] In the embodiments, the methods described herein are capable of purifying at least 0.1 kg, at least about 0.2 kg, at least about 0.3 kg, at least about 0.4 kg, at least about 0.5 kg, at least about 0.6 kg, at least about 0.7 kg, at least about 0.8 kg, at least about 0.9 kg, at least about 1 kg, at least about 2 kg, at least about 3 kg, at least about 4 kg, at least about 5 kg, at least about 6 kg, at least about 7 kg, at least about 8 kg, at least about 9 kg, at least about 10 kg or more of nucleic acids per day, including all values ​​and ranges between them.

[0220] In embodiments, the methods described herein are completed within approximately 0.5 hours to approximately 24 hours. In embodiments, the method is completed within approximately 0.5 hours, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours. In embodiments, the methods described herein are completed within approximately 0.5 hours to approximately 8 hours. In embodiments, the methods disclosed herein are completed within approximately 2 hours to approximately 6 hours.

[0221] In the embodiments, the methods described herein produce a final product containing a proportion of target nucleic acid based on the total nucleic acid content. In the embodiments, the proportion of target nucleic acid based on the total nucleic acid content can be used as a basis for calculating the purification yield. In the embodiments, the nucleic acid is a single-stranded nucleic acid.

[0222] In the examples, the purification yield of nucleic acids is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0223] In the examples, the nucleic acids are purified to at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0224] In the examples, the purification yield of the single-stranded nucleic acid is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% purity.

[0225] In the embodiments, single-stranded nucleic acids are purified to at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0226] In the examples, the purification yield of ssRNA is at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0227] In the examples, the ssRNA was purified to at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In the embodiments, the purified nucleic acids retain their biological activity and / or structure. In the embodiments, the purified nucleic acids have enhanced biological activity. In the embodiments, the purified single-stranded nucleic acids retain their biological activity and / or structure. In the embodiments, the purified single-stranded nucleic acids have enhanced biological activity. In the embodiments, ssRNA retains its biological activity and / or structure. In the embodiments, the purified ssRNA has enhanced biological activity.

[0228] In the examples, at least 400 g / m³ was purified daily. 2 (grams of nucleic acid / m) 2 (Filter membrane). In the examples, at least 400 g / m³ of material was purified daily. 2 At least 500 g / m 2 At least 600 g / m 2 At least 700 g / m 2 At least 800 g / m2 At least 900 g / m 2 or at least 1000 g / m 2 Nucleic acid. In the embodiments, the nucleic acid is, for example, mRNA, ssRNA, or a virus. In the embodiments, the nucleic acid is ssRNA.

[0229] In the examples, at least about 150 g / L (grams of nucleic acid / liter of fusion protein) is purified daily. In the examples, at least about 150 g / L, at least about 200 g / L, at least about 250 g / L, at least about 300 g / L, at least about 350 g / L, at least about 400 g / L, at least about 450 g / L, at least about 500 g / L, at least about 550 g / L, at least about 600 g / L, at least about 650 g / L, at least about 700 g / L, at least about 750 g / L, at least about 800 g / L, at least about 850 g / L, at least about 900 g / L, at least about 950 g / L, or at least about 1000 g / L are purified daily. In the examples, the nucleic acid is, for example, mRNA, ssRNA, or a virus. In the examples, the nucleic acid is ssRNA. In the embodiments, the product comprises about 70% to about 100% single-stranded nucleic acid. In the embodiments, the product comprises at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% single-stranded nucleic acid. In the embodiments, the single-stranded nucleic acid comprises ssRNA.

[0230] In embodiments, the final product contains about 10% or less of at least one contaminant. In embodiments, the product contains about 0.1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of at least one contaminant. In embodiments, the purification method includes removing at least about 3-log of contaminant from the final product compared to the amount of contaminant in the original composition. In embodiments, the purification method includes removing at least about 1-log, 2-log, 3-log, 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, or 10-log of contaminant compared to the amount of contaminant in the original composition. In embodiments, the purification method includes removing at least about 3-log to 10-log of contaminant compared to the amount of contaminant in the original composition. In embodiments, the contaminant comprises dsRNA.

[0231] In embodiments, the fusion protein is present at a concentration of about 1 µM to about 200 µM (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 µM, including all ranges and values ​​therein). In embodiments, the fusion protein is present at a concentration of about 30 µM to about 50 µM. In embodiments, the fusion protein is present at a concentration of about 40 µM. Environmental factors

[0232] In some embodiments, one or more environmental factors are applied to cause changes in a complex comprising a fusion protein and nucleic acid. In some embodiments, one or more environmental factors cause an increase in the size of the complex comprising the fusion protein and nucleic acid. In some embodiments, one or more environmental factors cause the aggregation of peptides exhibiting phase behavior. In some embodiments, one or more environmental factors cause the fusion protein to separate from the nucleic acid. In some embodiments, one or more environmental factors cause the fusion protein to separate from contaminants. In some embodiments, one or more environmental factors enable the nucleic acid to retain its native structure, function, and activity.

