Methods and compositions for single-stranded RNA purification

Abstract

Methods and compositions for purifying a nucleic acid preparation. The methods include contacting a nucleic acid preparation including single-stranded ribonucleic acid (ssRNA) with a double-stranded ribonucleic acid binding protein (dsRBP) that specifically binds to double-stranded ribonucleic acid (dsRNA) and is optionally immobilized on a solid support before or after the dsRBP-dsRNA complex is formed to remove dsRNA from a mixed nucleic acid preparation.

Description

PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1METHODS AND COMPOSITIONS FOR SINGLE-STRANDED RNA PURIFICATIONCROSS-REFERENCES TO RELATED APPLICATION(S)

[0001] None This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 701,854, filed October 1, 2024, and to U.S. Provisional Patent Application No. 63 / 843,549, filed July 14, 2025, the disclosures of which are considered part of, and incorporated in their entireties by reference in the disclosure of this application.SEQUENCE STATEMENT

[0001] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on September 23, 2025, is named TP387899WO1, and is 61,475 bytes in size.FIELD

[0002] The present disclosure relates to nucleic acid production, and more particularly to recombinant nucleic acids, compositions and methods for producing polynucleotides, as well as their use in a variety of applications.BACKGROUND

[0003] Messenger RNA (mRNA) based therapeutics offer various benefits, such as simplified manufacturing, reduced costs, accelerated development, and strong therapeutic potential. However, the purification of mRNA from in vitro transcription (IVT) reactions poses a challenge. Double-stranded RNA is formed as a byproduct in the IVT process and can trigger antagonistic immune responses if not adequately removed. This not only hampers the therapeutic potential of the mRNA but also leads to undesirable side effects.

[0004] Contaminating double-stranded RNA (dsRNA) fragments are difficult to separate from reaction mixtures containing a full-length, single-stranded RNA (ssRNA) product of interest. Existing methods for removing dsRNA from mRNA, such as ion-paired reverse-phase high- performance liquid chromatography (RP-HPLC), cellulose chromatography, and enzymatic (e.g., Rnase III) treatment, suffer from limitations that affect their scalability, reproducibility, and simplicity. Moreover, these methods often require harsh conditions, utilize toxic and volatile chemicals, and involve additional cleanup steps.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0005] There exists a substantial need for more efficient and less costly methods for purification of ssRNA for a variety of applications including therapeutics and cell and gene therapy development.SUMMARY

[0006] The technology set out herein is directed to improvements in ssRNA purification / isolation. Provided herein are systems, compositions, and purification methods for removing dsRNA contaminants from a nucleic acid preparation that includes a ssRNA (e.g., messenger RNA (mRNA)) product of interest. The purification methods of the present disclosure are based, at least in part, on experimental results showing that immobilized double-stranded ribonucleic acid binding protein (dsRBP) and dsRBP variants, as provided herein, bind specifically to dsRNA.

[0007] Thus, some aspects of the present disclosure provide methods of purifying a nucleic acid (e.g., RNA) preparation that includes contacting a nucleic acid preparation comprising messenger ribonucleic acid (mRNA) (e.g., an in vitro-transcribed mRNA) with a dsRBP that is immobilized on a solid support (e g., a resin or bead) before or after contacting the nucleic acid preparation. Typically, such a nucleic acid preparation comprises contaminating double-stranded RNA, therefore, affinity purification methods, as provided herein, may be performed under conditions that result in binding of the dsRBP to double-stranded RNA. In this manner, a purified nucleic acid preparation comprising the mRNA free, or substantially free, of dsRNA can be purified.

[0008] In some embodiments, the dsRBP comprises a B2 dsRBP as described herein.

[0009] Other aspects of the present disclosure provide methods that comprise performing an in vitro transcription reaction in the presence of a template nucleic acid (e.g., DNA) to produce an in vitro transcription product (e.g., RNA, such as mRNA), and contacting the in vitro transcription product with a dsRBP that is immobilized on a solid support (e.g., a resin or bead) before or after the dsRBP is contacted with the in vitro transcription product.

[0010] Further provided herein are compositions comprising RNA purified according to methods that comprise contacting a nucleic acid preparation comprising mRNA with a dsRBP. In some embodiments, the composition (e.g., comprising the purified RNA) is substantially free of doublestranded RNA.

[0011] Also provided are dsRBPs that specifically bind dsRNA, as well as kits including such dsRBP s.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 is a diagram illustrating an affinity purification workflow in embodiments of the disclosure.

[0013] FIG. 2 is a series of images depicting experimental results generated using the workflow depicted in FIG. 1 in embodiments of the disclosure.

[0014] FIG. 3 is a graph showing experimental data generated using the workflow depicted in FIG. 1 in embodiments of the disclosure.

[0015] FIG. 4 is a graph showing experimental data generated using the workflow depicted in FIG. 1 in embodiments of the disclosure.

[0016] FIG. 5 is a series of graphs showing experimental data generated using ssRNA purified by the affinity purification workflow shown in FIG. 1.

[0017] FIG. 6 is a flow chart depicting an overview of the workflow used in experiments performed in Example 2 of the disclosure.

[0018] FIG. 7 is a graphical representation depicting a B2 dsRBP splice variant including a streptavidin binding peptide (SBP) as a tag binding moiety. Dimerized helices of the splice variant are shown bound to dsRNA and the tag binding moiety is bound to a magnetic bead via streptavidin which is attached to the bead. Removal of the dsRNA from a mixture of RNA is facilitated by application of an external magnetic field to sequester the magnetic particles leaving ssRNA in the RNA preparation.

[0019] FIG. 8 is a series of images depicting experimental results generated using the workflow depicted in FIG. 6 in embodiments of the disclosure.

[0020] FIG. 9 is a series of graphs showing experimental data generated using a B2 dsRBP splice variant in embodiments of the disclosure.

[0021] FIG. 10 is a series of graphs showing experimental data generated using ssRNA affinity purified a B2 dsRBP splice variant in embodiments of the disclosure.DETAILED DESCRIPTION

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technologyPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the meanings provided herein.

[0023] The terminology used herein is for the purpose of describing particular instances only, and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] The product of ribonucleic acid (RNA) synthesis reactions, such as IVT reactions, often contains some amount of contaminating RNA, including double-stranded RNA. Such contaminants can adversely impact downstream molecular and therapeutic application, for example. Provided herein are methods of removing specifically dsRNA contaminants from an RNA preparation. More generally, the present disclosure provides methods of purifying a nucleic acid preparation. The methods may include, for example, contacting a nucleic acid preparation with a dsRBP that is immobilized on a solid support before or after it is contacted with the nucleic acid preparation.

[0025] To overcome these challenges, the disclosure provides a novel purification platform based on an affinity capture approach. By employing recombinantly generated dsRNA-binding proteins along with a specifically designed solid phase, effective capture and removal of dsRNA contaminants is possible. This approach offers several advantages over existing methods. It eliminates the need for harsh conditions and toxic chemicals, resolves scalability issues, and streamlines the purification process by eliminating the need for additional cleanup steps. Furthermore, the disclosure provides a higher level of specificity in recognizing dsRNA compared to existing methods, resulting in cleaner preparations of single-stranded mRNA for an improved therapeutic response.

[0026] Affinity Purification Using dsRBP s

[0027] RNA synthesis reactions, such as in vitro transcription reactions, typically produce an end product preparation that includes a mixture of different nucleic acid species. Purification of the intended single-stranded species is typically required prior to its use in any particular application (e.g., therapeutic application). Thus, affinity purification methods as provided herein are typically used to purify a “nucleic acid preparation,” which is simply a solution comprising nucleic acid, for example, a mixture of different nucleic acid species (e.g., full-length and truncated ssRNA, dsRNA, dsDNA, etc.). In some embodiments, a nucleic acid preparation is the end product of an in vitro transcription reaction (for example, using bacteriophage T7 RNA polymerase (e.g., asPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 described in Donzeet et al., Nucleic Acids Res 30:e46, 2002; and Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002)). RNA (e.g., mRNA) preparations produced by IVT often contain contaminants, such as nucleic acid contaminants (e.g., DNA template) and protein contaminants (e.g., T7 RNA polymerase). Methods of removing DNA contaminants (e.g., via digestion of the DNA by DNases) or protein contaminants (e.g., via pheno-chloroform precipitation) from an RNA preparation are known. Additional contaminants include truncated fragments of the ssRNA (e.g., mRNA) of interest generated by the T7 RNA polymerase. In some instances, these truncated fragments contain complementary species and form double-stranded RNA (dsRNA) species (e.g., as described in Kariko et al. (Nucleic Acids Research, 2011, Vol. 39, No. 21, el42, 2009) and loopback dsRNA as described in Dousis et al. (Nature Biology, 2023, Vol. 41, 560-568). It is difficult to separate such dsRNA contaminants from the desired ssRNA because the dsRNA contaminants and the ssRNA often have very similar biochemical and biophysical properties.

[0028] Provided herein are affinity purification methods that use immobilized dsRBP, as well as functional homologues, variants, and fragments of dsRBP, that specifically bind to (and thus capture) dsRNA species from an RNA preparation. The affinity purification methods make use of specific binding interactions between a dsRBP and double-stranded RNA. dsRBP is chemically immobilized or “coupled” to a solid support so that when a nucleic acid preparation is passed over the solid support (e.g., column), dsRNA molecules become bound to the dsRBP. The “flow through” fraction is essentially free of dsRNA contaminants. Alternatively, the dsRBP may be mixed with a nucleic acid preparation to facilitate binding of the dsRBP and subsequently the dsRBP is chemically immobilized or “coupled” to a solid support to enable purification of ssRNA from a mixture including dsRNA in the nucleic acid preparation.

