Method of separating nucleic acids

Abstract

Described herein are methods of separating nucleic acids using a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5. The pH and / or ionic strength of the loading buffer and eluting buffer(s) are adjusted to selectively bind and elute nucleic acids.

Description

METHOD OF SEPARATING NUCLEIC ACIDS TECHNICAL FIELD

[0001] The separating of nucleic acids from sample matrices are described. BACKGROUND

[0002] Nucleic acid therapeutics include all nucleic-acid-based approaches that modulate gene expression by inhibiting, adding, replacing, or editing at the DNA or RNA level. Research over the past 30 years has resulted in safe, effective delivery platforms enabling nucleic acid therapeutics. Several ex vivo and in vivo genetic drugs have been approved (or are in late-stage development) for treating a variety of inherited disorders. Significant technological breakthroughs and the unique circumstances of the COVID-19 pandemic led to FDA approval of mRNA medicines. The success of the Moderna and Pfizer / BioNTech COVID-19 vaccines and the unprecedented speed of their development has led to an explosion of new mRNA drugs and vaccines into clinical trials. Despite the very recent and rapid surge in development of mRNA- and other nucleic acid-based therapeutics, there is still considerable room for improvement in many aspects of their manufacture, and especially in the methods of their purification. Because mRNA is generated in an in vitro reaction, the purification matrix is well defined. In addition to the therapeutic mRNA reaction product, this mixture primarily includes leftover reaction components (nucleotide triphosphates, RNA polymerase, plasmid DNA, etc.) and aberrant RNA products (short abortive RNA molecules and double-stranded RNA (dsRNA)). Among these, dsRNAs are particularly problematic for separation from single stranded RNA due to their chemical similarity to the desired single-stranded (ss) mRNA products, and for patients due to their potential to trigger harmful interferon-mediated immune responses. SUMMARY

[0003] There are few purification methods for nucleic acids that are practical at large scale production. The current methods for dsRNA removal from ssRNA at manufacturing scale are cellulose chromatography in an ethanol solution and ion pair reverse phase (IP-RP) liquid chromatography. Although cellulose-based chromatography methods are simple and effective on laboratory scale, they have low reported dsRNA removal (90%) and ssRNA recovery (65%), while also requiring large amounts of ethanol. Alternatively, IP-RP chromatography is effective at removing dsRNA, but generates toxic acetonitrile waste and must be performed at high temperature. Thus, there is a desire to identify new solutions for the purification of nucleic acids,which are selective, non-toxic, and function at room temperature to improve the cost and / or efficiency of nucleic acid production and ensure the safety of the product.

[0004] In one aspect, a first method of separating a first nucleic acid and a second nucleic acid is disclosed. The method comprising the following steps in order: (a) providing a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5; (b) loading a sample matrix onto the solid phase, wherein the sample matrix comprises the first and second nucleic acids and a loading buffer that has a pH that is below or near the pKa and an ionic strength, which is sufficiently low to bind the first and second nucleic acids; (c) optionally washing the solid phase with a washing solution, wherein the washing solution has a pH that is below or near the pKa and an ionic strength, which is sufficiently low to bind the first and second nucleic acids: (d) passing a first eluting solution through the solid phase, wherein the first eluting solution comprises a ionic strength having a higher ionic strength than the first ionic strength, and optionally having a pH, which is higher than the pKa such that first nucleic acid is selectively eluted; and (e) passing a second eluting solution through the solid phase, wherein the second eluting solution comprises a ionic strength lower than the ionic strength of the first eluting solution and a pH, which is higher than the pKa, such that a second nucleic acid is eluted.

[0005] In one aspect, a second method of separating a first nucleic acid and a second nucleic acid is disclosed. The method comprising the following steps in order: (a) providing a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5; (b) loading a sample matrix onto the solid phase, wherein the sample matrix comprises the first and second nucleic acids and a loading buffer has a pH that is lower than the pKa and an ionic strength, which is sufficiently high to allow the first nucleic acid to flow through, while binding the second nucleic acid; (c) optionally washing the solid phase with a washing solution, wherein the washing solution has a pH that is lower the pKa and an ionic strength, which is sufficiently high to preserve binding of the second nucleic acid; (d) passing an eluting solution through the solid phase, wherein the eluting solution comprises a lower ionic strength than the ionic strength of the loading buffer and having a pH, which is higher than the pKa such that the second nucleic acid is selectively eluted.

[0006] In one aspect, a third method of separating a first nucleic acid and a second nucleic acid is disclosed. The method comprising the following steps in order:(a) providing a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5; (b) loading a sample matrix onto the solid phase, wherein the sample matrix comprises a plurality of nucleic acids and a loading buffer that has a pH that is higher than the pKa and an ionic strength, which is sufficiently low to bind the second nucleic acid and allow the first nucleic acid to flow through; (c) optionally washing the solid phase with a washing solution, wherein the washing solution has a pH that is higher than the pKa and an ionic strength, which is sufficiently low to bind the second nucleic acid and allow the first nucleic acid to flow through: (d) passing an eluting solution through the solid phase, wherein the eluting solution comprises a pH, which is higher than the pH of the loading buffer such that the second nucleic acid is selectively eluted.