[0233] In this embodiment, the environmental factor is the change in temperature. In the embodiments, the temperature is increased by about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In the embodiments, the temperature is reduced by about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0234] In this embodiment, the environmental factor is a change in pH. In this embodiment, the pH increases by approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and so on. Approximately 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 units. In the examples, the pH was reduced by approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9, and approximately... Approximately 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 units.

[0235] In this embodiment, the environmental factor is a change in ionic strength. In this embodiment, the change in ionic strength is caused by increasing the salt concentration. In this embodiment, the change in ionic strength is caused by decreasing the salt concentration. Non-limiting examples of salts include sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium citrate, copper sulfate, sodium iodide, ammonium sulfate, and sodium sulfate. In this embodiment, dialysis is used to change the salt concentration in a composition containing fusion proteins and nucleic acids and / or contaminants. In this embodiment, the salt is added at a concentration ranging from about 0.5 M to about 3 M (e.g., 0.5, 1, 1.5, 2, 2.5, or 3 M, including any range or value therein).

[0236] In the embodiments, the environmental factor is an added cofactor. Non-limiting examples of cofactors include calcium, magnesium, cobalt, copper, zinc, iron, manganese, selenium, molybdenum, potassium, coenzyme A (CoA), nucleoside triphosphates, and vitamins. In the embodiments, the cofactor is calcium. In the embodiments, the nucleoside triphosphate is adenosine triphosphate, uridine triphosphate, guanosine triphosphate, cytidine triphosphate, or thymidine triphosphate. In the embodiments, the vitamin is fat-soluble. In the embodiments, the vitamin is water-soluble. Non-limiting examples of vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, K1 and K2, folic acid, and biotin.

[0237] In this embodiment, the environmental factor is a change in the concentration of the fusion protein. In this embodiment, the environmental factor is a change in the concentration of the nucleic acid. In this embodiment, the environmental factor is a change in the concentration of the pollutant.

[0238] In one embodiment, the environmental factor is the pressure change of the composition containing the fusion protein and nucleic acid. In another embodiment, the environmental factor is the pressure change of the composition containing the fusion protein and a contaminant. In yet another embodiment, the pressure change can be achieved by increasing or decreasing the volume of the composition.

[0239] In the embodiments, the environmental factor is the addition of one or more surfactants. In the embodiments, one or more surfactants are selected from free fatty acid salts, soaps, fatty acid sulfonates such as sodium lauryl sulfate, ethoxylated compounds such as ethoxylated propylene glycol, lecithin, polygluconate, quaternary ammonium salts, lignin sulfonates, 3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate (CHAPS), sugars (including sucrose and glucose), Triton X-100, and NP-40. In the embodiments, the surfactant is anionic, nonionic, or amphoteric.

[0240] In the embodiments, the environmental factor is the addition of one or more molecular crowding agents. Non-limiting examples of molecular crowding agents include polyethylene glycol, dextran, and sucrose. Non-limiting examples of PEG include PEG400, PEG1450, PEG3000, PEG8000, and PEG10000.

[0241] In the embodiments, the environmental factor is the addition of one or more oxidants. Non-limiting examples of oxidants include hydrogen peroxide, hydrophilic or hydrophobic activated hydrogen peroxide, pre-formed peracids, monopersulfates, or hypochlorites.

[0242] In the embodiments, the environmental factor is the addition of one or more reducing agents. In the embodiments, the one or more reducing agents are selected from the group consisting of: dithiothreitol (DTT), 2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), hydrazine, borohydrides, amine boranes, lower alkyl-substituted amine boranes, triethanolamine, and N,N,N',N'-tetramethylethylenediamine (TEMED).

[0243] In the embodiments, the environmental factor is the addition of one or more denaturing agents. Non-limiting examples of denaturing agents include urea, guanidine hydrochloride, guanidine, sodium salicylate, dimethyl sulfoxide, and propylene glycol.

[0244] In the embodiments, the environmental factor is the addition of one or more enzymes. Non-limiting examples of enzymes include proteases, kinases, phosphatases, synthases, transferases, nucleases (such as restriction endonucleases), lyases, isomerases, dehydrogenases, decarboxylases, and lipases.