[0029] Thus, a nucleic acid preparation may be “purified” using affinity purification methods of the present disclosure, optionally in combination with other purification methods that remove DNA and protein contaminants. “Purification,” generally, refers to a process (one or more steps) of isolating one particular species (e.g., mRNA) or a subgroup of species from a larger group of species (e.g., a combination of RNA, DNA and protein). A purification process results in enrichment of the RNA of interest.

[0030] In some embodiments, purification can be partial (e.g., as in fractionation). In some embodiments, purification yields RNA of interest that is substantially free of other, chemically dissimilar types of molecules. For example, nucleic acids are purified from mixtures comprisingPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 proteins, lipids, carbohydrates, etc. In some embodiments, purification results in an RNA of interest that is in pure form, i.e., free or substantially free from all other substances, whether chemically similar or not. Being “substantially free of’ a substance (e.g., protein, carbohydrates, lipids, and other nucleic acids) means the RNA of interest comprises less than 20%, less than 10%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%, or less than 0.005%, or less than 0.004%, or less than 0.003%, or less than 0.002%, or less than 0.001%, or less than 0.0009%, or less than 0.0008%, or less than 0.0007%, or less than 0.0006%, or less than 0.0005% of the substance by weight or by molarity. In some embodiments, to reach its pure form, an RNA of interest may be subjected to more than one purification process (in addition to the affinity purification methods of as provided herein).

[0031] dsRBPs

[0032] In various embodiments, a dsRBP is an RNA binding protein that specifically binds dsRNA.

[0033] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, butthat function in a manner similar to a naturally occurring amino acid.

[0034] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0035] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may optionally be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0036] “Recombinant protein” refers to protein that is encoded by a nucleic acid that is introduced into a host cell. The host cell expresses the nucleic acid. The term “expressing a nucleic acid” is synonymous with “expressing a protein from an RNA encoded by a nucleic acid. “Protein” as used herein broadly refers to polymerized amino acids, e.g., peptides, polypeptides, proteins, lipoproteins, glycoproteins, etc.

[0037] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0038] The following eight groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Glycine (G); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (7) Serine (S), Threonine (T); and (8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0039] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignmentPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0040] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the disclosure or individual domains of the polypeptides of the disclosure), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms or by manual alignment and visual inspection. Such sequences that are at least about 80% identical are said to be “substantially identical.” In some embodiments, two sequences are 100% identical. In certain embodiments, two sequences are 100% identical over the entire length of one of the sequences (e.g., the shorter of the two sequences where the sequences have different lengths). In various embodiments, identity may refer to the complement of a test sequence. In some embodiments, the identity exists over a region that is at least about 10 to about 100, about 10 to about 75, about 10 to about 50 amino acids or nucleotides in length. In certain embodiments, the identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids in length.

[0041] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case ofPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0042] The terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein “corresponds” to a given residue when it occupies the same essential structural position within the protein as the given residue.

[0043] For specific proteins described herein (e.g., dsRBP), the named protein includes any of the protein’s naturally occurring forms, or fragments, or variants or homologs that maintain the protein’s dsRNA binding activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In aspects, variants or homologs have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In aspects, the protein is the protein as identified by its NCBI sequence reference. In aspects, the protein is the protein as identified by its NCBI sequence reference or functional fragment or homolog thereof.

[0044] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0045] A “comparison window” refers to a segment of any one of the number of contiguous positions (e.g., at least about 10 to about 100, about 20 to about 75, about 30 to about 50, 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250) in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. In various embodiments, a comparison window is the entire length of one or both of two aligned sequences. In some embodiments, two sequencesPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 being compared comprise different lengths, and the comparison window is the entire length of the longer or the shorter of the two sequences. In certain embodiments relating to two sequences of different lengths, the comparison window includes the entire length of the shorter of the two sequences. In some embodiments relating to two sequences of different lengths, the comparison window includes the entire length of the longer of the two sequences.

[0046] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0047] Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI), as is known in the art. An exemplary BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments;PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In certain embodiments, the NCBI BLASTN or BLASTP program is used to align sequences. In certain embodiments, the BLASTN or BLASTP program uses the defaults used by NCBI. In certain embodiments, the BLASTN program (for nucleotide sequences) uses as defaults: a word size (W) of 28; an expectation threshold (E) or 10; max matches in a query range set to 0; match / mismatch scores of 1, -2; linear gap costs; the filter for low complexity regions used; and mask for lookup table only used. In certain embodiments, the BLASTP program (for amino acid sequences) uses as defaults a word size (W) of 3; an expectation threshold (E) of 10; max matches in a query range set to 0; the BLOSUM62 matrix (see Henikoff and Henikoff 1992) Proc. Natl. Acad. Sci. USA 89: 10915); gap costs of existence: 11 and extension: 1; and conditional compositional score matrix adjustment.

[0048] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0049] In some embodiments, a dsRBP includes a double-stranded RNA binding domain (dsRBD). In some embodiments, a dsRBD is a well-characterized domain found in many proteins associated with various cellular functions including antiviral response, RNA editing, RNA processing, and RNA transport. A dsRBD (also referred to as dsRBM for double-stranded RNA- binding motif) is a conserved protein domain typically consisting of approximately 65-70 conserved amino acids, which binds double-stranded or highly structured RNAs as described in Baneijee et al. (RNA Biol. 2014 Oct; 11(10): 1226-1232) which is incorporated herein by reference. It was first recognized as a conserved protein domain from similarities between Drosophila Staufen, human TAR-RNA binding protein (TRBP) and Xenopus laevis RNA-binding protein A (XlrbpA). The central function of dsRBDs is to bind to dsRNA regions. This is primarily achieved by recognizing the RNA shape, such as for instance the shape of a regular A-form RNA helix or the shape of an RNA hairpin, even though some dsRBDs can bind to dsRNA in a sequence-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 specific manner. Illustrative embodiments of dsRBDs which may be utilized in embodiments of the disclosure include, but are not limited to Dicer and PKR.

[0050] In some embodiments, a dsRBP includes one or more helicase like domains that bind specifically to dsRNA. Illustrative embodiments of helicase like domains which may be utilized in embodiments of the disclosure include, but are not limited to DEAD box motifs such as those in RIG-I or MDA5.

[0051] In some embodiments, a dsRBP includes one or more zinc finger domains that bind specifically to dsRNA. Illustrative embodiments of zinc finger domains which may be utilized in embodiments of the disclosure , but are not limited to those of ADR proteins, EWS-FLI1 or J AZ.

[0052] In some embodiments, a dsRBP includes one or more pseudo-NTase domains that bind specifically to dsRNA. Illustrative embodiments of pseudo-NTase domains which may be utilized in embodiments of the disclosure include, but are not limited to those of OAS family proteins.

[0053] In some embodiments a dsRPB is a B2 dsRBP. A B2 dsRBP is an RNA binding protein that specifically binds dsRNA via one or more helicase domains. The protein is expressed in many organisms such as those in the Nodaviridae family and is highly conserved, especially among members of the Alphanodavirus genus and Betanodavirus genus. As further discussed herein, B2 dsRBP is capable of binding dsRNA as a monomer or dimer in which helicase domains are dimerized. As also discussed herein, fragments of full length (e.g., wild type) B2 dsRBP are capable of binding dsRNA as a monomer or dimer in which helicase domains are dimerized.

[0054] B2 dsRBPs that may be used in accordance with the present disclosure include B2 dsRBPs that specifically bind to dsRNA. A B2 dsRBP may be a viral protein. In some embodiments, a B2 dsRBP is a Nodaviridae family B2 dsRBP. In some embodiments, a B2 dsRBP is an Alphanodavirus genus B2 dsRBP. In some embodiments, a B2 dsRBP is a Betanodavirus genus B2 dsRBP. In some embodiments, a B2 dsRBP is a Flock house virus B2 dsRBP. In some embodiments, a B2 dsRBP is a Nodamura virus B2 dsRBP. In some embodiments, a B2 dsRBP is a Black beetle virus B2 dsRBP. In some embodiments, a B2 dsRBP is a Boolarra virus B2 dsRBP. In some embodiments, a B2 dsRBP is a Betanodavirus epinepheli, Betanodavirus pseudocarangis, Betanodavirus takifugui, or Betanodavirus verasperi virus B2 dsRBP.

[0055] As used herein, a dsRBP may refer to a functional homolog, a recombinantly generated variant, or a fragment thereof that is capable of specifically binding dsRNA.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0056] In various embodiments, a dsRBP can include one or more linkers, protease cleave sites, and / or tag moieties to assist in immobilizing the protein to a solid support. Such tags are well known in the art. By way of illustration only, tag binding moieties include, by way of illustration only, V5 epitope, c-myc, hemagglutinin (HA), FLAG™, polyhistidine (His) (e.g., 6X His (SEQ ID NO: 61)), glutathione-S-transferase (GST), maltose binding protein (MBP), streptavidin binding protein (SBP), biotin binding protein, avidin, biotinidase, imidazole binding protein and the like. Conjugate binding tags for such tag binding moieties are known in the art.

[0057] Immobilized dsRBP. In some embodiments, dsRBPs are immobilized on a solid support and then contacted with a nucleic acid preparation containing, for example, a mixture of singlestranded and double-stranded RNA. In some embodiments, dsRBPs are immobilized on a solid support after being contacted with a nucleic acid preparation containing, for example, a mixture of single-stranded and double-stranded RNA. In either instance, a dsRBP, when in contact with double- stranded RNA (dsRNA) binds to (captures) the dsRNA such that the dsRNA becomes associated with the solid support. For example, in some embodiments, a dsRBP binds to the dsRNA and subsequently the dsRBP is immobilized on a solid support such that the dsRNA bound by the dsRBP becomes associated with the solid support. In some embodiments, an immobilized dsRBP, when in contact with double-stranded RNA (dsRNA) binds to (captures) the dsRNA such that the dsRNA becomes associated with the solid support.