[0007] The above summary is not intended to describe each embodiment. The details of one or more embodiments of the invention are also set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims. DESCRIPTION OF FIGURES

[0008] Fig.1 is a chromatogram for Example 3.

[0009] Fig.2 is an overlaid chromatogram of Example 8 and Comparative Example 8a. DETAILED DESCRIPTION

[0010] As used herein, the term “a”, “an”, and “the” are used interchangeably and mean one or more; and “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes, (A and B) and (A or B).

[0011] As used herein, the terms “a”, “an”, “the”, and “at least one” are used interchangeably.

[0012] The term “and / or” means either or both. For example, “A and / or B” means A alone, B alone, or both A and B.

[0013] The term “alkyl” refers to a monovalent group that is a radical of an alkane. The alkyl group can have 1 to 32 carbon atoms, 1 to 20 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. The alkyl can be linear, branched, cyclic, or a combination thereof. A linear alkyl has at least one carbon atom while a cyclic or branched alkyl has at least 3 carbon atoms.

[0014] The term “alkylene” refers to a divalent group that is a radical of an alkane. The alkylene group can have 1 to 32 carbon atoms, 1 to 20 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbonatoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. The alkylene can be linear, branched, cyclic, or a combination thereof. A linear alkylene has at least one carbon atom while a cyclic or branched alkylene has at least 3 carbon atoms.

[0015] The term “heteroalkylene” refers to an alkylene having one or more of the carbon atoms replaced with a heteroatom. The heteroatom is typically nitrogen (e.g., -NH-), oxygen (-O-), or sulfur (-S-). Typically, there are not two heteroatoms adjacent to each other such as in a peroxide.

[0016] The term “(hetero)alkylene” refers to an alkylene, heteroalkylene, or both.

[0017] The term “alkoxy” refers to a monovalent group of formula -ORawhere Rais an alkyl as defined above.

[0018] The term “aryl” refers to a monovalent group that is a radical of an aromatic carbocyclic compound. The aryl group has at least one aromatic carbocyclic ring and can have 1 to 3 optional rings that are connected to or fused to the aromatic carbocyclic ring. The additional rings can be aromatic, aliphatic, or a combination thereof. The aryl group usually has 5 to 20 carbon atoms or 6 to 10 carbon atoms.

[0019] The term “heteroaryl” refers to aryl with at least one heteroatom in the ring. The term “heteroaryl” refers to an aryl having one or more of the ring carbons replaced with a heteroatom. The heteroatom is selected from nitrogen, oxygen, or sulfur typically. The ring often has 1 to 3 heteroatoms and typically has 5 or 6 ring members. The heteroaryl can have 1 to 3 optional rings that are connected to or fused to the heterocyclic ring. The additional rings can be aromatic, aliphatic, or a combination thereof and can include heteroatoms or be free of heteroatoms.

[0020] The term “(hetero)aryl” refers to an aryl or heteroaryl.

[0021] The term “catenated” refers to atoms in the main chain and / or ring of a compound. In the compound CH3-CH2-CH2-CH(CH3)-CH(CH3)-CH(CH3)-CH2-CH2-CH3, for example, there are 12 carbon atoms and 9 of them are catenated.

[0022] The term “grafted” is used to indicate that polymeric chains are covalently attached to the porous polymeric substrate. In most embodiments, the polymeric chains are grafted to a carbon atom in the polymeric backbone of the porous polymeric substrate.

[0023] The term “graft density” refers to the millimoles of monomeric units per gram grafted to a substrate. The millimoles are calculated by dividing the mass gain by the molecular weight of the monomer and multiplying by 1000. This value is then normalized by dividing by the original mass of the substrate (grams). The graft density is expressed as millimoles of monomeric units grafted per gram of substrate (mmoles / gram). For clarity, the material that is grafted is typically a polymeric material containing a plurality of monomeric units.

[0024] The term “pKa” refers to the acid dissociation constant. It indicates how easily a proton is released from a molecule.

[0025] The terms “polymer” and “polymeric material” are used interchangeably and refer to materials formed by reacting one or more monomers. The terms include homopolymers, copolymers, terpolymers, or the like. Likewise, the terms “polymerize” and “polymerizing” refer to the process of making a polymeric material that can be a homopolymer, copolymer, terpolymer, or the like.

[0026] The terms “in a range of” or “ranging from” are used interchangeably to refer to all values within the range plus the endpoints of the range.

[0027] Also herein, recitation of ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 10 includes 1.4, 1.9, 2.33, 5.75, 9.98, etc.).

[0028] Also herein, recitation of “at least one” includes all numbers of one and greater (e.g., at least 2, at least 4, at least 6, at least 8, at least 10, at least 25, at least 50, at least 100, etc.).