[0245] In this embodiment, the environmental factor is the applied electromagnetic wave. In this embodiment, the environmental factor is the applied light. In this embodiment, the electromagnetic wave has a wavelength between about 0.0001 nm and about 100 m. In this embodiment, the electromagnetic wave is selected from the group consisting of gamma rays, x-rays, ultraviolet light, visible light, infrared light, and radio waves. In this embodiment, the electromagnetic wave is gamma rays. In this embodiment, the gamma rays have a wavelength between about 0.0001 nm and about 0.01 nm, for example, 0.0001 nm, 0.0005 nm, 0.001 nm, 0.002 nm, 0.003 nm, 0.004 nm, 0.005 nm, 0.006 nm, 0.007 nm, 0.008 nm, 0.009 nm, and 0.01 nm. In the embodiments, the X-rays have wavelengths between about 0.01 nm and 10 nm, such as about 0.01 nm, 0.02 nm, 0.03 nm, 0.04 nm, 0.05 nm, 0.06 nm, 0.07 nm, 0.08 nm, 0.09 nm, 0.10 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or about 10 nm. In embodiments, ultraviolet radiation has wavelengths between about 10 nm and about 400 nm, such as about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 280 nm, about 300 nm, about 350 nm, or about 400 nm. In embodiments, visible light has wavelengths between about 400 nm and about 800 nm, such as about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm. In the embodiments, the infrared radiation has a wavelength between about 800 nm and about 0.1 cm, such as about 800 nm, about 1 µm, about 2 µm, about 3 µm, about 4 µm, about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, about 10 µm, about 20 µm, about 30 µm, about 40 µm, about 50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 200 µm, about 300 µm, about 400 µm, about 500 µm, about 600 µm, about 700 µm, about 800 µm, about 900 µm, or about 0.1 cm.In the embodiments, the radio waves have wavelengths between about 0.1 cm and 100 m, such as about 0.1 cm, about 1 cm, about 10 cm, about 100 cm, about 1000 cm, about 2000 cm, about 3000 cm, about 4000 cm, about 5000 cm, about 6000 cm, about 7000 cm, about 8000 cm, about 9000 cm, or about 100 m.

[0246] In this embodiment, the environmental factor is the applied sound wave. In this embodiment, the sound wave has a frequency between approximately 1 Hz and 2000 kHz. In the embodiments, the sound waves have frequencies of approximately 1 Hz, approximately 5 Hz, approximately 10 Hz, approximately 20 Hz, approximately 30 Hz, approximately 40 Hz, approximately 50 Hz, approximately 60 Hz, approximately 70 Hz, approximately 80 Hz, approximately 90 Hz, approximately 100 Hz, approximately 200 Hz, approximately 300 Hz, approximately 400 Hz, approximately 500 Hz, approximately 600 Hz, approximately 700 Hz, approximately 800 Hz, approximately 900 Hz, approximately 1 kHz, approximately 100 kHz, approximately 200 kHz, approximately 300 kHz, approximately 400 kHz, approximately 500 kHz, approximately 600 kHz, approximately 700 kHz, approximately 800 kHz, approximately 900 kHz, approximately 1000 kHz, approximately 1100 kHz, approximately 1200 kHz, approximately 1300 kHz, approximately 1400 kHz, and approximately 1500 kHz. Frequency of approximately kHz, approximately 1600 kHz, approximately 1700 kHz, approximately 1800 kHz, approximately 1900 kHz, or approximately 2000 kHz. Numbered Examples

[0247] In addition to the appended claims, the embodiments numbered below also form part of this disclosure.

[0248] 1. A fusion protein comprising a nucleic acid binding protein (NBP) and a polypeptide having phase behavior.

[0249] 2. The fusion protein of claim 1, wherein the NBP is selected from any one of the following: RNA-specific adenosine deaminase 1 (ADAR1), ADAR1 double-stranded RNA-binding domain 3 (dsRBD3), Bacillus subtilis cold shock protein B (Bs-CspB), cold shock domain Y-box protein (CSD-Y-box), eukaryotic translation initiation factor 4E (eIF4e), Fox-1 protein (FOX1), heterogeneous nucleoribonucleoprotein Q1 (hnRNPQ1), Homo sapiens zinc finger CCCH type 14 protein (HsZC3H14), polya-binding protein (PABP), polya-binding protein nucleus 1 (PABPN1), triangular pentapeptide repeat protein A (PPRpA), Pumilio-like repeat protein A (PUFpA), Staufen, 12-O-tetradecanoylphorbolol-13-acetate inducible sequence 11D (TIS11D), Z-DNA / RNA binding protein 1 (ZBP1), and zinc finger nuclease (ZNF).

[0250] 3. The fusion protein of claim 1, wherein the NBP has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 61-103.

[0251] 4. The fusion protein according to any one of claims 1-3, wherein the NBP is selected from any one of the following: ADAR1 double-stranded RNA-binding domain 3 (dsRBD3), heterogeneous nucleoribonucleoprotein Q1 (hnRNPQ1), human zinc finger CCCH type 14 protein (HsZC3H14), poly-A binding protein nucleus 1 (PABPN1), triangular pentapeptide repeat protein A (PPRpA), and Pumilio-like repeat protein A (PUFpA).