[0058] A dsRBP is considered “immobilized” on a solid support when the protein is covalently or non-covalently atached to the support such that the protein does not dissociate from the support when contacted with a pH neutral buffered solution. In some embodiments, the protein does dissociate from the support when in contact with a pH neutral buffered solution. In some embodiments, the protein is non-covalently attached to the solid support via a tag or binding peptide motif (e.g., histidine tag or streptavidin binding motif) and dissociates from the support when in contact with a pH neutral buffered solution containing a ligand competing for binding to the tad or binding motif of the dsRBP. In some embodiments, the protein does not dissociate from the support when in contact with a buffer solution lacking a competing ligand. For example, the protein may be non-covalently attached to a nickel resin support through a histidine tag or to a streptavidin resin through a streptavidin binding peptide. As such, in some embodiments, the protein does not dissociate from the support when in contact with a buffer solution unless it contains imidazole (for the histidine tag) or biotin (for the SBP tag).PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0059] A solid support may be a substance with a surface to which a dsRBP can be attached such that the polypeptide becomes immobilized with respect to the solid support. As used herein, the term “solid support” refers to each undissolved support that is capable of immobilizing a dsRBP or other intermediary reaction component on its surface. The solid support material is non- degradable and can be selected from, for example, agarose, modified agarose, sepharose, thiopropyl-sepharose, Sephadex™, polystyrene, cellulose and ferro- or ferrimagnetic particles, acrylamide, polysaccharides, nitrocellulose, metals or metal compounds (e.g., comprising gold, silver, steel, aluminum, or copper), inorganic glasses, silica, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polyactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, or any combination thereof.

[0060] A solid support may have different shapes, such as a slide, a membrane, a matrix, a plate, a chip, a resin or a bead. A solid support may further have holes or depressions to perform reactions at defined locations in an arrayed format on or within the solid support. Reactions on a solid support may be carried out in the presence of one or more additives. Such additives may help to keep beads in suspension or otherwise increase the fidelity of enzymes acting in close proximity to the solid support. The additive may be a chemical compound, a polymer, a polysaccharide, a protein, a chaperon, or any mixture thereof.

[0061] Methods and strategies for choosing appropriate solid supports and for coupling biomolecules to said solid supports are known in the art (see, e.g., Arndt-Jovin et al. 1 75. EUR. J. Biochem. 54 (2): 41 1-8; Kerrigan et al., 2001. Current Protocols in Molecular Biology. 24: 12.10.1-12.10.18; WO 1995 / 08626).

[0062] In various embodiments, the solid support is a bead, wherein the bead is of any convenient dimension and is constructed from any number of known materials. In some instances, the bead may be monodisperse. Examples of such materials include inorganic materials, natural polymers, and synthetic polymers. Specific examples of these materials include cellulose, cellulose derivatives, acrylic resins, glass; silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene, polyacrylamide, latex gel, polystyrene dextran, rubber, silicon, plastic, nitrocellulose, cellulose, natural sponge, silica gel, glass, metal, plastic, cellulose, methacrylate beads, cross-linked dextran (e.g., Sephadex™) andPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 agarose gels (Sepharose). In a specific embodiment, the solid support is a bead, wherein the bead is a magnetic bead or an agarose bead. In a specific embodiment, the solid support is an agarose resin. In a specific embodiment, the solid support is a metal (e.g., nickel, cobalt, copper, zinc and the like) agarose resin, such as a nickel agarose resin.

[0063] In some embodiments, magnetic beads may include microparticles or nanoparticles. In some examples, the magnetic beads may contain iron oxide. For example, magnetic nanoclusters as described in patent application No. GB2210796.5 which is hereby incorporated by reference may be used. For example, the magnetic beads may be selected from, but not limited to, Dynabeads™ MyOne™ Silane, Dynabeads™ MyOne™ Carboxylic Acid, Dynabeads™ M-270™ Carboxylic Acid, Dynabeads™, Oligo(dT)25 magnetic beads (all available from Thermo Fisher Scientific), SpeedBead™ (General Electric, Boston, MA), BioMagPlus COOH™ and ProMag 1 COOH™ (both Bangs Laboratories, INC Fishers), 4.4 pm fluorescent ferromagnetic beads or 2.0 pm ferromagnetic beads (both available from Spherotech INC Lake Forest, IL), 2 pm beads designated WHM-S001™ or 2 pm beads designated WHM-S002™ (both available from Creative Diagnostics, New York, NY), Silicon Hydroxyl Magnetic Microspheres or Carboxyl Magnetic Microspheres or Oligo(dT) Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), Carboxyl Adembeads (available at 100 nm, 200 nm, 300 nm or 500 nm from Ademtech, France) etc.

[0064] In some embodiments, a dsRBP is immobilized on nanoparticles. Nanoparticles include, but are not limited to, metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS coated with ZnS) and magnetic (e.g., ferromagnetic) colloidal materials.

[0065] Surface compositions that may be used to immobilize a dsRBP or a fragment or variant thereof (e.g., B2 dsRBP fragment) are available. For example, the surface of the support may comprise reactive functional groups that form covalent bonds with a dsRBP or a variant thereof. In some embodiments, the functional groups are chemical functionalities. That is, the binding surface may be derivatized such that a chemical functionality is presented at the binding surface, which can react with a chemical functionality on polypeptide to be attached, resulting in immobilization. Examples of functional groups for attachment that may be useful include, but are not limited to, amino-reactive groups, carboxyl -reactive groups, epoxide groups, maleimide groups, oxo groups, and thiol groups. Functional groups can be attached, either directly or indirectly through the use of a linker, the combination of which is sometimes referred to as aPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1“crosslinker.” Crosslinkers for attaching proteins to a support member are known in the art; for example, homo-or hetero-bifunctional crosslinkers as are well known (e.g., see 1994 Pierce Chemical Company catalog, technical section on crosslinkers, pages 155-200, or “Bioconjugate Techniques” by Greg T. Hermanson, Academic Press, 1996). Non-limiting example of crosslinkers include alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), esters, amide, amine, epoxy groups and ethylene glycol and derivatives. A linker may also be a sulfone group, forming a sulfonamide. In some embodiments, the functional group is a light-activated functional group. That is, the functional group can be activated by light to attach the capture component to the capture object surface. One example is PhotoLink™ technology available from SurModics, Inc. in Eden Prairie, Minn. The examples provided herein on the solid support and the surface composition are not meant to be limiting. Any solid support that are known in the art to be suitable for immobilization of polypeptides may be used in accordance with the present disclosure. Immobilization of a dsRBP to a solid support is carried out under conditions that maintains the structure and activity of a dsRBP. One skilled in the art is familiar with such conditions.

[0066] In some embodiments, the dsRBP includes a streptavidin binding protein. After the dsRBP is contacted with a mixture of RNAs and selectively bind dsRNA, the dsRNA-dsRBP complex is immobilized on a magnetic bead via binding of the SBP to streptavidin conjugated to the surface of the magnetic bead. An external magnet may then be applied to sequester the dsRNA-dsRBP complex to remove dsRNA from the mixture.

[0067] Exemplary B2 dsRBPs. Any B2 dsRBP, including homologs, variants and fragments thereof, that specifically binds to dsRNA may be used in accordance with the present disclosure. Non-limiting examples of B2 dsRBPs are listed in Table 1. In some embodiments, the B2 dsRBP specifically binds dsRNA as a dimer. In some embodiments, the B2 dsRBP specifically binds dsRNA as a monomer. In some embodiments, the B2 dsRBP specifically binds dsRNA as a dimer including 2 discrete dsRBPs. In some embodiments, the B2 dsRBP specifically binds dsRNA via a dsRNA binding region including dimerized helices. In some embodiments, the B2 dsRBP is encoded by an amino acid sequence that encodes helices that dimerize to form a dsRNA binding region that specifically binds dsRNA. In some embodiments, the B2 dsRBP is encoded by an amino acid sequence that encodes helices that dimerize to form a dsRNA binding region that specifically binds dsRNA and not ssRNA. In some embodiments, the B2 dsRBP is encoded by anPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 amino acid sequence that encodes a helix that dimerizes with a helix of a second B2 dsRBP to form a dsRNA binding region that specifically binds dsRNA. In some embodiments, the B2 dsRBP is encoded by an amino acid sequence that encodes a helix that dimerizes with a helix of a second B2 dsRBP to form a dsRNA binding region that specifically binds dsRNA and not ssRNA.

[0068] Illustrative Embodiments of B2 dsRBPs

[0069] Table 1 : Illustrative Embodiments of B2 dsRBPsPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1PCT / US25 / 47503 23 September 2025 (23.O9.2O25)Aty Docket No. TP387899WO1

[0070] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 25.

[0071] PSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 25)

[0072] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 26.

[0073] PSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSR MVTSLLEKPSVVAYLEGKAPEEAKPTLEERLRKLELSHSLPTTGSDPPPAKL (SEQ ID NO: 26)

[0074] In some embodiments, the B2 dsRBP is a splice variant including an amino acid sequence set forth in SEQ ID NO: 27 having two full length monomer sequences joined by an intervening linker sequence (underlined below).PCT / US25 / 47503 23 September 2025 (23.O9.2O25)Aty Docket No. TP387899WO1

[0075] PSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSR MVTSLLEKPSVVAYLEGKAGGGGGGGGGGGGGGGGGGGGPSKLALIOELPDRIQTAVE AAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 27)

[0076] In some embodiments, the B2 dsRBP is a splice variant including an amino acid sequence set forth in SEQ ID NO: 28 having a full length monomer sequence inserted within a second monomer sequence and joined by intervening linker sequences (underlined below).

[0077] PSKLALIQELPDRIQTAVEAAMGGGSGPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKAGGSGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 28)

[0078] In some embodiments, the B2 dsRBP is a splice variant including an amino acid sequence set forth in SEQ ID NO: 29 having a streptavidin binding peptide and linkers.

