Peptide ligands for affinity capture of nucleic acids

Abstract

An affinity membrane separator is provided that includes a separation substrate with a plurality of peptide ligands positioned thereon. The peptide ligands preferentially bind to one of a nucleic acid product and a waste nucleic acid product, e.g., produced by a mRNA synthesis bioreactor. The peptide ligands include fewer than 20 residues and at least one defined secondary structure, and have a global charge greater than about 1 and at least one lysine, arginine, or glutamine residue in order to preferentially bind ds-RNA relative to ss-RNA, or a global charge less than about 0 and at least one serine or asparagine residue in order to preferentially bind ss-RNA relative to ds-RNA. Peptide ligands can advantageously bind to ds-RNA byproducts from the production of ss-RNA vaccines, e.g., against SARS-CoV-2, while allowing the ss-RNA vaccines themselves to pass through the separator, providing an efficient system for production of purified ss-RNA vaccine products.

Description

PEPTIDE LIGANDS FOR AFFINITY CAPTURE OF NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 562,942. filed March 8, 2024, and 63 / 768,381, filed March 7, 2025, which are incorporated by reference as if disclosed herein in their entireties.INCORPORATION OF SEQUENCE LISTING

[0002] The contents of the file named “Sequence final.xml”, which was created on March 7, 2025 and is 420 KB in size, are hereby incorporated by reference in their entireties.BACKGROUND

[0003] The landscape of RNA-based therapeutics and vaccines has experienced a transformative surge, particularly following the success of messenger RNA (mRNA) vaccines in combating the COVID-19 pandemic. This achievement has positioned RNA as a powerful modality for therapeutic interventions across various indications, including cancer, infectious diseases, and beyond. RNA has the potential to target myriad genes of interest and significantly expand the scope of druggable targets beyond the 0.05% of the human genome currently addressed by protein-targeted therapeutics. This capability, coupled with the absence of the genetic risks associated with DNA-based therapies, challenges the traditional notion of undruggable targets and opens additional frontiers in drug discover}’.

[0004] The in vitro transcription process is a cell-free synthesis approach to the production of RNA therapeutics, particularly mRNA. Here, a linear DNA template undergoes transcription into mRNA using a single-subunit phage RNA polymerase, with the T7 phage polymerase (RNAP) predominantly employed. The quality of the generated mRNA stands as a determinant for therapeutic and research applications. Among the challenges encountered in this process, the presence of impurities and / or wastes including residual nucleoside triphosphates (NTPs), enzymes, abortive transcripts, incorrect and capless mRNAs. DNA plasmid templates, and double-stranded RNA (dsRNA) has emerged as a concern.

[0005] Formation of even a small amount of dsRNA is of particular concern during mRNA synthesis as RNA with double stranded regions longer than 40 bp can trigger undesirable cellular responses, including the activation of innate immune pathways, and impede efficient mRNA translation, thereby diminishing therapeutic efficacy. Consequently, the removal of most dsRNAfrom the final mRNA product is desired to meet stringent safety and efficacy thresholds in therapeutic applications that often utilize larger doses than vaccines. Several mechanisms have been proposed for the origin of dsRNA during transcription, all involving T7 RNA polymerase (T7 RNAP). The dsRNA generated is not a single, uniform molecule but rather a diverse population of molecules with varying sizes and degrees of annealing. The physicochemical properties of these dsRNA species are remarkably similar to those of target mRNA (except their negative charge), complicating their separation using conventional methods.

[0006] While significant efforts have been explored to improve the manufacturing process and minimize dsRNA formation, downstream removal of dsRNA still remains a challenge. Current strategies for dsRNA removal include cellulose-based purification and chromatographic methods such as reverse-phase high-pressure liquid chromatography (HPLC). Despite their effectiveness, these methods often suffer from drawbacks such as high cost, low yield, low ligand accessibility, the use of toxic organic chemicals, the potential degradation of long mRNA sequences, and product loss. Given these challenges, there is a pressing need for the development of cost-effective, scalable, and efficient methods for dsRNA removal.

[0007] One promising approach, affinity purification, involves the use of affinity ligands, where they enable selective capture of target molecules from complex mixtures. Oligo-dT ligands have shown promise for mRNA purification. However, their affinity for polyA tails limits their ability to differentiate dsRNA impurities from target mRNA. Thus, selective ligands for dsRNA removal are desired. Traditional natural ligands, such as enzyme substrates and antigens, offer strong binding but are costly, hard to elute, and susceptible to leakage.

[0008] Therefore, there remains a need in the art for improved materials, methods and systems for purification of nucleic acids.SUMMARY

[0009] Aspects of the present disclosure are directed to affinity membrane separators for purifying a nucleic acid product. In some embodiments, the affinity' membrane separators include a separation substrate and a plurality' of peptide ligands positioned on the separation substrate. In some embodiments, the separation substrate includes a flat sheet microporous membrane, a cellulose hollow fiber microporous affinity membrane, a tangential flow hollow fiber membrane, or combinations thereof. In some embodiments, the peptide ligands preferentially bind to one of a first nucleic acid product and a second nucleic acid product. In some embodiments, the ss-RNA is a vaccine against SARS-CoV-2, flu, respiratory' syncytialvirus (RSV), human metapneumovirus (HMPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV), human immunodeficiency viruses (HIV), norovirus, Lyme disease, zika virus, Mpox, or combinations thereof.

[0010] Tn some embodiments, the peptide ligands include fewer than 20 residues and at least one defined secondary structure. In some embodiments, the first nucleic acid product includes ss-RNA and the second nucleic acid product includes ds-RNA. In some embodiments, the peptide ligands include an animo acid sequence selected from the group consisting of SEQIDNO.: 1 - SEQ ID NO.: 490.

[0011] In some embodiments, the peptide ligands preferentially bind to ds-RNA relative to ss-RNA, and include a global charge greater than about 1. and at least one lysine, arginine, or glutamine residue. In some embodiments, the peptide ligands include an animo acid sequence selected from the group consisting of: SEQIDNO.: 1, SEQIDNO.: 2, SEQIDNO.: 3, SEQ ID NO.: 4, SEQIDNO.: 5, SEQIDNO.: 6, SEQIDNO.: 7, SEQIDNO.: 8, SEQIDNO.: 9. SEQIDNO.: 10, SEQIDNO.: 11, SEQIDNO.: 12, SEQIDNO.: 13, SEQIDNO : 14, SEQIDNO.: 15, SEQIDNO.: 16. SEQIDNO.: 17, SEQIDNO.: 18. SEQIDNO.: 19, SEQ ID NO : 20, SEQIDNO.: 21, SEQ ID NO.: 22, SEQIDNO.: 23, SEQIDNO.: 24, SEQ ID NO: 25, SEQIDNO.: 26, SEQIDNO.: 27, SEQIDNO.: 28, SEQIDNO.: 29, SEQ IDNO.: 30, SEQIDNO.: 31, SEQIDNO.: 32, SEQIDNO.: 33, SEQIDNO.: 34. SEQ IDNO.: 35, SEQIDNO.: 36, SEQIDNO.: 37. SEQIDNO.: 38, SEQIDNO.: 39. SEQ ID NO.: 40, SEQIDNO.: 41, SEQ ID NO.: 42, SEQIDNO.: 43, SEQIDNO.: 44, SEQ IDNO.: 45, SEQIDNO.: 46, SEQIDNO.: 47, SEQIDNO.: 48, and SEQ ID NO.: 49.