[0252] 5. The fusion protein according to any one of claims 1-4, wherein the NBP has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 89, 93, 95, or 97-99.

[0253] 6. The fusion protein according to any one of claims 1-5, wherein the polypeptide having phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 1-60 or 217.

[0254] 7. The fusion protein according to any one of claims 1-6, wherein the polypeptide having phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 57 or 60.

[0255] 8. The fusion protein according to any one of claims 1-7, wherein the fusion protein comprises a linker.

[0256] 9. The fusion protein of claim 8, wherein the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 143-216.

[0257] 10. The fusion protein of any one of claims 1-9, wherein the NBP is bound to RNA, DNA, or both.

[0258] 11. The fusion protein according to any one of claims 1-10, wherein the NBP binds to an RNA selected from any one of the following: double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), mRNA, precursor mRNA, polyadenosine (polyA) RNA, Z-conformation RNA (Z-RNA), or a combination thereof.

[0259] 12. The fusion protein of any one of claims 1-11, wherein the NBP binds to the 3' end of mRNA, the 3' end of mRNA, the 3' untranslated region (UTR) of mRNA, the poly-A tail of mRNA, or an AU-rich element of mRNA, or a combination thereof.

[0260] 13. The fusion protein according to any one of claims 1-11, wherein the NBP binds to the precursor mRNA.

[0261] 14. The fusion protein of claim 13, wherein the NBP binds to an intron, an exon, a poly-A tail, or a combination thereof of the precursor mRNA.

[0262] 15. The fusion protein of any one of claims 1-10, wherein the NBP binds to DNA.

[0263] 16. The fusion protein of claim 15, wherein the NBP is bound to: single-stranded DNA, double-stranded DNA, polyadenosine (polyA) DNA, Z-conformation DNA (Z-DNA), or a combination thereof.

[0264] 17. A nucleic acid encoding a fusion protein as described in any one of claims 1-16.

[0265] 18. The nucleic acid of claim 17, wherein the nucleic acid has a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid of any one of SEQ ID NO: 119-133.

[0266] 19. A vector encoding a fusion protein as described in any one of claims 1-16.

[0267] 20. A vector comprising the nucleic acid as described in any one of claims 17-18.

[0268] 21. A vector having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid of any one of SEQ ID NO: 104-118, 134, and 135.

[0269] 22. A method for purifying nucleic acids, the method comprising: (i) contacting a composition comprising the nucleic acid and at least one contaminant with a fusion protein as claimed in any one of claims 1-16, wherein the fusion protein binds to the nucleic acid to form a complex; (ii) contacting the complex with a first environmental factor to increase the size of the complex; (iii) separating the complex from at least one contaminant; and (iv) separating the nucleic acid from the fusion protein by contacting the complex with a second environmental factor.

[0270] 23. The method of claim 22, wherein the complex is separated from the at least one contaminant based on size.

[0271] 24. The method of claim 23, wherein the size-based separation is performed using a method selected from any of the following: tangential flow filtration, membrane chromatography, analytical ultracentrifugation, high performance liquid chromatography, membrane chromatography, conventional flow filtration, acoustic separation, centrifugation, countercurrent centrifugation, and rapid protein liquid chromatography.

[0272] 25. The method of any one of claims 22-24, wherein the first environmental factor comprises one or more of the following: (a) a change in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of viral particles, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturing agents; or (c) the application of electromagnetic waves or sound waves.

[0273] 26. The method of any one of claims 22-25, wherein the second environmental factor comprises one or more of the following: (a) a change in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of viral particles, or pressure; (b) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturing agents; or (c) the application of electromagnetic waves or sound waves.

[0274] 27. The method of any one of claims 22-26, wherein the at least one contaminant is selected from solvents, proteins, peptides, carbohydrates, nucleic acids, viruses, cells (e.g., bacteria, yeast, or mammalian cells), carbohydrates, lipids, or lipopolysaccharides. Example Example 1. Design of fusion proteins containing nucleic acid-binding proteins and peptides with phase behavior.

[0275] A fusion protein comprising an NBP and a peptide exhibiting phase behavior is generated and characterized. The NBP comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 1-60 or 217. The peptide exhibiting phase behavior comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 1-60 or 217. A fusion protein containing NBP and a phase-behaving peptide was expressed according to a standard protocol. The affinity of the fusion protein for nucleic acids was assessed using a capture assay. The transition temperature of the fusion protein was determined using UV-Vis spectrophotometry. Example 2. Purifying nucleic acids using the fusion protein from Example 1

[0276] The fusion protein from Example 1 was used to purify the target nucleic acid. The fusion protein was mixed with a sample containing the target nucleic acid and contaminants. The fusion protein bound to the target nucleic acid. A first environmental factor was added to the composition to increase the size of the complex. TFF was used to separate the complex from the contaminants. A second environmental factor was added to the solution containing the purified complex to separate the fusion protein from the nucleic acid. Example 3. Reagents containing different peptides with phase behaviors generated by ITC for nonspecific binding comparison.