[0079] MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPGGGGPSKLALIQELPDRIQTAVEAAMGGGSGPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHA CLNKAKLTVSRMVTSLLEKPSVVAYLEGKAGGSGMSYQDAPNNVRRDLDNLHACLNK AKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 29)

[0080] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 30 having a 6XHis tag (SEQ ID NO: 61), V5 tag, TEV / protease cleavage site and linkers.

[0081] MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTKKKKGGGGGGGGGGGGPSKLALIQELPDRIQTAVEAAMGGGSGPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSWAYLEGKAGGSGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 30)

[0082] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 31 having a 6XHis (SEQ ID NO: 61) tag, V5 tag, TEV / protease cleavage site and a linker.

[0083] MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTKKKKGGGGGGGGGGGGPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSWAYLEGKA (SEQ ID NO: 31)

[0084] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 32 having a 6XHis (SEQ ID NO: 61) tag, V5 tag, TEV / protease cleavage site and linkers.

[0085] MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTKKKKGGGGGGGGGGGGPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPPCT / US25 / 47503 23 September 2025 (23.O9.2O25)Aty Docket No. TP387899WO1SWAYLEGKAGGGGGGGGGGGGGGGGGGGGPSKLALIQELPDRIQTAVEAAMGMSYQ DAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 32)

[0086] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 33.

[0087] PSKLALIQELPDRIQTAVEAAMG (SEQ ID NO: 33)

[0088] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 34

[0089] MSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 34)

[0090] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 35.

[0091] NNVRRDLDNLHACLNKAKLTVSRMVTSLLEK (SEQ ID NO: 35)

[0092] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 36.

[0093] CLNKAKLTVSRMVTSLLEK (SEQ ID NO: 36)

[0094] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 37.

[0095] CLNKAKLTVRRMVTSLLEK (SEQ ID NO: 37)

[0096] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 38.

[0097] CLNKAKQTVNRMVTSLLDK (SEQ ID NO: 38)

[0098] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 39.

[0099] CLAKLETTCFRATDSLLSK (SEQ ID NO: 39)

[0100] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 40.

[0101] CLNKAKLTVSRMVTSLLEK (SEQ ID NO: 40)

[0102] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 41.

[0103] CLAGLSAKAERATKALLAK (SEQ ID NO: 41)PCT / US25 / 47503 23 September 2025 (23.O9.2O25)Aty Docket No. TP387899WO1

[0104] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 42.

[0105] CLNKARLTVNRMVTALLDK (SEQ ID NO: 42)

[0106] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 43.

[0107] CIHKGRTAIIRGIETTLEK (SEQ ID NO: 43)

[0108] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 44.

[0109] CLNKAKLTVSRMVTSLLEK (SEQ ID NO: 44)

[0110] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 45.

[0111] CLNKAKLTVGRMVTSLLEK (SEQ ID NO: 45)

[0112] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 46.

[0113] CLNKARLTASRMVTSLLEK (SEQ ID NO: 46)

[0114] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 47.

[0115] CLAKTKQTTSRMVTALLQK (SEQ ID NO: 47)

[0116] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 48.

[0117] CAQKALDTLERMVHSQLEK (SEQ ID NO: 48)

[0118] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 49.

[0119] CLTKAKQRVERMVTSLLTK (SEQ ID NO: 49)

[0120] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 50.

[0121] CLNKARLTVNRMVTALLDK (SEQ ID NO: 50)

[0122] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 51.

[0123] CLRKQETTVRNATRSLLEK (SEQ ID NO: 51)PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0124] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 52.

[0125] CLNKAKLTVSRMVTSLLEK (SEQ ID NO: 52)

[0126] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 53.

[0127] CLLGMREKAQKSTQSLLQK (SEQ ID NO: 53)

[0128] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 54.

[0129] CASLSVTKVTKAVTSLLEK (SEQ ID NO: 54)

[0130] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 55.

[0131] CLNKAKQTVNRMVTSLLDK (SEQ ID NO: 55)

[0132] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 56.

[0133] CLNKAKLTVSRMVTSLLEK (SEQ ID NO: 56)

[0134] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NO: 57.

[0135] CLNKAKLTVGRMVTSLLEK (SEQ ID NO: 57)

[0136] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of binding dsRNA. In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of dimerization with a helix of a second B2 dsRBP to specifically bind dsRNA. In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of dimerization with a helix of a second B2 dsRBP, wherein the dimer forms a dsRNA binding region that specifically binds dsRNA. In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of dimerization with a helix of a second B2 dsRBP, wherein the dimer forms a dsRNA binding region that specifically binds dsRNA and not ssRNA.

[0137] In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of dimerization with a helix ofPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 a second B2 dsRBP including an amino acid sequence set forth in SEQ ID NOs: 34-57 to specifically bind dsRNA. In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of dimerization with a helix of a second B2 dsRBP including an amino acid sequence set forth in SEQ ID NOs: 34-57, wherein the dimer forms a dsRNA binding region that specifically binds dsRNA. In some embodiments, the B2 dsRBP includes an amino acid sequence set forth in SEQ ID NOs: 34-57 that forms a three dimensional helix structure capable of dimerization with a helix of a second B2 dsRBP including an amino acid sequence set forth in SEQ ID NOs: 34-57, wherein the dimer forms a dsRNA binding region that specifically binds dsRNA and not ssRNA.

[0138] In some embodiments the B2 dsRBP includes a first amino acid sequence set forth in SEQ ID NOs: 34-57 and a second amino acid sequence set forth in SEQ ID NOs: 34-57, wherein the first and second amino acid sequences are joined by an intervening sequence. In some embodiments, the first amino acid sequence is identical to the second amino acid sequence and the sequences form a dsRNA binding region that specifically binds dsRNA, the region including a first helix and a second helix that are dimerized.

[0139] In some embodiments, the B2 dsRBP is a B2 dsRBP variant including two helices, each helix being encoded by an amino acid sequence including a sequence as set forth in SEQ ID NO: 35, 36, or 37 joined by an intervening sequence. In some embodiments, the helices dimerize to form a dsRNA binding region that specifically binds dsRNA. In some embodiments, the helices dimerize to form a dsRNA binding region that specifically binds dsRNA and not ssRNA.

[0140] In some embodiments, a B2 dsRBP used in the affinity purification methods as provided herein includes the amino acid sequence identified by any of SEQ ID NOs: 1-59 or fragment thereof. In some embodiments, a B2 dsRBP includes an amino acid sequence that is at least 80% identical to the amino acid sequence identified by any of SEQ ID NOs: 1-59 or fragment thereof. For example, a B2 dsRBP may include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence identified by any of SEQ ID NOs: 1-59 or fragment thereof. In some embodiments, a B2 dsRBP includes an amino acid sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence identified by any of SEQ ID NOs: 1-59 or fragment thereof.

[0141] dsRBP Homologs, Variants and FragmentsPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0142] dsRBP homologs, variants and fragments are also within the scope of the present disclosure. In some embodiments, provided herein are dsRBP fragments (polypeptide sequences at least one amino acid residue shorter than a full-length dsRBP but otherwise identical) having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids. Any dsRBP polypeptide that includes a stretch of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any of the dsRBP sequences described herein may be utilized in an affinity purification method of the present disclosure, provided the polypeptide binds specifically to dsRNA.

[0143] In some embodiments, provided herein are B2 dsRBP fragments (polypeptide sequences at least one amino acid residue shorter than a full-length B2 dsRBP (e.g., SEQ ID NOs: l-59) but otherwise identical) having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids. Any B2 dsRBP polypeptide that includes a stretch of 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the B2 dsRBP sequences described herein may be utilized in an affinity purification method of the present disclosure, provided the polypeptide binds specifically to dsRNA.

[0144] In some embodiments, the B2 dsRBP includes a stretch of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to any of SEQ ID NOs: 1-59 and specifically binds dsRNA.

[0145] dsRBP may be recombinantly produced and purified. Methods of expressing and purifying a dsRBP are known. For example, a nucleic acid sequence encoding a dsRBP may be cloned into expression vectors, for the expression of the dsRBP in a variety of host cells, e.g., bacterial cells, insect cells, or mammalian cells.

[0146] Ribonucleic Acid

[0147] Affinity purification methods as provided herein are used, in some embodiments, to remove double-stranded RNA (dsRNA) from a preparation containing single-stranded RNA (ssRNA), such as in vitro transcribed mRNA. A “single-stranded RNA” is a polymeric strand of contiguous ribonucleotides. A “double-stranded RNA” is comprised of two polymeric strands of contiguous ribonucleotides bound to each other through complementary ribonucleotide base pairing. Single-stranded RNA includes, without limitation, mRNA, ribosomal RNA (see, e.g., Widmann et al., Nucleic Acids Res. 35 (10): 3339-54), transfer RNA (tRNA), tmRNA (see, e.g., Felden et al., RNA. 3 (1): 89-103), microRNA (miRNA), short-hairpin RNA (shRNA), and non-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 coding RNA (ncRNA). In some embodiments, a ssRNA is a messenger RNA (mRNA), such as a therapeutic mRNA. ssRNAs described herein may form intramolecular secondary structures and may be partially double-stranded. An RNA molecule that is partially double-stranded due to intramolecular structures may be considered a “partially double-stranded” or a “partially singlestranded” molecule.

[0148] Modified Ribonucleic Acid. RNA molecules of the present disclosure (e.g., mRNA molecules), and nucleic acid (e.g., DNA) molecules encoding the RNA molecules, may include a chemical modification (are chemically modified). The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties (5' cap). With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.

[0149] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e g., a modified mRNA polynucleotide), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).