[0012] A feature of peptides exhibiting selective binding to dsRNA consistent with some embodiments of the present disclosure is the abundance of positively charged amino acids. Both phage display and rational design libraries yielded peptides predominantly carrying a positive charge under the binding conditions. This observation is in line with the behavior of RNAs, whose narrow and deep major grooves generate strong electrostatic fields. Without wishing to be bound by theory, the geometric complementarity between the positively charged residues in the peptides and the negatively charged regions of RNA can contribute to the enhanced binding specificity and stability of the peptide-RNA complexes. Interestingly, this feature aligns with the binding mechanisms observed in natural dsRNA-binding proteins (dsRBPs). dsRBPs can stabilize their interactions by orienting basic residues towards the RNA's phosphate backbone, leveraging the stable double-helical structure of dsRNA, which is characterized by extensivehydrogen bonding between complementary base pairs. This structural integrity, less prominent in ssRNA, allows dsRBPs to selectively bind to dsRNA through a combination of electrostatic forces and precise hydrogen bond formation.

[0013] In some embodiments, the peptide ligands preferentially bind to ss-RNA relative to ds-RNA, and include a global charge less than about 0, and at least one serine or asparagine residue. In some embodiments, the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO.: 50. SEQ ID NO.: 51, SEQ ID NO.: 52. SEQ ID NO : 53, SEQ ID NO.: 54, SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NO.: 57, SEQ IDNO.: 58, SEQ ID NO.: 59, SEQ ID NO.: 60, SEQ ID NO.: 61, SEQ ID NO.: 62, SEQ IDNO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67. SEQ IDNO : 68, SEQ ID NO.: 69, SEQ ID NO.: 70. SEQ ID NO.: 71, SEQ ID NO.: 72. SEQ IDNO.: 73, SEQ ID NO.: 74, SEQ ID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 77, SEQ IDNO : 78, SEQ ID NO.: 79, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ IDNO.: 83, and SEQ ID NO.: 84.

[0014] Aspects of the present disclosure are directed to a method for purification of a nucleic acid product. In some embodiments, the method includes generating a concentration of a nucleic acid product and contacting a medium including the nucleic acid product and a waste product with an affinity membrane separator, binding one of the waste product or the nucleic acid product via peptide ligands to form a concentration of bound product, and eluting from the affinity membrane separator an effluent including a concentration of the bound product. In some embodiments, eluting a concentration of the bound product includes rinsing the bound product with a solution having a pH between about 8-10. As discussed above, in some embodiments, the affinity membrane separator includes a separation substrate and a plurality of peptide ligands positioned on the separation substrate. In some embodiments, the peptide ligands preferentially bind to one of the waste product or the nucleic acid product. In some embodiments, the nucleic acid product includes ss-RNA and the waste product includes ds-RNA.

[0015] Aspects of the present disclosure are directed to a system for continuous affinity membrane manufacturing. In some embodiments, the system includes a bioreactor including components sufficient to promote synthesis of a nucleic acid product and an affinity' membrane separator in fluid communication with the bioreactor. In some embodiments, the affinity membrane separator includes a separation substrate and a plurality of peptide ligands positioned on the separation substrate, wherein the peptide ligands preferentially bind to one of a waste product from the bioreactor or the nucleic acid product. In some embodiments, the nucleic acid product includes ss-RNA and the waste product includes ds-RNA.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings show embodiments of the disclosed subject matter for the purpose of illustrating the invention. However, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

[0017] FIG. 1 is a schematic representation of an affinity membrane separator for purifying a nucleic acid product according to some embodiments of the present disclosure;

[0018] FIG. 2 is a schematic representation of a system for continuous affinity membrane manufacturing according to some embodiments of the present disclosure;

[0019] FIG. 3 is a chart of a method for purification of a nucleic acid product according to some embodiments of the present disclosure;

[0020] FIG. 4 is a graph portraying protein template distribution of ssRNA / dsRNA selective peptide ligands according to some embodiments of the present disclosure;

[0021] FIG. 4B is a graph portraying an amino acid fraction and global charge comparison between a candidate library and dsRNA selective peptides according to some embodiments of the present disclosure;

[0022] FIG. 4C is a graph portraying amino acid fraction and global charge comparison between dsRNA selective peptides and ssRNA selective peptides according to some embodiments of the present disclosure;

[0023] FIG. 5A is a graph portraying static binding kinetics for peptide ligands according to some embodiments of the present disclosure with dsRNA and ssRNA;

[0024] FIG. 5B includes binding variables including kaand qe, and compositions of kaand qcbetween dsRNA and ssRNA binding for peptide ligands according to some embodiments of the present disclosure;

[0025] FIG. 6A is a graph portraying the dynamic characterization of dsRNA / ssRNA binding to peptide-functionalized cellulose disks according to some embodiments of the present disclosure;

[0026] FIG. 6B is a graph portraying the overlay of dynamic characterization of dsRNA / ssRNA binding E3-50 peptide functionalized cellulose disks according to some embodiments of the present disclosure;

[0027] FIG. 7 includes dsRNA binding capacity, recovery percentage, and selectivity calculations based on the mass and molar quantities of bound and eluted dsRNA / ssRNA data for peptide ligands according to some embodiments of the present disclosure;

[0028] FIG. 8 is a graph portraying quantitative characterization of dsRNA and ssRNA binding kinetics to peptide-functionalized surfaces according to some embodiments of the present disclosure via Quartz Crystal Microbalance (QCM);

[0029] FIG. 9 is a representation of structure prediction for peptide ligands according to some embodiments of the present disclosure from phage display;

[0030] FIG. 10 is a schematic representation of the docking of peptide ligands according to some embodiments of the present disclosure to representative dsRNA;

[0031] FIG. 11 A is a schematic representation of a TAV2B-dsRNA binding complex, including a zoomed in view of the binding interface of TAV2B-6 and representative dsRNA (left) front view of the peptide-dsRNA binding interface and (right) side view of the peptide- dsRNA binding interface;

[0032] FIG. 1 IB is a schematic representation of TAV2B-dsRNA binding complex of surface mapping of a TAV2B-6 peptide-dsRNA complex based on hydrophobicity. H-bonds, and charge; and

[0033] FIG. 12 is a schematic representation of a binding interface of Phage-5 peptide ligands consistent with embodiments of the present disclosure with representative dsRNA.DETAILED DESCRIPTION

[0034] Referring now to FIG. 1. some aspects of the disclosed subject matter are directed to an affinity membrane separator 100 for the production of nucleic acid products. In some embodiments, separator 100 is configured for purifying a nucleic acid product, such as ss-RNA vaccines. In some embodiments, the nucleic acid products are purified after synthesis at scale in and / or feed from a bioreactor, e.g., a cell-free or in vitro transcription (IVT) bioreactor, as will be discussed in greater detail below. While embodiments described herein demonstrate and are configured for the purification of ss-RNA products via removal of ds-RNA, the systems and methods of the present disclosure are not intended to be limited to this particular application, as useful applications further include, but are not limited to: quality analysis of ss-RNA; qualify analysis of ds-RNA; prediction of ds-RNA and / or ss-RNA associated or co-eluted impurities;prediction of concentration of ds-RNA and / or ss-RNA; preventing ss-RNA from forming ds- RNA; RNA interference (RNAi) therapy; antiviral therapy; immunomodulation; cancer therapy; drug delivery7, e.g., RNA-based therapeutics, dsRNA-specific binding peptides used to deliver other types of drugs or imaging agents to cells or tissues where dsRNA is present, such as viral- infected cells or cancer cells with dsRNA overexpression; research tools, e.g., use as a probe for in-vitro detection of dsRNA by electrophoresis mobility shift assay (EMSA), northwestern blotting or by direct fluorescence labeling of fixed samples; use as a prove for in vivo detection of dsRNA; spatio-temporal analysis of dsRNA virus infection in plant / insect / mammalian cells; profiling dsRNA binding proteins inside different cells (dsRNA peptide capture dsRNA-dsRNA binding protein complex), etc., or combinations thereof.