[0277] Objective: The objective of this study is to identify the most efficient peptides with phase behavior for use in the purification of single-stranded nucleic acids.

[0278] Methods: This study evaluated the nonspecific binding affinity of individual phase-behavioral peptides to a variety of nucleic acids. The affinity of phase-behavioral peptides for nucleic acids was assessed as described in Example 2. Nucleic acids were synthesized, mixed with different phase-behavioral peptides, and then purified as described in Example 2 (e.g., using a reversible phase-transition cycle). The final concentrations of the phase-behavioral peptides, nucleic acids, and salts in the capture reaction were 40 µM, 0.1 mg / ml, and 1.5 M, respectively. Subsequently, 10 µl of the capture reaction was loaded into wells for analysis. In addition to linearized plasmid DNA, the binding of RNA templates of different lengths and strand densities to four different phase-behavioral peptides (20A80 (SEQ ID NO: 262), 50A80 (SEQ ID NO: 263), 100V80 (SEQ ID NO: 56), and 40L80 (SEQ ID NO: 264)) was tested. The capture reactants were loaded directly onto a 1% natural agarose gel, so that the successful binding of phase-behaving peptides to nucleic acid species would be indicated by their retention within the pores.

[0279] Result: The result is in Figure 1 As shown in the figure, 20A80, ​​50A80, ​​and 100V80 exhibited almost no nonspecific binding, while 40L80 showed nonspecific binding to a wide range of nucleic acid species and therefore lower efficiency in nucleic acid capture. Among the peptides exhibiting phase behavior, 100V80 also demonstrated the optimal operating temperature for reversible phase transition cycling during production. Therefore, 100V80 was identified as the most successful candidate peptide with phase behavior for the development of fusion proteins. Example 4. Efficient elution of single-stranded RNA during in vitro transcription using reagents containing Bs-CspB and a phase-behaving peptide.

[0280] Objective: This study aimed to evaluate the purification of single-stranded RNA (ssRNA) during in vitro transcription using a reagent containing a fusion protein (SEQ ID NO: 225) comprising NBP Bs-CspB (SEQ ID NO: 90) and a phase-behaving polypeptide known as 100V80 (SEQ ID NO: 56).

[0281] Methods: This experiment was performed using an ssRNA purification reagent (referred to as “Isotag”) generated in shake flasks and purified by reversible phase transition cycling (ITC). RNA capture reactions were then performed using the ssRNA purification reagent from in vitro transcription (IVT) reactions that transcribed a range of mRNA template sizes, including 1 kb, 4 kb, and 8 kb.

[0282] Direct IVT capture was performed using ssRNA purification reagent generated by ITC. 50 µM of reagent was added to each IVT reaction to capture the desired ssRNA present in the reaction. NaCl was added to achieve a final concentration of 1.5 M, allowing for phase separation of the captured RNA within the droplets from other IVT impurities based on size using centrifugation at room temperature (RT). After aspirating the capture supernatant, the RNA was dissociated from the droplets, which remained intact in the presence of heat, using water. Another round of centrifugation separated the droplets from the finally purified RNA. To determine the nucleic acid concentration within the IVT reaction, the RNA present in the IVT reaction was precipitated and purified using lithium acetate precipitation and subsequent ethanol washing, then resuspended in water. RNA concentration was calculated by assessing absorbance at 260 nm and 280 nm using a UV spectrometer and calculating the concentration using Beer-Lambert's law. The percentage of total RNA eluted for each mRNA template (including 1 kb, 4 kb, and 8 kb) was then calculated. The elution percentage reflects the final yield after elution compared to the total RNA present in the initial IVT reaction.

[0283] Result: The result is in Figure 2 In summary, the ssRNA purification reagent achieved over 80% ssRNA recovery during the ITC reaction. These findings demonstrate that target ssRNA was successfully and efficiently purified using a fusion protein comprising Bs-CspB and the phase-behaving peptide 100V80, employing size-based separation. Example 5. Using the reagent generated by the ITC in Example 4, purified RNA was captured with high selectivity.

[0284] Objective: This study aimed to evaluate the specificity of the reagent generated by ITC in Example 4 for selectively capturing target ssRNA by quantifying the isolation of contaminants (exemplified by dsRNA).

[0285] Methods: This study examined the ability of the ssRNA purification reagent from Example 3 to selectively capture purified RNA. For this study, the separation of dsRNA from ssRNA was evaluated at a range of ssRNA purification reagent:RNA molar ratios.