[0150] Modifications of polynucleotides (e.g. RNA polynucleotides, such as mRNA polynucleotides) include, but are not limited to the following: 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6- methyladenosine; N6-threonylcarbamoyladenosine; l,2'-O-dimethyladenosine; 1- methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO12'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis- hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6- hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2- methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1 -methyladenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2- (alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2- (halo)adenine; 2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2'-Deoxy- 2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7- deaza-8-aza-adenosine; 7-methyladenine; 1 -Deazaadenosine TP; 2'Fluoro-N6-Bz- deoxyadenosine TP; 2'-OMe-2-Amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a- Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2'-a- Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b-Ethynyladenosine TP; 2- Bromoadenosine TP; 2'-b-Trifluoromethyl adenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'- difluoroadenosine TP; 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2'-a- thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b- fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine TP; 2'- Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2- Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3 -Deaza-3 -bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3 -Deaza-3 -fluoroadenosine TP; 3 -Deaza-3 -iodoadenosine TP; 3 -Deazaadenosine TP; 4 '-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5 '-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluorom ethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6- diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6- diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3 -methylcytidine;PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO15-formylcytidine; 5 -hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O- methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5 -formyl-2'-O-m ethylcytidine; Lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4- Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolecytidine; a-thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxy-CTP; 2'- Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-O- dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5- (propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1- methyl-l-deaza-pseudoisocytidine; 1 -methyl -pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2- methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-l-methyl-pseudoisocytidine; 4-methoxy- pseudoisocytidine; 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine; 4-thio- 1 -methyl - pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo- pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4- Acetyl-cytidine TP; 2'0-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a- Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy- 2',2'-difluorocytidine TP; 2'-Deoxy-2'-a-mercaptocytidine TP; 2'-Deoxy-2'-a- thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'- Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2’-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP; T-Deoxy-T-b- thiomethoxycytidine TP; 2'-O-Methyl-5-(l-propynyl)cytidine TP; 3 '-Ethynyl cytidine TP; 4'- Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(l-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5- Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5'- Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methyl guanosine; N2,2'-O- dimethylguanosine; N2-methylguanosine; Wyosine; l,2'-O-dimethyl guanosine; 1- methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7- cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O- trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O- trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; Nl-methyl- guanosine; a-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP; 2'- Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8- (alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8- (thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N- (methyl)guanine; l-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza- guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7- methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me- GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a- Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP; 2'-b-Ethynylguanosine TP; 2'-b- Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a- mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP; 2'-Deoxy-2'-b- chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'- Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5 '-Homo-guanosine TP; 8-bromo- guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1 -methylinosine; Inosine; 1,2'- O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3 -methyluridine; 5- carboxymethyluridine; 5 -hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5- taurinom ethyluridine; Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1- methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl- pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O- methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2’-O-dimethyluridine; 3-Methyl-pseudo-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5- carbamoylmethyl-2'-O-methyluridine; 5-carbamoylmethyluridine; 5- carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5- carboxymethylaminomethyl-2’-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5- carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl- 2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5- methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5 -Methyldihydrouridine; 5- Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; Nl-methyl-pseudo- uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3- carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thioutidine TP; 5-(iso- Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5- propynyl uracil; a-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonyiethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)- pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; l-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil; 1- Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; l-Methyl-3-(3-amino-3- carboxypropyl)pseudo-UTP; 1 -Methyl -pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2' methyl, 2'amino, 2'azido, 2'fluro- guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy-UTP; 2'-Azido-deoxyuridine TP; 2'-O- methylpseudouridine; 2' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP; 2'- Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil, 4-thiouracil; 5 (l,3-diazole-l-alkyl)uracil;5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2- aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5- (guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(l,3-diazole-l-alkyl)uracil; 5-(methoxy)uracil; 5- (methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2- (thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5- (methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)- 2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5- (trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; ally amino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-l-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1- carboxymethyl-pseudouridine; 1 -methyl- 1 -deaza-pseudouridine; 1 -propynyl-uridine; taurinom ethyl- 1 -methyl -uridine; l-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-l -methyl- 1 -deaza-pseudouridine; 2 -thio- 1 -m ethylpseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio- pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-l -methylpseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (±)1(2- Hydroxypropyl)pseudouridine TP; (2R)-l-(2-Hydroxypropyl)pseudouridine TP; (2S)-l-(2- Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo- vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; 1- (2,2,2-Trifluoroethyl)-pseudo-UTP; l-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; l-(2,2- Diethoxyethyl)pseudouridine TP; l-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1 -(2,4,6- Trimethyl-benzyl)pseudo-UTP; l-(2,4,6-Trimethyl-phenyl)pseudo-UTP; l-(2-Amino-2- carboxyethyl)pseudo-UTP; l-(2-Amino-ethyl)pseudo-UTP; 1 -(2-Hydroxyethyl)pseudouridine TP; 1 -(2 -Methoxy ethyl)pseudouri dine TP; l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; l-(3,4-Dimethoxybenzyl)pseudouridine TP; l-(3-Amino-3-carboxypropyl)pseudo-UTP; l-(3- Amino-propyl)pseudo-UTP; l-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; l-(4-Amino-4-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 carboxybutyl)pseudo-UTP; l-(4-Amino-benzyl)pseudo-UTP; l-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP; l-(4-Azidobenzyl)pseudouridine TP; l-(4- Bromobenzyl)pseudouridine TP; l-(4-Chlorobenzyl)pseudouridine TP; l-(4- Fluorobenzyl)pseudouridine TP; l-(4-Iodobenzyl)pseudouridine TP; l-(4- Methanesulfonylbenzyl)pseudouridine TP; l-(4-Methoxybenzyl)pseudouridine TP; l-(4-Methoxy-benzyl)pseudo-UTP; 1 -(4-Methoxy-phenyl)pseudo-UTP; 1 -(4-Methylbenzyl)pseudouridine TP; l-(4-Methyl-benzyl)pseudo-UTP; l-(4-Nitrobenzyl)pseudouridine TP; l-(4-Nitro-benzyl)pseudo-UTP; l(4-Nitro-phenyl)pseudo-UTP; l-(4-Thiomethoxybenzyl)pseudouridine TP; l-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1- (4-Trifluoromethylbenzyl)pseudouridine TP; l-(5-Amino-pentyl)pseudo-UTP; l-(6-Amino- hexyl)pseudo-UTP; 1 ,6-Dimethyl-pseudo-UTP; l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]- ethoxy}-ethoxy)-propionyl]pseudouridine TP; l-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-Acetylpseudouridine TP; l-Alkyl-6-(l-propynyl)-pseudo-UTP; l-Alkyl-6- (2-propynyl)-pseudo-UTP; l-Alkyl-6-allyl-pseudo-UTP; 1 -Alkyl -6-ethynyl-pseudo-UTP; 1- Alkyl-6-homoallyl-pseudo-UTP; l-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1- Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1 -Benzyloxymethylpseudouridine TT; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2 -pseudouridine TP; 1-Biotinylpseudouridine TP; 1- Butyl-pseudo-UTP; 1 -Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1- Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1- Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1-Cyclooetylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1- Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1 -Ethyl -pseudo-UTP; 1-Hexyl- pseudo-UTP; 1 -Homoallylpseudouridine TP; 1 -Hydroxymethylpseudouridine TP; 1 -iso-propyl - pseudo-UTP; l-Me-2-thio-pseudo-UTP; l-Me-4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo- UTP; 1 -Methanesulfonylmethylpseudouridine TP; 1 -Methoxymethylpseudouridine TP; 1 -Methyl - 6-(2,2,2-Trifluoroethyl)pseudo-UTP; l-Methyl-6-(4-morpholino)-pseudo-UTP; l-Methyl-6-(4- thiomorpholino)-pseudo-UTP; l-Methyl-6-(substituted phenyl)pseudo-UTP; l-Methyl-6-amino- pseudo-UTP; l-Methyl-6-azido-pseudo-UTP; l-Methyl-6-bromo-pseudo-UTP; l-Methyl-6- butyl-pseudo-UTP; l-Methyl-6-chloro-pseudo-UTP; l-Methyl-6-cyano-pseudo-UTP; 1 -Methyl - 6-dimethylamino-pseudo-UTP; l-Methyl-6-ethoxy-pseudo-UTP; l-Methyl-6-ethylcarboxylate- pseudo-UTP; l-Methyl-6-ethyl-pseudo-UTP; l-Methyl-6-fluoro-pseudo-UTP; l-Methyl-6-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 formyl-pseudo-UTP; 1 -Methyl -6-hydroxyamino-pseudo-UTP; 1 -Methyl-6-hydroxy-pseudo- UTP; l-Methyl-6-iodo-pseudo-UTP; l-Methyl-6-iso-propyl-pseudo-UTP; l-Methyl-6-methoxy- pseudo-UTP; l-Methyl-6-methylamino-pseudo-UTP; 1 -Methyl -6-phenyl-pseudo-UTP; 1- Methyl-6-propyl-pseudo-UTP; 1 -Methyl-6-tert-butyl-pseudo-UTP; 1 -Methyl-6- trifluorom ethoxy -pseudo-UTP; 1 -Methyl-6-trifluoromethyl-pseudo-UTP; 1 -Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1 -Phenyl -pseudo-UTP; 1- Pivaloylpseudouridine TP; 1 -Propargylpseudouridine TP; 1-Propyl-pseudo-UTP; 1-propynyl- pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1-Thiom ethoxymethylpseudouridine TP; 1 -Thiomorpholinomethylpseudouridine TP; 1- Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2 -deoxy-UTP; 2'-OMe-5- Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b- Ethynyluridine TP; 2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy- 2'-a-mercaptoutidine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O- Methyl-5-(l-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(l-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5- Cyanouridine TP; 5-Dimethylaminouridine TP; 5 '-Homo-uridine TP; 5 -iodo-2 '-fluorodeoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5- Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6-(4- Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)-pseudo- UTP; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo- UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy- pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro-pseudo-UTP; 6- Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo- UTP; 6-iso-Propyl -pseudo-UTP; 6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6- Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6- tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy -pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine l-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1(4-methylbenzoic acid) TP; Pseudouridine TP l-[3-(2-ethoxy)]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2- [2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2- [2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1- methylphosphonic acid diethyl ester; Pseudo-UTP-Nl-3-propionic acid; Pseudo-UTP-Nl-4- butanoic acid; Pseudo-UTP-Nl-5-pentanoic acid; Pseudo-UTP-Nl-6-hexanoic acid; Pseudo- UTP-Nl-7-heptanoic acid; Pseudo-UTP-Nl-methyl-p-benzoic acid; Pseudo-UTP-Nl-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxy wybutosine; 4-demethylwyosine; 2,6-(diamino)purine; l-(aza)-2-(thio)-3-(aza)- phenoxazin-l-yl; l,3-(diaza)-2-(oxo)-phenthiazin-l-yl; l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; l,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido, 2'fluro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'- azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified bases; 2'-O-methyl-ribose; 2- oxo-2-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3- (methyl)-7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6- (methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrityl; 5-nitroindole; 6- (aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl; 7- (aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)-l,3- (diaza)-2-(oxo)-phenoxazin- 1 -yl ; 7-(aminoalkythy droxy)- 1 , 3 -(diaza)-2-(oxo)-phenthiazin- 1 -yl ; 7-(aminoalkylhy droxy)- 1 , 3 -(diaza)-2-(oxo)-phenoxazin- 1 -yl ; 7 -(aza)indolyl; 7 -(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(guanidiniumalkyl-hydroxy)- 1 ,3-(diaza)-2-(oxo)-phenthiazin- 1 -yl; 7-(guanidiniumalkylhy droxy)- 1 ,3-(diaza)-2-(oxo)- phenoxazin-l-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7- (aza)indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2- amino-purine; N6- substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purities; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho- sub stituted- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrol opyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2- amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'- OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(19-Amino- pentaoxanonadecyl)adenosine TP.