[0035] In some embodiments, separator 100 includes a separation substrate 102. In some embodiments, separation substrate 102 is porous. In some embodiments, separation substrate 102 includes a fibrous network configured to allow diffusion and / or transport across the substrate. In some embodiments, separation substrate 102 includes one or more materials capable of being conjugated to polypeptide ligands, themselves configured to bind nucleic acid products, as will be discussed in greater detail below. In some embodiments, separation substrate 102 includes cellulose, regenerated cellulose, or combinations thereof. In some embodiments, separation substrate 102 includes a flat sheet microporous membrane, a cellulose hollow fiber microporous affinity membrane, a tangential flow hollow fiber membrane, or combinations thereof. In some embodiments, separation substrate 102 has a total membrane surface area of about 1 m2or greater. In some embodiments, separation substrate 102 has a total membrane surface area of about 2 m2or greater. In some embodiments, separation substrate 102 has a total membrane surface area of about 4 m2or greater. In some embodiments, separation substrate 102 has a total membrane surface area of about 5 m2or greater.

[0036] Still referring to FIG. 1, in some embodiments, a plurality of peptide ligands 104 are positioned on separation substrate 102. In some embodiments, peptide ligands 104 are provided in a layer L on a surface S of separation substrate 102. In some embodiments, peptide ligands 104 are provided within pores of separation substrate 102. i.e., on an interior surface (not pictured) of the substrate. In some embodiments, peptide ligands 104 are provided on separation substrate 102 by reacting desired nucleic acid-binding peptides to the substrate. In some embodiments, this reaction includes forming a covalent bond between peptide ligands 104 and separation substrate 102. In some embodiments, this reaction includes forming a peptide bond between peptide ligands 104 and separation substrate 102. In some embodiments, the peptidebond is mediated by diisopropylcarbodiimide (DIC) and ethyl (hydroxyimino)cyanoacetate (Oxyma).

[0037] In some embodiments, peptide ligands 104 covalently bond to the membrane via a heterobifunctional crosslinker or a homobifunctional crosslinker. In some embodiments, peptide ligands 104 are provided on separation substrate 102 by reacting a functional group of the substrate with a first end of a crosslinker; and reacting a second end of the crosslinker with peptides ligands 104. In some embodiments, peptides ligands 104 are attached to separation substrate 102, e.g., flat sheet regenerated cellulose membranes, using standard EDC-NHS coupling chemistry. Other crosslinking bonds are contemplated in some embodiments as well, e.g., amine to sulfhydry l. In some embodiments, peptides ligands 104 are covalently bound to separation substrate 102 via a heterobifunctional crosslinker that is suitable for the solubility of the peptide, mRNAs, and membrane functional groups, e g., heterobifunctional crosslinkers available from ThermoFisher Scientific and G-Biosciences, including 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC), SMCC, sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sulfo-SMCC), succinimidyl 3-(2- pyridyldithio)propionate (SPDP), sulfosuccinimidyl 6-(3'-(2- pyridyldithio)propionamido)hexanoate (sulfo-LC-SPDP), and Pierce™ branded reagents. In some embodiments, peptide ligands 104 are covalently bound to separation substrate 102 via a homobifunctional crosslinker, e.g., homobifunctional crosslinkers available from ThermoFisher Scientific, including disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), PEGylated bis(sulfosuccinimidyl)suberate (BS(PEG)9), dithiobis(succimmidyl propionate) (DSP). 3.3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), SMCC, sulfo-SMCC, SPDP, and LC-SPDP.

[0038] In some embodiments, peptide ligands 104 are provided on separation substrate 102 by reacting a carboxyl group on the substrate, e.g.. of a regenerated cellulose membrane, with 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to form an O-acylisourea, and subsequently reacting an N-hydroxysuccinimide-modified peptide ligand 104 with the O- acylisourea.

[0039] In some embodiments, peptide ligands 104 preferentially bind to a first nucleic acid product relative to a second nucleic acid product. In some embodiments, the first nucleic acid product is a polynucleotide produced by a bioreactor, e.g., an IVT bioreactor. In some embodiments, the second nucleic acid product is a polynucleotide produced in the same bioreactor as the first nucleic acid product. In some embodiments, the second nucleic acidproduct is a polynucleotide produced as a byproduct of producing the first nucleic acid product. In some embodiments, the second nucleic acid product is a polynucleotide produced during a preprocessing or postprocessing step of producing and / or isolating the first nucleic acid product, e.g., a purification step.

[0040] Tn some embodiments, the first nucleic acid product includes ss-RNA, ds-RNA, DNA, or a derivative or conjugate thereof. In some embodiments, the first nucleic acid product includes a non-natural nucleic acid, L-sugars, and / or synthetic nucleotides / nucleosides. In some embodiments, the second nucleic acid product includes ss-RNA, ds-RNA, DNA, or a derivative or conjugate thereof. In some embodiments, the second nucleic acid product includes a nonnatural nucleic acid, L-sugars, and / or synthetic nucleotides / nucleosides.

[0041] In some embodiments, the first nucleic acid product includes ss-RNA and the second nucleic acid product includes ds-RNA. In some embodiments, the ss-RNA is a vaccine against SARS-CoV-2, flu, respiratory syncytial virus (RSV), human metapneumo virus (HMPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicellazoster virus (VZV), human immunodeficiency viruses (HIV), norovirus, Lyme disease, zika virus. Mpox, or combinations thereof.

[0042] In some embodiments, each of peptide ligands 104 have the same amino acid sequence, or a functional equivalent thereof. In some embodiments, peptides 104 are composed of a first set of peptides having a first amino acid sequence and a second set of peptides having a second amino acid sequence, where the first amino acid sequence and the second amino acid sequence are different.

[0043] In some embodiments, peptide ligands 104 include fewer than 20 residues. In some embodiments, peptide ligands 104 include at least one defined secondary structure, e.g., an alpha helix, a beta sheet, etc. In some embodiments, each of peptide ligands 104 include fewer than 20 residues and at least one defined secondary' structure. In some embodiments, peptide ligands 104 have an animo acid sequence according to SEQ ID NO.: 1 - SEQ ID NO.: 490.

[0044] In some embodiments, peptide ligands 104 preferentially bind to ds-RNA relative to ss-RNA. In some embodiments, peptide ligands 104 have a global charge greater than about 1 and at least one lysine (K), arginine (R), or glutamine (Q) residue. In some embodiments, peptide ligands 104 have an ammo acid sequence according to SEQ ID NO.: I, SEQ ID NO.: 2. SEQ ID NO : 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.:13, SEQIDNO.: 14, SEQIDNO.: 15. SEQIDNO.: 16, SEQIDNO.: 17. SEQIDNO.: 18, SEQIDNO : 19, SEQIDNO.: 20, SEQIDNO.: 21, SEQIDNO.: 22, SEQIDNO.: 23, SEQ ID NO.: 24, SEQIDNO.: 25, SEQIDNO.: 26, SEQIDNO.: 27, SEQIDNO.: 28, SEQ ID NO.: 29, SEQIDNO.: 30, SEQIDNO.: 31, SEQIDNO.: 32, SEQIDNO.: 33. SEQ ID NO: 34, SEQIDNO.: 35, SEQIDNO.: 36. SEQIDNO.: 37, SEQIDNO.: 38. SEQ IDNO.: 39, SEQIDNO.: 40, SEQIDNO.: 41, SEQIDNO.: 42, SEQIDNO.: 43, SEQ IDNO: 44, SEQIDNO.: 45, SEQIDNO.: 46, SEQIDNO.: 47, SEQIDNO.: 48, and SEQ IDNO.: 49, as shown in Table 1 below.