[0286] To determine the Log10 removal value (LRV) of dsRNA, the concentration of dsRNA in solution was assessed before and after purification with reagents generated using ITC. RNA was generated using the HiScribe® T7 Rapid High-Yield RNA Synthesis Kit (NEB) and a linearized eGFP template carrying 5' and 3' UTRs with 120 nucleotide poly-A tails. IVT was performed at 20° CO / N to ensure sufficient dsRNA generation. To test the specificity for ssRNA in the presence of elevated dsRNA impurities, ssRNA was generated via IVT using Hi-T7® RNA polymerase (NEB), and subsequently enriched using cellulose-based ssRNA purification. Simultaneously, dsRNAs of sequence and size matching the ssRNA were also generated and purified, as described in Baiersdorfer et al., Mol. Ther NucleicAcids [Molecular Therapy - Nucleic Acids], 2019. ssRNA concentration was assessed using UV spectroscopy, while dsRNA concentration was tested using a multi-species dsRNA ELISA kit (Novus Biologicals). Furthermore, the elution percentage of total dsRNA was evaluated in total RNA containing a range of dsRNA incorporation percentages (including 0%, 0.01%, 0.1%, 1%, and 10%).

[0287] Result: The result is in Figure 3A and Figure 3B As shown in the diagram. In summary, the ssRNA purification reagents exhibit high selectivity for ssRNA. For example... Figure 3A As shown, the RNA capture of purified RNA incorporating dsRNA demonstrates that, within a certain range of ssRNA purification reagent:RNA molar ratios, this reagent retains selectivity for ssRNA and is capable of removing greater than 4-log dsDNA. Furthermore, Figure 3B The results showed that even with dsRNA incorporation at values ​​up to 10% of total RNA, selectivity was maintained in the capture of purified RNA. Regardless of the amount of added dsRNA, the percentage eluted during purification remained less than 4%. These findings indicate that reagents containing Bs-CspB and 100V80 are highly selective for target nucleic acids and are able to separate most contaminants from the final product. Example 6. Comparison of Bs-CspB and 100V80 reagents with other nucleic acid binding protein reagents for binding ssRNA and other sequences.

[0288] Objective: This study aimed to evaluate the ability of the ITC-generated reagents (containing Bs-CspB and 100V80) described in Examples 4 and 5 to bind to a wide range of ssRNA templates and other sequences, and to compare this ability with other ITC-generated reagents containing different nucleic acid-binding proteins and 100V80.

[0289] Methods: This study evaluated nucleic acid capture of purified RNA using several different ITC-generating reagents. RNA was synthesized from PCR-generated DNA templates or linearized plasmids using the HiScribe T7 ARCA mRNA Kit (NEB), followed by IVT tailing of transcripts carrying poly-A tails. A 513 bp dsRNA was generated using a luciferase template following the method described in Baiersdorfer et al., Mol Ther Nucleic Acids, 2019. RNA was purified by phenol-chloroform extraction followed by ethanol precipitation with ammonium acetate. The final RNA precipitate was resuspended in ultrapure water at 1 mg / ml. The purity and length of the mRNA transcript were confirmed using UV-Vis spectroscopy and gel electrophoresis. The final concentrations of biopolymer, RNA, and salt in the capture reaction were 40 µM, 0.1 mg / ml, and 1.5 M, respectively. After centrifugation at 5000 g for 5 min, the capture supernatant was aspirated. Successful capture was assessed by gel electrophoresis of samples individually loaded onto a 1% natural agarose gel supplemented with 1x Sybr safe DNA gel staining agent.

[0290] The ability of ssRNA-binding candidates, composed of various nucleic acid-binding proteins coupled to 100V80, to capture ssRNA and dsRNA targets in independent capture reactions was screened. The presence of nucleic acid species in the capture supernatant was assessed using agarose gel electrophoresis. The presence of bands on the gel indicated uncaptured nucleic acids.

[0291] Result: The result is in Figure 4 As shown in the figure, neither the Bs-CspB nor the 100V80 reagents exhibited any bands of ssRNA template, demonstrating that a large amount of dsRNA remained uncaptured. Overall, the Bs-CspB reagent showed significant capture of a wide range of ssRNA species without retaining dsRNA within the droplet. In conclusion, these findings demonstrate the successful purification of single-stranded nucleic acids using a fusion protein comprising Bs-CspB and 100V80. By incorporating references

[0292] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. However, any mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be, considered an admission or implication of any kind that it constitutes valid prior art or forms part of common general knowledge in any country of the world. The following patent documents are incorporated herein by reference in their entirety for all purposes: International Patent Publication No. 2021 / 168270 and International Patent Publication No. 2022 / 178537.

Claims

1. A fusion protein comprising Bacillus subtilis cold shock protein B (Bs-CspB) and a polypeptide exhibiting phase behavior.

2. The fusion protein of claim 1, wherein the Bs-CspB has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO:

90.

3. The fusion protein of claim 1 or 2, wherein the phase-behaving polypeptide comprises P and G motifs, the P and G motifs comprising a plurality of proline residues and a plurality of glycine residues.