[0151] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0152] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (xp), N1 -methylpseudouridine (mly), N1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2-thio-l -methylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyl uridine. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0153] When the purified ssRNA of the present disclosure is a purified mRNA, it may comprise any additional modifications known to one of skill in the art and as described in US Patent Publications US20120046346 and US20120251618, and PCT Publication WO 2012 / 019168. Other such components include, for example, a 5' cap, a polyA tail, a Kozak sequence; a 3' untranslated region (3' UTR); a 5' untranslated region (5' UTR); one or more intronic nucleotide sequences capable of being excised from the nucleic acid, or any combination thereof.

[0154] In some embodiments, the purified ssRNAs of the present disclosure include a natural 5’ cap, or any conventionally known and / or commercially available 5’ cap. In some embodiments, the purified mRNAs of the present disclosure include a natural 5' cap. In some embodiments, a 5' cap may be a 5' cap analog, such as, e.g., a 5' diguanosine cap, tetraphosphate cap analogs having a methylene-bis (phosphonate) moiety, cap analogs having a sulfur substitution for a non-bridging oxygen, N7-benzylated dinucleoside tetraphosphate analogs, or anti-reverse cap analogs. In some embodiments, the 5' cap is 7mG(5')ppp(5')NlmpNp. In some embodiments, the 5 'cap analog is a 5'diguanosine cap. In some embodiments, the synthetic, modified mRNA of the present disclosure does not comprise a 5' triphosphate.

[0155] Both the 5 'UTR and the 3 'UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5 '-cap and the 3 '-poly (A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. The 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3'- end of the transcribed mRNA. In some embodiments the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the individual mRNA. A poly-A tail (SEQ ID NO: 60) may be greater than 30 nucleotides in length, greater than 35 nucleotides in length, at least 40 nucleotides, at least 45 nucleotides, at least 55 nucleotides, at least 60 nucleotide, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, or more.

[0156] In some embodiments, the purified mRNA of the present disclosure encodes a protein. In some embodiments, the protein is an antigen, e.g., a viral antigen. In some embodiments, the modified mRNA purified using the methods described herein may be used as a mRNA vaccine.

[0157] MethodsPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0158] Method of purifying a nucleic acid (e.g., mRNA) preparation of the present disclosure generally include contacting the nucleic acid preparation with a dsRBP that is immobilized on a solid support.

[0159] An affinity purification method may, in some embodiments, include: (1) incubating a nucleic acid preparation (e.g., a solution containing in vitro-transcribed mRNA) with a solid support to which a dsRBP is immobilized under conditions that result in binding of doublestranded RNA (dsRNA) to the immobilized dsRBP; (2) eluting unbound preparation components from the support using appropriate buffers that maintain the binding interaction between the dsRBP and the dsRNA to produce a preparation enriched for single-stranded RNA (ssRNA) (a “ssRNA-enriched preparation”); and (3) optionally performing at least one additional purification process to isolate a ssRNA (e.g., mRNA) from the preparation.

[0160] An affinity purification method may, in some embodiments, include: (1) incubating a nucleic acid preparation (e.g., a solution containing in vitro-transcribed mRNA) with a dsRBP or variant thereof under conditions that result in binding of double-stranded RNA (dsRNA) to the dsRBP; (2) immobilizing the dsRBP to a solid support; (3) eluting unbound preparation components from the support using appropriate buffers that maintain the binding interaction between the dsRBP and the dsRNA to produce a preparation enriched for single-stranded RNA (ssRNA) (a “ssRNA-enriched preparation”); and (3) optionally performing at least one additional purification process to isolate a ssRNA (e.g., mRNA) from the preparation.

[0161] Methods may be performed under conditions that result in binding of the dsRBP to doublestranded RNA. These conditions are readily determined by a skilled artisan and include, for example, temperature conditions, buffer (e.g., salt and pH) conditions, and reaction / process time.

[0162] Incubation (contact) times may vary. In some embodiments, a nucleic acid preparation (e.g., an IVT preparation) is contacted with dsRBP optionally immobilized on a solid support for 1 minutes to 3 hours, or longer. For example, a nucleic acid preparation may be contacted with dsRBP optionally immobilized on a solid support for 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or longer.

[0163] In some embodiments, affinity purification methods further comprise separating solid phase dsRBP-dsRNA complexes from the liquid phase ssRNA-enriched preparation. This separation step(s) depends on the type of solid support used in an affinity purification method. For example, for dsRBP immobilized on a resin, the ssRNA-enriched preparation may be separated byPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 centrifugation. For dsRBP immobilized on magnetic beads, a magnet may be used to remove the beads from the ssRNA-enriched preparation. Separation of the solid phase from the liquid phase yields ssRNA that is substantially free of dsRNA contaminants. Following or preceding performance of an affinity purification method using a dsRBP, the nucleic acid preparation may be subjected to one or more additional purification methods to remove DNA and / or protein contaminants. Methods of removing DNA or protein contaminants from a preparation containing RNA are known. The order in which the different purification methods are performed may be varied.

[0164] Compositions

[0165] The present disclosure also encompasses compositions comprising ssRNA (e.g., mRNA) prepared, for example, via IVT and purified according to the affinity purification methods as provided herein. In some embodiments, the compositions are therapeutic compositions. For example, the RNA (e.g., mRNA) purified using the methods of the present disclosure may be used as a gene editing reagent used in the generation of a cell or gene therapy product or to produce a vaccine composition to treat or prevent a disease or disorder.

[0166] In some embodiments, a composition includes a RNA (e.g., mRNA) purified by a method of the present disclosure having an open reading frame comprising at least one chemical modification or optionally no nucleotide modification, a 5' terminal cap that is 7mG(5')ppp(5')NlmpNp, and a polyA tail. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1 -methyl pseudouridine. In some embodiments, 100% of the uracil in the open reading frame have a Nl- methyl pseudouridine in the 5-position of the uracil.

[0167] In some embodiments, the RNA (e.g., mRNA) purified using the methods of the present disclosure may be formulated in a nanoparticle, such as a lipid particle described, for example, in any one of International Application No. PCT / US 16 / 58327, International Application No. PCT / US16 / 583140, and International Application No. PCT / US 16 / 58324, each of which was filed Oct. 21, 2016 and is herein incorporated by reference. In some embodiments, the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the lipid nanoparticle includes a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle includes a molar ratio ofPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid. In some embodiments, a lipid nanoparticle includes a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, a cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z) — N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine (L608), and N,N-dimethyl-l- [(1 S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

[0168] Although the present disclosure has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description and the preferred versions contained within this specification. Various aspects of the present disclosure will be illustrated with reference to the following non-limiting examples.EXAMPLES

[0169] Example 1. Affinity resin for the capture of double-stranded RNA (dsRNA) contaminants in nucleic acid preparation

[0170] This example depicts use of a Flock house virus (FHV) B2 dsRBP. It is a small protein (~10 kDa) that tightly binds dsRNA (KD~1 nM) in a sequence-independent manner and does not specifically bind single-stranded RNA. The B2 dsRBP contains two helices that in coordination with another B2 dsRBP recognize dsRNA as a dimer.

[0171] Three different variants of the protein were expressed, purified, and conjugated onto a bead-based resin: 1) monomer comprised of a single B2 unit, 2) B2 dimer comprised of two monomers linked by a glycine linker, and 3) B2 splice variant in which a B2 monomer was added between helices of one B2 unit (amino acid sequences shown below). The proteins were conjugated to a nickel agarose resin through addition of a terminal 6xHis tag (SEQ ID NO: 61).