[0045] In some embodiments, peptide ligands 104 preferentially bind to ss-RNA relative to ds-RNA. In some embodiments, peptide ligands 104 have a global charge less than about 0 and at least one serine (S) or asparagine (N) residue. In some embodiments, peptide ligands 104 have an amino acid sequence according to SEQ ID NO.: 50, SEQ ID NO.: 51, SEQ ID NO.: 52, SEQIDNO.: 53, SEQIDNO.: 54. SEQ ID NO.: 55, SEQIDNO.: 56. SEQIDNO.: 57, SEQIDNO: 58, SEQIDNO.: 59, SEQIDNO.: 60, SEQIDNO.: 61, SEQ ID NO.: 62, SEQ ID NO.: 63, SEQIDNO.: 64, SEQIDNO.: 65, SEQIDNO.: 66, SEQIDNO.: 67, SEQ IDNO.: 68, SEQIDNO.: 69, SEQIDNO.: 70, SEQIDNO.: 71, SEQ ID NO.: 72. SEQ IDNO: 73, SEQIDNO.: 74, SEQIDNO.: 75. SEQIDNO.: 76, SEQIDNO.: 77. SEQ IDNO.: 78, SEQIDNO.: 79, SEQIDNO.: 80, SEQIDNO.: 81, SEQ ID NO.: 82, SEQ IDNO.: 83, and SEQ ID NO.: 84, as shown in Table 1 below.Table 1; Peptide ligands according to embodiments of the present disclosure.

[0046] Referring now to FIG. 2, some embodiments of the present disclosure are directed to a system 200 for continuous affinity membrane manufacturing. In some embodiments, system 200 is configured for the production of purified nucleic acid products, such as ss-RNA vaccines. In some embodiments, system 200 includes a bioreactor 202 including components sufficient to promote synthesis of a nucleic acid product. In some embodiments, bioreactor 202 is an IVT bioreactor.

[0047] Still referring to FIG. 2, in some embodiments, system 200 includes an affinity membrane separator 204 in fluid communication with bioreactor 202. In some embodiments, affinity membrane separator 204 is configured for purifying a nucleic acid product, such as ss- RNA vaccines. In some embodiments, nucleic acid products from bioreactor 202 are first provided to one or more processing modules, e.g., intermediate filtering systems, before being transported to separator 204. In some embodiments, effluent from these processing modules, having a reduced concentration of nucleic acid products, is recycled to bioreactor 202 to aid in the production of additional nucleic acid product. In some embodiments, affinity' membrane separator 204 includes a separation substrate 204A. As discussed above, in some embodiments, separation substrate 204A is porous. In some embodiments, separation substrate 204A includes a fibrous network configured to allow diffusion and / or transport across the substrate. In some embodiments, separation substrate 204 A includes one or more materials capable of being conjugated to polypeptide ligands, themselves configured to bind nucleic acid products, e.g., ss- RNA vaccines. In some embodiments, separation substrate 204A includes cellulose, regenerated cellulose, or combinations thereof. In some embodiments, separation substrate 204A includes aflat sheet microporous membrane, a cellulose hollow fiber microporous affinity membrane, a tangential flow hollow fiber membrane, or combinations thereof. In some embodiments, separation substrate 204A has a total membrane surface area of about 1 m2or greater. In some embodiments, separation substrate 204A has a total membrane surface area of about 2 m2or greater. In some embodiments, separation substrate 204A has a total membrane surface area of about 4 m2or greater. In some embodiments, separation substrate 204A has a total membrane surface area of about 5 m2or greater.

[0048] Referring again to FIG. 2, in some embodiments, a plurality of peptide ligands 204B are positioned on separation substrate 204A As discussed above, in some embodiments, peptide ligands 204B preferentially bind to a first nucleic acid product 206A relative to a second nucleic acid product 206B. In some embodiments, first nucleic acid product 206A includes ss-RNA, ds- RNA. DNA, or a derivative or conjugate thereof. In some embodiments, first nucleic acid product 206A includes a non-natural nucleic acid. L-sugars. and / or synthetic nucleotides / nucleosides. In some embodiments, second nucleic acid product 206B includes ss- RNA, ds-RNA, DNA, or a derivative or conjugate thereof. In some embodiments, second nucleic acid product 206B includes a non-natural nucleic acid, L-sugars, and / or synthetic nucleotides / nucleosides.

[0049] In some embodiments, first nucleic acid product 206A is a polynucleotide produced by bioreactor 202, e.g., an IVT bioreactor. In some embodiments, second nucleic acid product 206B is a polynucleotide produced in bioreactor 202. In some embodiments, second nucleic acid product 206B is a polynucleotide produced as a byproduct of producing first nucleic acid product 206 A. In some embodiments, second nucleic acid product 206B is a polynucleotide produced during a preprocessing or postprocessing step of producing first nucleic acid product 206 A, e.g.. a purification step. In some embodiments, peptide ligands 204B preferentially bind to one of a nucleic acid product, e.g.. first nucleic product 206A, and a waste product, e.g., second nucleic acid product 206B. In some embodiments, peptide ligands 204B preferentially bind to one of a nucleic acid product from bioreactor 202, e.g., first nucleic acid product 206B, and a waste product from bioreactor 202, e.g., second nucleic acid product 206B. In some embodiments, the nucleic acid product includes ss-RNA and the waste product includes ds-RNA.

[0050] In some embodiments, peptide ligands 204B are provided in a layer on a surface of separation substrate 204A. In some embodiments, peptide ligands 204B are provided within pores of separation substrate 204A, i.e., on an interior surface (not pictured) of the substrate.

[0051] In some embodiments, peptide ligands 204B are provided on separation substrate 204A by reacting desired nucleic acid-binding peptides to the substrate. In some embodiments, this reaction includes forming a covalent bond between peptide ligands 204B and separation substrate 204A. In some embodiments, this reaction includes forming a peptide bond between peptide ligands 204B and separation substrate 204A. In some embodiments, the peptide bond is mediated by diisopropylcarbodiimide (DIC) and ethyl (hydroxyimino)cyanoacetate (Oxyma).

[0052] In some embodiments, peptide ligands 204B covalently bond to peptide ligands 204B via a heterobifunctional crosslinker or a homobifunctional crosslinker. In some embodiments, peptide ligands 204B are provided on separation substrate 204A by reacting a functional group of the substrate with a first end of a crosslinker and reacting a second end of the crosslinker with peptides ligands 204B. In some embodiments, peptide ligands 204B are attached to separation substrate 204A, e.g., flat sheet regenerated cellulose membranes, using standard EDC-NHS coupling chemistry. Other crosslinking bonds are contemplated in some embodiments as well, e.g., amine to sulfhydryl. In some embodiments, peptide ligands 204B are covalently bound to separation substrate 204A, e.g., a membrane, via a heterobifunctional crosslinker that is suitable for the solubility of the peptide, mRNAs. and membrane functional groups, e.g., heterobifunctional crosslinkers available from ThermoFisher Scientific and G- Biosciences, including l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sulfo-SMCC), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), sulfosuccinimidyl 6-(3'-(2- pyridyldithio)propionamido)hexanoate (sulfo-LC-SPDP). and Pierce™ branded reagents. In some embodiments, peptide ligands 204B are covalently bound to separation substrate 204A, e.g., membrane, via a homobifunctional crosslinker, e.g., homobifunctional crosslinkers available from ThermoFisher Scientific, including disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), PEGylated bis(sulfosuccinimidyl)suberate (BS(PEG)9), dithiobis(succinimidyl propionate) (DSP), 3,3'- dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), SMCC, sulfo-SMCC. SPDP, and LC-SPDP.

[0053] In some embodiments, peptide ligands 204B are provided on separation substrate 204A by reacting a carboxyl group on the substrate, e.g., of a regenerated cellulose membrane, with l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to form an O- acylisourea, and subsequently reacting an N-hydroxysuccinimide-modified peptide ligand 204B with the O-acylisourea.