4. The fusion protein of any one of 1-3, wherein the P and G motifs comprise at least about 10% proline residues and at least about 20% glycine residues.

5. The fusion protein according to any one of claims 1-4, wherein the phase-behaving polypeptide comprises a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 217), or a random, disordered analogue thereof; wherein Xaa may be any amino acid other than proline.

6. The fusion protein of claim 5, wherein n is an integer from 1 to 360, including endpoints.

7. The fusion protein of any one of claims 1-6, wherein the phase-behaving polypeptide comprises an amino acid sequence selected from: a.(GRGDSPY) n (SEQ ID NO: 1); b.(GRGDSPH) n (SEQ ID NO: 2); c.(GRGDSPV) n (SEQ ID NO: 3); d.(GRGDSPYG) n (SEQ ID NO: 4); e.(RPLGYDS) n (SEQ ID NO: 5); f.(RPAGYDS) n (SEQ ID NO: 6); g.(GRGDSYP) n (SEQ ID NO: 7); h.(GRGDSPYQ) n (SEQ ID NO: 8); i.(GRGNSPYG) n (SEQ ID NO: 9); j.(GVGVP) n (SEQ ID NO: 10); k.(GVGVPGLGVPGVGVPGLGVPGVGVP) m (SEQ ID NO: 11); l.(GVGVPGVVGPGVVGPGVVVP) m (SEQ ID NO: 12); m.(GVGVPGVGVPGWGVPGVGVP) m (SEQ ID NO: 13); n.(GVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP) m (SEQ ID NO: 14); o.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) m (SEQ ID NO: 15); and p.(GAGVPGVGVPGAGVPGVGVPGAGVP) m (SEQ ID NO: 16); Or its random, disordered counterparts; in: n is an integer in the range of 20-360, inclusive; and m is an integer in the range of 4-25, inclusive.

8. The fusion protein of any one of claims 1-5, wherein the phase-behaving polypeptide comprises an amino acid sequence selected from the following: a.(GVGVP) m (SEQ ID NO: 22); b.(ZZPXXXXGZ) m (SEQ ID NO: 23) c.(ZZPXGZ) m (SEQ ID NO: 24); d.(ZZPXXGZ) m (SEQ ID NO: 25); or e.(ZZPXXXGZ) m (SEQ ID NO: 26), where m is an integer between 10 and 160, including the endpoints, where X is any amino acid other than proline or glycine, and where Z is any amino acid, if present.

9. The fusion protein according to any one of claims 1-5, wherein the phase-behaving polypeptide comprises an amino acid sequence selected from: a. (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 17); or b.(GVGVPGVGVPGLGVPGVGVPGVGVP) m (SEQ ID NO: 18); Where m is an integer between 2 and 32, inclusive.

10. The fusion protein of any one of claims 1-5, wherein the phase-behaving polypeptide comprises an amino acid sequence selected from: a.(GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 19), where m is 8 or 16; b.(GVGVPGAGVP) m (SEQ ID NO: 20), where m is an integer between 5 and 80, inclusive; or c.(GXGVP) m (SEQ ID NO: 21), where m is an integer between 10 and 160, including the endpoints, and where each repeating X is independently selected from the group consisting of: glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine.

11. The fusion protein according to any one of claims 1-5, wherein the polypeptide having phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 1-60, 217, or 262-264.

12. The fusion protein according to any one of claims 1-5, wherein the polypeptide having phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of any one of SEQ ID NO: 56 or 262-264.

13. The fusion protein according to any one of claims 1-5, wherein the polypeptide having phase behavior has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO:

56.

14. The fusion protein of claim 1 or 2, wherein the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 90 and at least 90% identity with the polypeptide of any one of SEQ ID NO: 56 and 262-264.

15. The fusion protein according to any one of claims 1-2 or 14, wherein the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 90 and at least 90% identity with the polypeptide of SEQ ID NO:

56.

16. The fusion protein according to any one of claims 1-15, wherein the fusion protein comprises a linker.

17. The fusion protein of claim 16, wherein the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 143-216 or 261.

18. The fusion protein of claim 16 or 17, wherein the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO:

261.

19. The fusion protein of claim 1, wherein the fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NO: 225 or 226.

20. The fusion protein of claim 1, wherein the fusion protein has at least 90% identity with the polypeptide of SEQ ID NO: 225 or 226.

21. The fusion protein according to any one of claims 1-20, wherein the Bs-CspB is bound to RNA, DNA, or both.

22. The fusion protein according to any one of claims 1-21, wherein the Bs-CspB binds to an RNA selected from any one of: double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), mRNA, precursor mRNA, polyadenosine (polyA) RNA, Z-conformation RNA (Z-RNA), or a combination thereof.