[0172] B2 monomer (N-terminal 6xHis tag (SEQ ID NO: 61) not shown)

[0173] PSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSR MVTSLLEKPSVVAYLEGKA (SEQ ID NO: 25)

[0174] B2 dimer (N-terminal 6xHis tag (SEQ ID NO: 61) not shown)PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0175] PSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSR MVTSLLEKPSVVAYLEGKAGGGGGGGGGGGGGGGGGGGGPSKLALIQELPDRIQTAVE AAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 27)

[0176] B2 splice variant (N-terminal 6xHis tag (SEQ ID NO: 61) not shown)

[0177] PSKLALIQELPDRIQTAVEAAMGGGSGPSKLALIQELPDRIQTAVEAAMGMSYQD APNNVRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKAGGSGMSYQDAPNN VRRDLDNLHACLNKAKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 28)

[0178] All three resin variants were tested for dsRNA binding using a ssRNA ladder, dsRNA ladder, or a 4:1 mixture of the ssRNA and dsRNA ladders. Solutions of the ladders were added to the resins, mixed well, and incubated at room temperature for 10 minutes. The supernatant was separated from the resin and loaded onto an agarose gel to detect the RNA species. The gel showed that all three resin variants specifically recognize dsRNA over ssRNA. It also showed that all three resins can be used to remove dsRNA from ssRNA.

[0179] To show that the dsRNA of the disclosure can be used to remove dsRNA from IVT mRNA and provide better-performing mRNA for downstream applications, the splice variant conjugated to resin (referred to as ‘TF -Resin’) was used for further studies. A four-step workflow that used the TF-resin in a spin column was performed to prepare IVT mRNA depleted of dsRNA as shown in FIG. 1.

[0180] The flow through for the dsRNA removal step (#3) contained the mRNA depleted of dsRNA. An 8M urea elution was performed to get additional mRNA off the resin. This elution was combined with the flow through. This workflow was performed for three different IVT mRNAs: 1) red fluorescent protein (RFP) labeled mRNA which is 1 kb long, 2) firefly luciferase (fLuc) labeled mRNA which is 2 kb long, and 3) a 5 kb long mRNA. The amount of dsRNA removed from each mRNA was measured by dot blot using an antibody that specifically recognizes dsRNA and compared to two common methods to remove dsRNA: cellulose or HPLC (FIGS. 2 and 3).

[0181] The input was the mRNA sample from the Clean-up Step (#2) in the workflow described above. The data above shows that the TF-Resin removes >70% of the input dsRNA (red line) and gives a prep that is cleaner than the cellulose method. The TF-resin gives a purity that is veryPCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 similar to HPLC for the RFP and fLuc mRNAs. The HPLC prep of the 5 kb mRNA is cleaner than the TF-Resin; however, as shown in the graph of FIG. 4, its recovery is very low.

[0182] The mRNA recovery with the TF-Resin is >70% of the input mRNA which is on par with cellulose and significantly better than HPLC. The data shows that TF-Resin can give higher yields and cleaner mRNA preps compared to two commonly used methods: cellulose and HPLC.

[0183] Next, the RFP mRNA was tested for function in vitro by transfecting the mRNA into the JAWSII cell line which was found to be very sensitive to mRNA purity (mlT RFP mRNA cell culture transfection with Lipfectamine™ MessengerMax™ Transfection Reagent (Thermo Fisher Scientific, Catalog No. LMRNA001)). Results of the functional testing are shown in FIG. 5.

[0184] Example 2. ssRNA affinity purification with B2 dsRBP splice variant

[0185] The B2 dsRBP splice variant of SEQ ID NO: 28 was recombinantly modified to include a streptavidin binding protein (SBP) tag moiety (‘B2 dsRBP splice variant / SBP’). The amino acid sequence of the generated B2 dsRBP splice variant / SBP is shown below. Inclusion of the SBP allows for binding to a solid support having streptavidin conjugated to the solid support to separate ssRNA and dsRNA molecules in a mixture within an affinity purification workflow.

[0186] B2 dsRBP splice variant / SBP

[0187] MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPGGGGPSKLALIQELP DRIQTAVEAAMGGGSGPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHA CLNKAKLTVSRMVTSLLEKPSVVAYLEGKAGGSGMSYQDAPNNVRRDLDNLHACLNK AKLTVSRMVTSLLEKPSVVAYLEGKA (SEQ ID NO: 29)

[0188] The B2 dsRBP splice variant / SBP (‘SBP-B2 Spliced’ as referred to in FIG. 6) was compared to five different ligands: 1) J2 anti-dsRNA antibody (‘J2’ as referred to in FIG. 6; Thermo Fisher Scientific, Catalog No. 10010200-200UG); 2) B2 dsRBP monomer described in Example 1 with a 6xHis (SEQ ID NO: 61) tag (SEQ ID NO: 31; ‘6xHis(SEQ ID NO: 61)-B2 Monomer’ as referred to in FIG. 6); 3) AVIPure® dsRNA Clear OPUS resin (‘Repligen’ as referred to in FIG. 6); 4) custom generated anti-dsRNA camelid antibody labeled with biotin (‘Biotin-BPG-4’ as referred to in FIG. 6); and 5) CaptureSelect™ Biotin Anti-AAVX Conjugate (‘AAVX’ as referred to in FIG. 6; Thermo Fisher Scientific, Catalog No. 7103522100) as a negative control. The input for all ligands / resins was a solution mixture composed of ssRNA RiboRuler™ High Range RNA Ladder (Thermo Fisher Scientific, Catalog No. SM1821) at 20PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 ng / uL, dsRNA Ladder (New England Biolabs, Catalog No. N0363S) at 3 ng / uL, and custom generated 2 kb dsRNA modified with mlT at 0.5 ng / uL.

[0189] An overview of the affinity purification experiment is shown in FIG. 6. A decreasing titration of each ligand / resin was added to mixture and incubated at room temperature for 10 minutes to allow the ligand / resin to bind the dsRNA. Next, reagent was added to capture the ligand and remove the ligand via magnet or spin column (Repligen). Dynabeads™ anti-mouse IgG (Thermo Fisher Scientific) were used to capture and remove the J2 antibody. Agarose nickel resin was used to capture and remove the 6xHis (SEQ ID NO: 61) tagged B2 monomer. Streptavidin conjugated magnetic beads (Pierce™ Streptavidin Magnetic Beads; Thermo Fisher Scientific, Catalog No. 88816) were used to capture the B2 dsRBP splice variant / SBP, the AAVX ligand, or the camelid. The Repligen slurry was separated from the mixture using a spin column containing a cellulose acetate filter. The separated solutions were purified using the GeneJET RNA™ Cleanup and Concentration Micro Kit (Thermo Fisher Scientific, Catalog No. K0842) and run in a 2% agarose gel.

[0190] FIG. 8 shows the results of the affinity purification experiment. The dsRNA fragments can be seen in the input by comparing to the lanes with ssRNA only or dsRNA only. All the dsRNA fragments were observed in the AAVX negative control, indicating that any dsRNA removal was specific to the ligand used. Samples treated with the J2 antibody showed dsRNA depletion. However, trace amounts of dsRNA were easily observed for all samples. The same observation was made for the camelid, especially for the smaller fragments. The Repligen resin appeared to bind nonspecifically to ssRNA as suggested by depletion of ssRNA when using higher amounts of the resin (lanes 1 & 2). At lower amounts of the resin (lanes 3 & 4), there is depletion of dsRNA. However, trace amounts of dsRNA was easily observed. Both B2 dsRBP variants (i.e., splice and monomer) displayed effective dsRNA removal as indicated by lanes 2 of the respective gels. High amounts of either B2 dsRBP variant may bind nonspecifically to ssRNA as suggested by lanes 1 of the respective gels. However, it was clear that they selectively bind dsRNA over ssRNA. The data showed that less B2 dsRBP splice variant / SBP was needed than the B2 dsRBP monomer to achieve effective dsRNA removal, suggesting that the splice variant performs better than the monomer variant.

[0191] Without being bound by any theory, the reduced amount of the splice variant required as compared to the monomer variant may be due to the binding kinetics of the interaction. ThePCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1 monomer requires two steps: 1) search and bind another monomer to dimerize, and then 2) search and bind the dsRNA substrate. The splice variant is a simple one step interaction: search and bind the dsRNA substrate. In all, the data suggested that both B2 dsRBP variants (i.e., splice and monomer) performed better in removing dsRNA from the mixture as compared to the other reagents, with the splice variant performing best. Furthermore, both B2 dsRBP variants were able to efficiently remove the modified dsRNA fragment, showing that RNA modification did not significantly affect the interaction with the B2 dsRBP variants. This was in contrast to the Repligen resin which clearly showed difficulties removing the modified dsRNA.

[0192] Example 3. Affinity purification of mRNA using B2 dsRBP splice variant / SBP

[0193] To show that B2 dsRBP splice variant / SBP can be used to remove dsRNA from IVT mRNA and provide beter-performing mRNA for downstream applications, B2 dsRBP splice variant / SBP was used to remove dsRNA from IVT reactions. Four samples of mlT-modified GFP mRNA were prepared using different methods including: 1) ‘Crude’ which was purified by LiCl precipitation and contains dsRNA contaminant; 2) ‘Cellulose’ which was purified using the cellulose protocol to remove dsRNA as described in Baiersdorfer et al. (Mol Ther Nucleic Acids. 2019; 15:26-35); 3) ‘HPLC’ which was purified using ion-paired reverse phase HPLC as described in Weissman et al. (Methods Mol Biol. 2013; 969:43-54.); and 4) ‘Splice’ which was treated with the B2 dsRBP splice variant / SBP to remove dsRNA.

[0194] FIG. 9 shows the recovery of mRNA for each sample. The recovery when using the B2 splice variant was -80% which is in between the Cellulose and HPLC samples. The dsRNA level of each sample was measured by ELISA (FIG. 9), and the data clearly showed that the spliced variant removes dsRNA much better than cellulose and is on par with HPLC which is the gold standard for dsRNA removal although quite costly and time consuming.