[0054] Still referring to FIG. 2, in some embodiments, each of peptide ligands 204B have the same amino acid sequence, or a functional equivalent thereof. In some embodiments, peptides 204B are composed of a first set of peptides having a first amino acid sequence and a second set of peptides having a second amino acid sequence, where the first amino acid sequence and the second amino acid sequence are different.

[0055] In some embodiments, peptide ligands 204B include fewer than 20 residues. In some embodiments, peptide ligands 204B include at least one defined secondary structure, e.g., an alpha helix, a beta sheet, etc. In some embodiments, each of peptide ligands 204B include fewer than 20 residues and at least one defined secondary structure. In some embodiments, peptide ligands 204B have an animo acid sequence according to SEQ ID NO.: 1 - SEQ ID NO.: 490.

[0056] In some embodiments, peptide ligands 204B preferentially bind to ds-RNA relative to ss-RNA. In some embodiments, peptide ligands 204B have a global charge greater than about 1 and at least one lysine (K), arginine (R), or glutamine (Q) residue. In some embodiments, peptide ligands 204B have an amino acid sequence according to SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO. : 1 1 , SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, SEQ ID NO.: 24, SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.: 29, SEQ ID NO.: 30, SEQ ID NO.: 31, SEQ ID NO.: 32, SEQ IDNO.: 33, SEQ ID NO.: 34, SEQ ID NO.: 35, SEQ ID NO.: 36, SEQ ID NO.: 37, SEQ IDNO.: 38, SEQ ID NO.: 39, SEQ ID NO.: 40, SEQ ID NO.: 41, SEQ ID NO.: 42, SEQ IDNO.: 43, SEQ ID NO.: 44, SEQ ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47. SEQ IDNO.: 48. and SEQ ID NO.: 49.

[0057] In some embodiments, peptide ligands 204B preferentially bind to ss-RNA relative to ds-RNA. In some embodiments, peptide ligands 204B have a global charge less than about 0 and at least one serine (S) or asparagine (N) residue. In some embodiments, peptide ligands 204B have an amino acid sequence according to SEQ ID NO.: 50, SEQ ID NO.: 51 , SEQ ID NO.: 52, SEQ ID NO.: 53, SEQ ID NO.: 54, SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NO.: 57, SEQ ID NO.: 58, SEQ ID NO.: 59, SEQ ID NO.: 60, SEQ ID NO : 61, SEQ ID NO.: 62, SEQ ID NO.: 63, SEQ ID NO.: 64. SEQ ID NO.: 65, SEQ ID NO.: 66. SEQ ID NO.: 67, SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71, SEQ IDNO.: 72. SEQ ID NO.: 73, SEQ ID NO.: 74. SEQ ID NO.: 75, SEQ ID NO.: 76. SEQ ID NO : 77, SEQ ID NO.: 78, SEQ ID NO.: 79, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.: 83, and SEQ ID NO.: 84.

[0058] In some embodiments, system 200 is configured for continuous affinity membrane manufacturing and purification of a ss-RNA vaccine against coronaviruses. In some embodiments, bioreactor 202 is configured to produce one or more ss-RNA vaccines against coronaviruses. As discussed above, in some embodiments, the ss-RNA is a vaccine against coronaviruses, e.g.. SARS-CoV-2, flu. RSV. HMPV, CMV. EBV, HSV, VZV, HIV, norovirus, Lyme disease, zika virus, Mpox, and other viruses.

[0059] In some embodiments, system 200 is operated to maximize yield of product, e.g., ss- RNA. In some embodiments, system 200 is operated to maximize purity of product. In some embodiments, system 200 is operated to balance yield and purity of product.

[0060] Referring now to FIG. 3, some embodiments of the present disclosure are directed to a method 300 for purification of a nucleic acid product. In some embodiments, at 302, a concentration of a nucleic acid product is generated. In some embodiments, the nucleic acid product is generated in a bioreactor. In some embodiments, the nucleic acid product includes ss- RNA, ds-RNA, DNA, or a derivative or conjugate thereof. In some embodiments, the nucleic acid product is a ss-RNA vaccine against coronaviruses. In some embodiments, the bioreactor is configured to produce one or more ss-RNA vaccines against coronaviruses. As discussed above, in some embodiments, the ss-RNA is a vaccine against, e.g., SARS-CoV-2, flu, RSV, HMPV, CMV, EBV, HSV, N7N, HIV, norovirus, Lyme disease, zika virus, Mpox, and other viruses. In some embodiments, the bioreactor also produces a waste product along with the nucleic acid product.

[0061] In some embodiments, at 304, a medium including the nucleic acid product and the waste product is contacted with an affinity membrane separator. As discussed above, in some embodiments, the affinity membrane separator includes a separation substrate. In some embodiments, the separator includes a plurality of peptide ligands positioned on the separation substrate. In some embodiments, the peptide ligands preferentially bind to one of the waste product or the nucleic acid product. In some embodiments, the peptide ligands include fewer than 20 residues. In some embodiments, the peptide ligands include at least one defined secondary structure, e.g.. an alpha helix, a beta sheet, etc. In some embodiments, each peptide ligand includes fewer than 20 residues and at least one defined secondary structure. In someembodiments, the peptide ligands have an animo acid sequence according to SEQ ID NO.: 1 - SEQIDNO: 490.

[0062] In some embodiments, the peptide ligands preferentially bind to ds-RNA relative to ss-RNA. In some embodiments, the peptide ligands have a global charge greater than about 1 and at least one lysine (K), arginine (R), or glutamine (Q) residue. In some embodiments, the peptide ligands have an amino acid sequence according to SEQ ID NO.: 1, SEQ ID NO.: 2, SEQIDNO.: 3, SEQIDNO.: 4, SEQIDNO.: 5, SEQIDNO.: 6, SEQIDNO.: 7, SEQ ID NO: 8, SEQIDNO.: 9, SEQIDNO.: 10, SEQIDNO.: 11, SEQ ID NO.: 12, SEQIDNO.: 13, SEQIDNO.: 14, SEQIDNO.: 15, SEQIDNO.: 16, SEQIDNO.: 17, SEQIDNO.: 18, SEQIDNO.: 19, SEQIDNO.: 20, SEQIDNO.: 21, SEQIDNO.: 22, SEQIDNO.: 23, SEQ ID NO.: 24, SEQIDNO.: 25, SEQIDNO.: 26, SEQIDNO.: 27, SEQIDNO.: 28, SEQ ID NO.: 29, SEQIDNO.: 30, SEQIDNO.: 31, SEQIDNO.: 32, SEQIDNO.: 33. SEQ IDNO.: 34. SEQIDNO.: 35, SEQIDNO.: 36. SEQIDNO.: 37, SEQIDNO.: 38. SEQ IDNO.: 39, SEQIDNO.: 40, SEQIDNO.: 41, SEQ ID NO.: 42, SEQIDNO.: 43, SEQ IDNO.: 44, SEQIDNO.: 45, SEQIDNO.: 46, SEQIDNO.: 47, SEQIDNO.: 48, and SEQ IDNO.: 49.

[0063] In some embodiments, the peptide ligands preferentially bind to ss-RNA relative to ds-RNA. In some embodiments, the peptide ligands have a global charge less than about 0 and at least one serine (S) or asparagine (N) residue. In some embodiments, the peptide ligands have an amino acid sequence according to SEQ ID NO.: 50, SEQ ID NO.: 51. SEQ ID NO.: 52, SEQIDNO.: 53, SEQIDNO.: 54, SEQIDNO.: 55, SEQIDNO.: 56, SEQIDNO.: 57, SEQIDNO.: 58, SEQIDNO.: 59, SEQIDNO.: 60, SEQIDNO.: 61, SEQ ID NO.: 62, SEQ ID NO.: 63, SEQIDNO.: 64, SEQIDNO.: 65, SEQIDNO.: 66, SEQIDNO.: 67, SEQ IDNO.: 68, SEQIDNO.: 69, SEQIDNO.: 70, SEQIDNO.: 71, SEQ ID NO.: 72. SEQ IDNO.: 73. SEQIDNO.: 74, SEQIDNO.: 75. SEQIDNO.: 76, SEQIDNO.: 77. SEQ IDNO.: 78, SEQIDNO.: 79, SEQIDNO.: 80, SEQIDNO.: 81, SEQ ID NO.: 82, SEQ IDNO.: 83, and SEQ ID NO.: 84.