23. The fusion protein according to any one of claims 1-22, wherein the Bs-CspB binds to ssRNA.

24. The fusion protein of any one of claims 1-23, wherein the Bs-CspB binds to the 3' end of the mRNA, the 5' end of the mRNA, the coding region of the mRNA, the non-coding region of the mRNA, or a combination thereof.

25. The fusion protein according to any one of claims 1-23, wherein the Bs-CspB binds to the precursor mRNA.

26. The fusion protein of claim 25, wherein the Bs-CspB binds to an intron, an exon, a 5' UTR, a 3' UTR, or a combination thereof of the precursor mRNA.

27. The fusion protein according to any one of claims 1-21, wherein the Bs-CspB binds to DNA.

28. The fusion protein of claim 27, wherein the Bs-CspB binds to single-stranded DNA, double-stranded DNA, polyadenosine (polyA) DNA, Z-conformation DNA (Z-DNA), or a combination thereof.

29. A nucleic acid encoding a fusion protein as described in any one of claims 1-28.

30. The nucleic acid of claim 29, wherein the nucleic acid has a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid of any one of SEQ ID NO: 119-133.

31. A vector encoding a fusion protein as described in any one of claims 1-28.

32. A vector comprising the nucleic acid as described in any one of claims 29-30.

33. A vector having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the nucleic acid of any one of SEQ ID NO: 104-118, 134, and 135.

34. A method for purifying single-stranded nucleic acids, the method comprising: (i) Contacting a composition comprising the single-stranded nucleic acid and at least one contaminant with a fusion protein as described in any one of claims 1-28, wherein the fusion protein binds to the single-stranded nucleic acid to form a complex; (ii) Adding a first environmental factor to the composition comprising the complex, thereby increasing the size of the complex; (iii) Separating the complex from at least one contaminant; as well as (iv) Separating the single-stranded nucleic acid from the fusion protein by contacting the complex with a second environmental factor, thereby forming a product containing the single-stranded nucleic acid.

35. The method of claim 34, wherein the single-stranded nucleic acid comprises ssRNA.

36. The method of claim 34 or 35, wherein the complex is separated from the at least one contaminant based on size.

37. The method of claim 36, wherein the size-based separation is performed using a method selected from any of the following: tangential flow filtration, membrane chromatography, analytical ultracentrifugation, high performance liquid chromatography, conventional flow filtration, acoustic separation, centrifugation, countercurrent centrifugation, and rapid protein liquid chromatography.

38. The method of claim 37, wherein the size-based separation is performed using a method including centrifugation.

39. The method of any one of claims 34-38, wherein the first environmental factor comprises one or more of the following: (a) Changes in one or more of the following: temperature, pH, salt concentration, concentration of purification matrix, concentration of viral particles, or pressure; (b) Adding one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturants; or (c) Apply electromagnetic waves or sound waves.

40. The method of any one of claims 34-39, wherein the second environmental factor comprises one or more of the following: (a) Changes in one or more of the following: temperature, pH, salt concentration, concentration of purification matrix, concentration of viral particles, or pressure; (b) Adding one or more surfactants, cofactors, vitamins, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturants; or (c) Apply electromagnetic waves or sound waves.

41. The method of any one of claims 34-40, wherein the at least one contaminant is selected from solvents, proteins, peptides, carbohydrates, double-stranded nucleic acids, viruses, cells (e.g., bacteria, yeast, or mammalian cells), carbohydrates, lipids, or lipopolysaccharides.

42. The method of claim 41, wherein the at least one contaminant is a double-stranded nucleic acid.

43. The method of claim 42, wherein the double-stranded nucleic acid is dsRNA.

44. The method of claim 39 or 40, wherein the salt is added at a concentration of about 0.5 M to about 3 M.

45. The method of any one of claims 39-40 or 44, wherein the salt is added at a concentration comprising about 1.2 M to about 1.7 M.

46. ​​The method of any one of claims 34-45, wherein, based on the total nucleic acid content in the product of step (iv), the product comprises about 70% to about 100% of the single-stranded nucleic acid.

47. The method of claim 46, wherein the single-stranded nucleic acid comprises ssRNA.

48. The method of any one of claims 34-47, wherein, based on the total nucleic acid content in the product, the product contains about 10% or less of the at least one contaminant.

49. The method of claim 48, wherein the at least one contaminant comprises dsRNA.

50. The method of any one of claims 34-49, wherein the purification method comprises removing at least about 1-log of the contaminant compared to the amount of contaminant in the composition of step (i).

51. The method of any one of claims 34-50, wherein the purification method comprises removing 1 to 10-log of the contaminant compared to the amount of contaminant in the composition of step (i).

52. The method of any one of claims 34-51, wherein in step (i), the fusion protein is present at a concentration of about 1 µM to about 200 µM.

53. The method of any one of claims 34-52, wherein in step (i), the fusion protein is present at a concentration of about 30 µM to about 60 µM.

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