[0195] These mRNA samples were then transfected into the murine JAWS II dendritic cell line to assess the mRNA performance as these cells are very sensitive to mRNA purity. FIG. 10 shows that the mRNA purified using the spliced variant performed like the HPLC mRNA as shown by GFP transfection efficiency (% cells with GFP signal), GFP expression (GFP mean fluorescent imaging (GFP MFI)), and the inflammation response (pg / mL TNFa level). In all, this experiment demonstrated that the B2 dsRBP splice variant / SBP performed better than cellulose in terms of dsRNA removal and is comparable to HPLC but with better recovery.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1

[0196] After reading this description it will become apparent to one skilled in the art how to implement the disclosed technology in various alternative instances and alternative applications. However, all the various instances of the present technology will not be described herein. It will be understood that the instances presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative instances should not be construed to limit the scope or breadth of the present technology as set forth below.

[0197] The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.

Claims

PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO1WHAT IS CLAIMED IS:

1. A method of purifying a nucleic acid preparation, comprising: contacting a nucleic acid preparation comprising messenger ribonucleic acid (mRNA) and double-stranded ribonucleic acid (dsRNA) with a double-stranded ribonucleic acid binding protein (dsRBP) that specifically binds the dsRNA, wherein the dsRBP is a B2 dsRBP; and isolating a purified nucleic acid preparation comprising the mRNA.

2. The method of claim 1, wherein the purified nucleic acid preparation is substantially free of dsRNA.

3. The method of claim 1, wherein the dsRBP is immobilized on a solid support.

4. The method of claim 3, wherein the dsRBP is immobilized on a solid support before binding the dsRNA or the dsRBP is immobilized on a solid support after binding the dsRNA.

5. The method of claim 1, wherein the mRNA is an in vitro-transcribed mRNA.

6. The method of claim 1, wherein the B2 dsRBP is a Nodaviridae family B2 dsRBP.

7. The method of claim 1, wherein the B2 dsRBP is an Alphanodavirus genus B2 dsRBP.

8. The method of claim 1, wherein the B2 dsRBP is of a species selected from Black beetle virus, Boolarra virus, Flock house virus, Nodamura virus, and Pariacoto virus.

9. The method of claim 1, wherein the B2 dsRBP comprises an amino acid sequence as set forth in SEQ ID NOs: 1-59.

10. The method of claim 1, wherein the B2 dsRBP comprises a tag binding moiety.

11. The method of claim 10, wherein the tag binding moiety is a streptavidin binding protein, imidazole binding protein, biotin binding protein or a histidine tag.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO112. The method of claim 11, wherein the B2 dsRBP is immobilized on a solid support via binding of the tag binding moiety to the solid support comprising a conjugate binding tag.

13. The method of claim 11, wherein the tag binding moiety is a histidine tag and the solid support is a resin comprising nickel.

14. The method of claim 11, wherein the tag binding moiety is a streptavidin binding protein and the solid support is a head comprising streptavidin.

15. The method of claim 14, wherein the bead is magnetic.

16. The method of claim 15, further comprising applying a magnetic field to sequester dsRNA specifically bound to dsRNA in the nucleic acid preparation.

17. The method of claim 3, wherein the solid support is a chromatography resin.

18. A method, comprising: performing an in vitro transcription reaction in the presence of a template nucleic acid to produce an in vitro transcription product; and contacting the in vitro transcription product with a double-stranded ribonucleic acid binding protein (dsRBP) that specifically binds the dsRNA, wherein the dsRBP is a B2 dsRBP that is optionally immobilized on a solid support.

19. The method of claim 18, wherein the in vitro transcription product comprises messenger RNA (mRNA).

20. The method of claim 18, wherein the in vitro transcription product further comprises doublestranded RNA (dsRNA).

21. The method of claim 18, wherein the dsRBP is immobilized on a solid support before contacting the dsRBP with the in vitro transcription product.

22. The method of claim 18, wherein the dsRBP is immobilized on a solid support after contacting the dsRBP with the in vitro transcription product.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO123. The method of claim 18, wherein the B2 dsRBP is aNodaviridae family B2 dsRBP.

24. The method of claim 18, wherein the B2 dsRBP is an Alphanodavirus genus B2 dsRBP.

25. The method of claim 18, wherein the B2 dsRBP is of a species selected from Black beetle virus, Boolarra virus, Flock house virus, Nodamura virus, and Pariacoto virus.

26. The method of claim 18, wherein the B2 dsRBP comprises an amino acid sequence as set forth in SEQ ID NOS: 1-59.

27. The method of claim 26, wherein the B2 dsRBP comprises a tag biding moiety.

28. The method of claim 27, wherein the tag binding moiety is is a streptavidin binding protein, imidazole binding protein, biotin binding protein or a histidine tag.

29. The method of claim 18, wherein the solid support comprises a resin or bead.

30. The method of claim 29, wherein the solid support is a resin.

31. The method of claim 30, wherein the resin comprises nickel.

32. The method of claim 30, wherein the resin is a chromatography resin.

33. The method of claim 29, wherein the solid support is a bead.

34. The method of claim 33, wherein the bead is magnetic.

35. The method of claim 33, wherein the bead comprises biotin, imidazole or streptavidin.

36. A method of purifying a nucleic acid preparation, comprising: contacting a nucleic acid preparation comprising single-stranded ribonucleic acid (ssRNA) and double-stranded ribonucleic acid (dsRNA) with a double- stranded ribonucleic acid binding protein (dsRBP) that specifically binds the dsRNA; and isolating a purified nucleic acid preparation comprising the ssRNA.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO137. The method of claim 36, wherein the ssRNA is a messenger ribonucleic acid (mRNA), a micro ribonucleic acid (microRNA), a circular ribonucleic acid, or a self-amplifying ribonucleic acid.

38. The method of claim 37, wherein the self-amplifying ribonucleic acid is a replicon.

39. A method, comprising: performing an in vitro transcription reaction in the presence of a template nucleic acid to produce an in vitro transcription product; and contacting the in vitro transcription product with a double-stranded ribonucleic acid binding protein (dsRBP) that specifically binds the dsRNA.

40. The method of claim 39, wherein the in vitro transcription product is a single-stranded ribonucleic acid (ssRNA).

41. The method of claim 40, wherein the ssRNA is a messenger ribonucleic acid (mRNA), a micro ribonucleic acid (microRNA), a circular ribonucleic acid, or a self-amplifying ribonucleic acid.

42. The method of claim 41, wherein the self-amplifying ribonucleic acid is a replicon.

43. A double-stranded ribonucleic acid binding protein (dsRBP) having an amino acid sequence including at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence set forth in SEQ ID NOs: 1-59, wherein the dsRBP specifically binds double-stranded ribonucleic acid (dsRNA).

44. The dsRBP of claim 43, further comprising a tag binding moiety.

45. The dsRBP of claim 43, wherein the dsRBP comprises 2 amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence set forth in SEQ ID NOs: 35-57.

46. The dsRBP of claim 45, wherein the 2 amino acid sequences are joined by a linker sequence.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO147. The dsRBP of claim 45, wherein the 2 amino acid sequences are identical.

48. The dsRBP of claim 45, wherein the 2 amino acid sequences form helices that dimerize and form a dsRNA binding region that specifically binds dsRNA.

49. The dsRBP of claim 45, wherein the 2 amino acid sequences are set forth in SEQ ID NOs: 35-37.

50. A composition comprising: a double- stranded ribonucleic acid binding protein (dsRBP) of any one of the preceding claims; and a solid support.

51. The composition of claim 40, wherein the dsRBP is immobilized to a solid support.

52. The composition of claim 41, wherein the dsRBP is a dsRBP-double-stranded ribonucleic acid (dsRNA) complex.

53. The composition of claim 41, wherein the solid support is a resin or bead.

54. The composition of claim 53, wherein the solid support is a resin comprising nickel.

55. The composition of claim 53, wherein the solid support is a bead.

56. The composition of claim 55, wherein the bead is magnetic.

57. The composition of claim 56, wherein the bead comprises a binding tag.

58. The composition of claim 57, wherein the binding tag is streptavidin, biotin or imidazole.

59. A composition comprising: a single-stranded ribonucleic acid (ssRNA) purified by a method of any one of the preceding claims; and a gene editing reagent or a pharmaceutical excipient.PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO160. The composition of claim 59, wherein the ssRNA is a messenger ribonucleic acid (mRNA), a micro ribonucleic acid (microRNA), a circular ribonucleic acid, or a self-amplifying ribonucleic acid.

61. The composition of claim 60, wherein the self-amplifying ribonucleic acid is a replicon.

62. A kit comprising: a double- stranded ribonucleic acid binding protein (dsRBP) of any one of the preceding claims; and a solid support.

63. The kit of claim 62, wherein the solid support is a resin or bead.

64. The kit of claim 63, wherein the solid support is a resin.

65. The kit of claim 64, wherein the solid support comprises nickel.

66. The kit of claim 63, wherein the solid support is a bead.

67. The kit of claim 66, wherein the bead comprises biotin, imidazole or streptavidin.

68. A method of determining an amount of double-stranded ribonucleic acid (dsRNA) in a nucleic acid preparation comprising: contacting a nucleic acid preparation comprising dsRNA with a double-stranded ribonucleic acid binding protein (dsRBP) that specifically binds the dsRNA; isolating the dsRNA; and determining the amount of isolated dsRNA.

69. The method of claim 70, wherein the nucleic acid preparation further comprises singlestranded ribonucleic acid (ssRNA).PCT / US25 / 47503 23 September 2025 (23.09.2025)Aty Docket No. TP387899WO170. The method of claim 69, wherein the ssRNA is a messenger ribonucleic acid (mRNA), a micro ribonucleic acid (microRNA), a circular ribonucleic acid, or a self-amplifying ribonucleic acid.

71. The method of claim 70, wherein the self-amplifying ribonucleic acid is a replicon.

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