[0064] Still referring again to FIG.3, at 306, one of the waste product or the nucleic acid product is bound via the peptide ligands to form a concentration of bound product. In some embodiments, the nucleic acid product includes ss-RNA and the waste product includes ds-RNA. Since the peptide ligands preferentially bind to one of the nucleic acid product or the waste product, the unbound product is allowed to pass through the separation substrate, purifying the bound product of the unbound product and vice-a-versa. At 308, an effluent including aconcentration of the bound product, e.g.. bound ds-RNA or bound ss-RNA. is eluted from the affinity membrane separator. In some embodiments, eluting 308 includes rinsing the bound product with a solution having a pH between about 8-10.EXAMPLES

[0065] Referring now7to FIGs. 4A-4C, in an exemplary7embodiment of the present disclosure, peptides selected from rational design approach (as detailed in Table 1) were further validated using competitive microarray screening. The peptides selected for additional screening included SEQ ID NO. 1 , SEQ ID NO 2, SEQ ID NO. 3 SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, and SEQ ID NO. 32. Peptides displaying consistent selective binding to ssRNA or dsRNA were further analyzed to assess their amino acid composition, protein template contribution, and global charge in comparison to initial candidate peptide libraries and to each other. As illustrated in FIGs. 4B-4C, dsRNA selective peptides exhibited a significant increase in R content, as well as higher K and Q residues, which contributed to a more positive global charge compared to the original candidate library. In contrast, ssRNA selective peptides showed a higher abundance of serine (S) and asparagine (N), leading to an alternative charge profile. These differences in amino acid propensity7and charge underscore molecular characteristics driving selectivity for dsRNA and ssRNA.

[0066] As discussed above, the ds-RNA-selective binding peptides consistent with embodiments of the present disclosure are beneficial for use as affinity ligands for the removal of ds-RNA during mRNA purification. To evaluate their effectiveness in a purification setting, three peptides, phage_5 (SEQ ID NO 3). TAV2b_6 (SEQ ID NO 1), and E3_50 (SEQ ID NO 1). as detailed in Table 2 below, were selected for further testing. These peptides were grafted onto cellulose filter papers, providing a practical platform for assessing their potential in dsRNA removal.Table 2; Selected dsRNA specific binding peptides for purification testing.

[0067] Referring now to FIGs. 5 A-5B, a static binding assay was performed to examine the kinetic profiles of the peptide-functionalized cellulose disks when interacting with dsRNA and ssRNA. Referring specifically to FIG. 5B, the binding curves were fitted using a pseudo first order kinetics model, with binding variables including kaand qe, and R2 being obtained for the averaged data. Comparisons of kaand qebetween dsRNA and ssRNA binding are provided as well.

[0068] TAV2b-6 demonstrated a well-balanced profile, with a high binding rate and capacity for dsRNA, and a slower binding rate and lower binding capacity for ssRNA. This suggests TAV2b-6's strong dsRNA selectivity. All peptides demonstrated selectivity to dsRNA, with E3-50 showing the highest selectivity in terms of binding rate and binding capacity. E3-50 exhibited a significantly faster binding rate for dsRNA than for ssRNA. Phage-5, while showing selectivity for dsRNA in both binding rate and capacity, was outperformed by TAV2b-6 and ESSO. The static assay confirmed that all peptides demonstrated dsRNA selectivity, with E3-50 standing out in terms of binding kinetics and capacity.

[0069] Referring now to FIG. 6A-6B, a dynamic binding assay was performed using a custom-designed membrane holder module and HPLC to further evaluate the peptide- functionalized cellulose disks under flow conditions. The dynamic performance of the peptides was assessed by challenging the peptide-cellulose disk systems with sufficient dsRNA / ssRNAto quantify dynamic binding capacity. This method allowed for continuous monitoring of binding and elution. Clear elution peaks were observed for both dsRNA and ssRNA, but dsRNA exhibited significantly higher binding capacity7across all peptide disks. Distinct elution peaks at about 25 minutes w ere observed, indicating successful binding and elution, confirming the feasibility7of a bind-and-elute purification process. Notably, both TAV2b-6 and E3-50 displayednear-full recovery of bound dsRNA during elution, demonstrating their practical utility in dynamic settings. The dynamic assay confirmed the performance of TAV2b-6 and E3-50, with both showing high dsRNA binding capacity and recovery efficiency, and strong selectivity compared to ssRNA (see also FIG. 7).

[0070] Both static and dynamic binding studies highlighted the selectivity of the tested peptides for dsRNA over ssRNA. Among the tested candidates, TAV2b-6 and E3-50 advantageously demonstrated combined high binding capacity, selectivity, and recovery' rates. These findings suggest that these peptides could serve as effective affinity ligands in dsRNA purification processes, particularly in applications where dsRNA needs to be selectively removed and / or in complex, dsRNA / ssRNA, IVT mixtures, etc.

[0071] Referring now to FIG. 8. in another exemplary’ embodiment, TAV2b-6. identified from rational design was further assessed for its diagnostic potential using Quartz Crystal Microbalance (QCM) to evaluate the dynamic binding and elution kinetics of dsRNA and ssRNA to peptide-functionalized surfaces.

[0072] The preparation of the QCM sensor involved two steps: immobilizing the peptide and passivating the remaining surface with free cysteine. A frequency drop of 15 Hz was observed after peptide immobilization, indicating successful mass loading, followed by a minor 2 Hz increase after blocking with free cysteine, suggesting some displacement of loosely bound peptides. The packing density of the peptide, determined via the Sauerbrey equation, was calculated as 4.72* 1010mol / cm2(or 2.83xl014molecules / cm2), corresponding to an average cross-sectional area of about 0.35 nm2per peptide, suggesting dense packing. The subsequent buffer rinse showed minimal frequency changes, confirming strong peptide attachment to the surface. Cysteine was then introduced to block the unoccupied surface areas, ensuring specificity in dsRNA detection. To assess the peptide's binding ability, LMW poly(EC) dsRNA was introduced at a concentration of 0.45 ng / pL. A rapid and significant frequency drop of -8.72 Hz was recorded, corresponding to 154 ng / cm2of dsRNA bound (9.7x 1013mol / cm2), demonstrating substantial dsRNA capture. The fast binding kinetics confirmed dsRNA's high affinity for the peptide. After the equilibrium was reached, a washing step with HEPES buffer removed non-specifically bound material, and no significant frequency change was observed, indicating strong and selective binding. Elution with a pH 9 buffer successfully restored the original frequency, confirming dsRNA removal from the sensor. When ssRNA was introduced under the same conditions and molar concentration, the sensor showed reduced binding, as evidenced by a smaller frequency drop of 1.75 Hz, corresponding to 31 ng / cm2(4.0x 1014mol / cm2). This finding demonstrates the peptide’s selectivity for dsRNA over ssRNA. which is beneficial for its diagnostic application. To determine the limit of detection (TOD), dsRNA was introduced at concentrations ranging from 0.45 ng / pL to 0.45 pg / pL to evaluate the lowest detectable concentration using the QCM sensor. The peptide TAV2b-6 demonstrated robust dsRNA binding even at the lowest concentration tested (0.45 ng / pL), showcasing its potential for sensitive dsRNA detection applications.

[0073] Referring now to FIG. 9. structure assumes a pivotal role in ligand design, and in the case of rationally designed dsRNA-selective peptides, protein templates were selected based on a noticeable prevalence of the alpha helix secondary' structure. To validate the persistence of this alpha helix propensity' in the rationally designed peptides, structure predictions were conducted for randomly phage-selected peptides. FIG. 9 illustrates that enriched peptides from phage panning, exhibiting a marked dominance of the alpha helix structure also observed in many dsRNA selective binding proteins. Prediction was carried out in PEP-FOLD4 (pH 7.5, NaCl 150 mM).

[0074] This consistent observation across both rationally designed and randomly selected peptides underscores incorporation of the alpha helix in conferring selectivity in and mediating specific interactions with dsRNA targets by peptide ligands consistent with embodiments of the present disclosure.

[0075] To gain deeper insights into the binding mechanisms of the peptide ligands according to some embodiments of the present disclosure, molecular simulations were employed. These simulations offer a way to explore the specific interactions between the peptides and dsRNA at a molecular level, aiming to provide a better understanding of how these peptides achieve their selective binding.

[0076] Referring now to FIG. 10, for each peptide-RNA pair, the top 10 binding poses were isolated, and the top binding pose was rendered in Discovery Studio. Most peptides were found to bind within the grooves of dsRNA, interacting primarily with the phosphate backbones. Two peptides from the rational design library' (TAV2B_6, E3_50) were positioned adjacent to the backbone of the dsRNA helix.

[0077] Referring now to FIGs. 11 A-l IB, the TAV2b_6 peptide demonstrated a binding mode similar to its parent protein's interaction with dsRNA. Surface mapping based on charge was created for the receptor surface. The peptide sat inside of the charged pocket created by the phosphate backbone. Despite being a fragment, the peptide maintained its binding capability. The binding interface showed a series of electrostatic interactions between basic amino acids and acidic phosphates. The helical peptide oriented itself within the negatively charged backbonepocket of dsRNA. As shown in FIG. 1 IB. surface mapping of the peptide and dsRNA. including hydrogen bond, charge, and hydrophobicity mappings, highlighted the interactions facilitating this binding, which is H-bond and electrostatic interactions.

[0078] Referring now to FIG. 12, the Phage-5 peptide exhibited a binding behavior with two positively charged residues (arginine and lysine) positioned at opposite ends of the peptide. A surface mapping based on charge distribution was performed to elucidate the receptor surface properties. The peptide is shown to adopt a conformation that snugly fits into the dsRNA groove, with lysine and arginine residues from the peptide each anchoring to one strand of the dsRNA, colored yellow and grey respectively. This positioning allows for effective charge complementarity and enhances the stability of the complex. Additional stabilization is provided by other polar residues in the peptide, which form hydrogen bonds with the dsRNA, further securing the peptide within the binding groove. Each positively charged residue engaged with a different strand of the dsRNA, stabilizing the peptide within the dsRNA groove.

[0079] To further validate the selective binding to dsRNA of peptides ligands consistent with embodiments of the present disclosure and to explore the underlying mechanisms, molecular dynamics (MD) simulations w ere conducted on the highest-ranking peptide-dsRNA complexes from the molecular docking results. The simulations for both TAV2b-6 and Phage-5 peptides demonstrated stable associations with dsRNA throughout most of the simulation time. While minor fluctuations occurred, the peptides consistently returned to stable conformations around the dsRNA, reinforcing the observed stability and specificity of their interactions. These findings align with the docking results.

[0080] Systems and methods of the present disclosure advantageously provide nucleic acid product binding peptides with demonstrated specificity for different classes of nucleic acids. The peptide ligands according to embodiments of the present disclosure exhibit strong selective binding, e.g., to dsRNA over ssRNA, for use in targeted applications and underscoring their candidacy for use in therapeutic and diagnostic contexts. The advantages of cellulose substrate can be combined with conjugated peptide ligands of the present disclosure. The peptide ligands can be synthesized in a high throughput manner. Then, use of membrane surface grafting can be used to attach the selective binding peptides to microporous hydrophilic surfaces using grafting methods, e.g., standard EDC-NHS coupling.

[0081] Without wishing to be bound by theory, further molecular dynamic modeling with natural AUGC dsRNA showed consistent groove-binding interactions, indicating that these peptides would likely bind to other dsRNA species beyond poly(I:C), with backbone interactions reinforcing sequence independence. Using two distinct selection methods, peptides withconvergent structural and binding properties were identified. Notably, most high-binding peptides possessed a positive global charge and shared an alpha-helical structure, which can enhance flexibility and facilitate binding to the rigid dsRNA structure. Dominant groovebinding interactions, mainly mediated by the phosphate backbone, indicate sequenceindependent binding. Without wishing to be bound by theory, this selective recognition likely stems from the increased groove frequency in dsRNA, leading to faster binding kinetics and greater capacity for dsRNA compared to ssRNA. A 100 ns MD simulation confirmed the stability of this binding mode. The use of polydispersed poly(I:C) (1.5 kb to 8 kb) as the screening target allowed selection of peptides with broad affinity to dsRNA structural features rather than sequence-specific dsRNA species. This broader binding range aligns well with applications for dsRNA byproduct removal in mRNA in vitro transcription (IVT) processes, where byproducts also vary in size.

[0082] The systems and methods of the present disclosure are useful, e.g., for purification of mRNA products and their commercialization. Today, besides purifying mRNA vaccines, many mRNA products are being developed at Modema (and Pfizer, BioNTech and Greenlight Biosciences among many others) for treatment of many diseases and agricultural applications. Peptide affinity ligands provide high specificity, tunability, low immunogenicity, and chemical stability’, presenting a versatile and practical alternative for biotechnology applications.

[0083] Although the invention has been described and illustrated with respect to exemplary embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions can be made therein and thereto, without parting from the spirit and scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. An affinity membrane separator for purifying a nucleic acid product, comprising: a separation substrate; and a plurality of peptide ligands positioned on the separation substrate, wherein the peptide ligands preferentially bind to one of a first nucleic acid product and a second nucleic acid product, wherein the peptide ligands include fewer than 20 residues and at least one defined secondary structure, wherein the first nucleic acid product includes ss-RNA and the second nucleic acid product includes ds-RNA.

2. The separator including claim 1, wherein the separation substrate includes a flat sheet microporous membrane, a cellulose hollow fiber microporous affinity membrane, a tangential flow hollow fiber membrane, or combinations thereof.

3. The separator according to claim 1, wherein the ss-RNA is a vaccine against SARS-CoV- 2, flu, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV), human immunodeficiency viruses (HIV), norovirus, Lyme disease, zika virus, Mpox, or combinations thereof.

4. The separator according to claim 1, wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO.: 1 - SEQ ID NO.: 490.

5. The separator according to claim 4, wherein the peptide ligands preferentially bind to ds- RNA relative to ss-RNA, and include: a global charge greater than about 1, and at least one lysine, arginine, or glutamine residue.

6. The separator according to claim 5, wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.:

8. SEQ ID NO.:

9. SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ IDNO.: 13, SEQIDNO.: 14, SEQIDNO.:

15. SEQIDNO.: 16, SEQIDNO.:

17. SEQ ID NO.: 18, SEQIDNO.: 19, SEQIDNO.: 20, SEQIDNO.: 21, SEQIDNO.: 22, SEQIDNO.: 23, SEQIDNO.: 24, SEQIDNO.: 25, SEQIDNO.: 26, SEQIDNO.: 27, SEQIDNO.:

28. SEQIDNO.: 29, SEQIDNO.: 30, SEQIDNO.: 31, SEQ ID NO.: 32, SEQIDNO.: 33, SEQIDNO.: 34, SEQIDNO.: 35, SEQIDNO.:

36. SEQ ID NO.: 37, SEQIDNO.: 38, SEQIDNO.: 39, SEQIDNO.: 40, SEQIDNO.: 41, SEQIDNO.: 42, SEQIDNO.: 43, SEQIDNO.: 44, SEQIDNO.: 45, SEQIDNO.: 46, SEQIDNO.: 47, SEQIDNO.: 48, and SEQ ID NO.: 49.

7. The separator according to claim 4, wherein the peptide ligands preferentially bind to ss- RNA relative to ds-RNA, and include: a global charge less than about 0. and at least one serine or asparagine residue.

8. The separator according to claim 7, wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO. : 50, SEQ ID NO. : 51, SEQIDNO.: 52, SEQIDNO.: 53, SEQIDNO.: 54, SEQIDNO.: 55, SEQIDNO.: 56, SEQIDNO.: 57, SEQIDNO.: 58, SEQIDNO.: 59, SEQIDNO.: 60, SEQ ID NO.: 61, SEQIDNO.: 62, SEQIDNO.: 63, SEQIDNO.: 64, SEQIDNO.: 65, SEQ ID NO.: 66, SEQIDNO.:

67. SEQIDNO.: 68, SEQIDNO.:

69. SEQIDNO.: 70, SEQIDNO.: 71, SEQ ID NO.: 72, SEQ ID NO : 73, SEQIDNO.: 74, SEQIDNO.: 75, SEQIDNO.: 76, SEQIDNO.: 77, SEQIDNO.: 78, SEQIDNO.: 79, SEQ ID NO.: 80, SEQIDNO.: 81, SEQIDNO.: 82, SEQIDNO.: 83, and SEQ ID NO.: 84.

9. A method for purification of a nucleic acid product, comprising: generating a concentration of a nucleic acid product; contacting a medium including the nucleic acid product and a waste product with an affinity membrane separator, the affinity membrane separator including: a separation substrate, and a plurality7of peptide ligands positioned on the separation substrate, wherein the peptide ligands preferentially bind to one of the waste product or the nucleic acid product,binding one of the waste product or the nucleic acid product via the peptide ligands to form a concentration of bound product; and eluting from the affinity membrane separator an effluent including a concentration of the bound product. wherein the nucleic acid product includes ss-RNA and the waste product includes ds-RNA.

10. The method according to claim 9, wherein eluting includes: rinsing the bound product with a solution having a pH between about 8-10.

11. The method according to claim 9, wherein the ss-RNA is a vaccine against SARS-CoV-2, flu, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV). human immunodeficiency viruses (HIV), norovirus. Lyme disease, zika virus, Mpox, or combinations thereof.

12. The method according to claim 9, wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQIDNO.: 1 -SEQIDNO.: 490.

13. The method according to claim 9, wherein the peptide ligands include fewer than 20 residues and at least one defined secondary structure.

14. The method according to claim 13, wherein the peptide ligands preferentially bind to ds- RNA relative to ss-RNA, and include: a global charge greater than about 1, and at least one lysine, arginine, or glutamine residue.

15. The method according to claim 14. wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQIDNO.: 4, SEQIDNO.: 5, SEQIDNO.: 6, SEQIDNO.: 7, SEQ ID NO.: 8, SEQIDNO.: 9, SEQIDNO.: 10, SEQIDNO.:

11. SEQ ID NO.: 12, SEQ ID NO.: 13, SEQIDNO.: 14, SEQIDNO.: 15, SEQIDNO.: 16, SEQIDNO.:

17. SEQ ID NO.: 18, SEQIDNO.: 19, SEQIDNO.: 20, SEQIDNO.: 21, SEQ ID NO.: 22, SEQIDNO.: 23, SEQIDNO.: 24, SEQIDNO.: 25, SEQIDNO.: 26, SEQIDNO.: 27, SEQIDNO.: 28, SEQIDNO.: 29, SEQIDNO.: 30, SEQIDNO.: 31, SEQ IDNO.: 32, SEQIDNO.: 33, SEQIDNO.:

34. SEQIDNO.: 35, SEQIDNO.:

36. SEQ ID NO.: 37, SEQIDNO.: 38, SEQIDNO.: 39, SEQIDNO.: 40, SEQIDNO.: 41, SEQIDNO.: 42, SEQIDNO.: 43, SEQIDNO.: 44, SEQIDNO.: 45, SEQIDNO.: 46, SEQIDNO.:

47. SEQIDNO.: 48, and SEQ ID NO.: 49.1 . The method according to claim 13, wherein the peptide ligands preferentially bind to ss- RNA relative to ds-RNA, and include: a global charge less than about 0. and at least one serine or asparagine residue.

17. The method according to claim 16. wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO. : 50, SEQ ID NO. : 51, SEQIDNO.: 52, SEQIDNO.: 53, SEQIDNO.: 54, SEQIDNO.: 55, SEQIDNO.: 56, SEQIDNO.:

57. SEQIDNO.: 58, SEQIDNO.: 59, SEQIDNO.: 60, SEQ ID NO.: 61, SEQIDNO.: 62, SEQIDNO.: 63, SEQIDNO.: 64, SEQIDNO.:

65. SEQ ID NO.: 66, SEQIDNO.: 67, SEQIDNO.: 68, SEQIDNO.: 69, SEQIDNO.: 70, SEQIDNO.: 71, SEQ ID NO.: 72, SEQIDNO.: 73, SEQIDNO.: 74, SEQIDNO.: 75, SEQIDNO.: 76, SEQIDNO.: 77, SEQIDNO.: 78, SEQIDNO.: 79, SEQ ID NO.: 80, SEQIDNO.: 81, SEQIDNO.: 82, SEQIDNO.: 83, and SEQ ID NO.: 84.

18. A system for continuous affinity membrane manufacturing, comprising: a bioreactor including components sufficient to promote synthesis of a nucleic acid product, and an affinity membrane separator in fluid communication with the bioreactor, the affinity membrane separator including: a separation substrate; and a plurality of peptide ligands positioned on the separation substrate, wherein the peptide ligands preferentially bind to one of a waste product from the bioreactor or the nucleic acid product, wherein the nucleic acid product includes ss-RNA and the waste product includes ds-RNA.wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO.: 1- SEQ ID NO.: 490.

19. The system according to claim 18, wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO : 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.:

19. SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, SEQ ID NO.: 24, SEQ ID NO. : 25, SEQ ID NO.: 26, SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.: 29, SEQ ID NO.: 30, SEQ ID NO.: 31, SEQ ID NO.: 32, SEQ ID NO.: 33, SEQ ID NO.: 34, SEQ ID NO.: 35, SEQ ID NO.: 36, SEQ ID NO.: 37, SEQ ID NO.:

38. SEQ ID NO.: 39, SEQ ID NO.: 40, SEQ ID NO.: 41, SEQ ID NO.: 42, SEQ ID NO.: 43, SEQ ID NO.: 44, SEQ ID NO.:

45. SEQ ID NO.: 46, SEQ ID NO.: 47, SEQ ID NO.: 48, and SEQ ID NO : 49.

20. The system according to claim 18, wherein the peptide ligands include an animo acid sequence selected from the group consisting of: SEQ ID NO. : 50, SEQ ID NO. :

51. SEQ ID NO.: 52, SEQ ID NO.: 53, SEQ ID NO : 54, SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NO.: 57, SEQ ID NO.: 58, SEQ ID NO.: 59, SEQ ID NO.: 60, SEQ ID NO.: 61, SEQ ID NO.: 62, SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.:

67. SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71, SEQ ID NO.: 72, SEQ ID NO.: 73, SEQ ID NO.: 74, SEQ ID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 77, SEQ ID NO.: 78, SEQ ID NO.: 79, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.: 83, and SEQ ID NO.: 84.

Images