[0029] As used herein, “comprises at least one of” A, B, and C refers to element A by itself, element B by itself, element C by itself, A and B, A and C, B and C, and a combination of all three.

[0030] In the present disclosure, it has been found that a solid phase comprising a multimodal ligand, preferably an aliphatic multimodal ligand, comprising a nitrogen-containing heterocycle having a pKa value of 3.5 to 9.5 can be used to selectively separate nucleic acids with varying the pH and / or ionic strength of the requisite solutions.

[0031] The separation method of the present invention employs a solid phase comprising a multimodal ligand. The multimodal ligand comprises at least one nitrogen-containing heterocycle as described below. In a preferred embodiment, the solid phase is a support matrix having the multimodal ligands grafted thereto.

[0032] The multimodal ligands are often grafted to a substrate that is a solid. Hence, the term “solid phase” means that the substrate is not a liquid and is not dissolved in a solution. Small particles grafted with the multimodal ligands suspended in a liquid are not considered to be dissolved in the liquid. That is, a suspension is not considered to be a solution herein and the suspended particles are solids as the term is used herein. In many embodiments, however, the solid phase is not a small particle but is selected to have a larger form such as those described further below.

[0033] In some embodiments, the solid phase is a porous polymeric substrate with the multimodal ligand attached thereon. The pores of the polymeric substrate can have any desired average size. In some embodiments, the pores are macro-porous, mesoporous, microporous, or a mixture thereof. As used herein, the term “macro-porous” refers to a polymeric substrate having pores with diameters greater than 50 nanometers, the term “meso-porous” refers a polymericsubstrate having pores with diameters in a range of 2 nanometers to 50 nanometers, and the term “micro-porous” refers to a material having pores with diameters less than 2 nanometers.

[0034] The porous polymeric substrate can have any desired size, shape, and form. For example, the porous polymeric substrate can be in the form of particles, fibers, films, non-woven webs, woven webs, membranes, sponges, or sheets. In some examples, the polymeric substrate is a porous membrane or a porous non-woven web. To prepare large separation articles or many separation articles and for ease of manufacturing, the polymeric substrate can be in the form of or formed from a roll such as a roll of a film, non-woven web, woven web, membrane, sponge, or sheet. This allows the use of roll-to-roll processing to prepare the separation articles. The porous polymeric substrate can include a single layer or multiple layers of the same or different polymeric materials.

[0035] The porous polymeric substrate is often formed from a thermoplastic material. Suitable thermoplastics include, but are not limited to, polyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates) and copolymers thereof such as poly(ethylene)-co- poly(vinyl acetate), polyesters such as poly(lactic acid), poly(vinyl alcohol) and copolymers thereof such as poly(ethylene)–co-poly(vinyl alcohol), poly(vinyl esters), poly(vinyl ethers), poly(carbonates), polyurethanes, poly((meth)acrylates) and copolymers thereof, and combinations thereof.

[0036] Suitable polyolefins for the porous polymeric substrate include poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, alpha olefin copolymers (such as copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and / or 1- decene), poly(ethylene-co-1-butene), poly(ethylene-co-1-butene-co-1-hexene), poly(butadiene) and copolymers thereof, and combinations thereof.

[0037] Suitable fluorinated polymers for the porous polymeric substrate include poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (such as poly(vinylidene fluoride-co-hexafluoropropylene)), copolymers of chlorotrifluoroethylene (such as poly(ethylene- co-chlorotrifluoroethylene)), and combinations thereof.

[0038] Suitable polyamides for the porous polymeric substrate include various nylon compositions such as, for example, poly(iminoadipoyliminohexamethylene), poly(iminoadipoyliminodecamethylene), polycaprolactam, and combinations thereof. Suitable polyimides include poly(pyromellitimide), and combinations thereof.

[0039] Suitable poly(ether sulfones) for the porous polymeric substrate include poly(diphenylether sulfone), poly(diphenylsulfone-co-diphenylene oxide sulfone), and combinations thereof.

[0040] Suitable copolymers of vinyl acetate for the porous polymeric substrate include copolymers of ethylene and vinyl acetate as well as terpolymers of vinyl acetate, vinyl alcohol, and ethylene.

[0041] In some embodiments, the porous polymeric substrate is a porous membrane having an average pore size (average longest diameter of the pore) that is often greater than 0.1 micrometer to minimize size exclusion separations, minimize diffusion constraints, and maximize surface area and separation. Generally, the average pore size can be in the range of 0.1 to 10 micrometers. For example, the average pore size is at least 0.2, 0.4, 0.6, or even 0.8 micrometers and up to 8, 6, 4, or even 2 micrometers.

[0042] The porous polymeric substrate can be a macro-porous membrane such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are often prepared by forming a solution of a thermoplastic material and a second material above the melting point of the thermoplastic material. Upon cooling, the thermoplastic material crystallizes and phase separates from the second material. The crystallized material is often stretched. The second material is optionally removed either before or after stretching. Some exemplary TIPS membranes include poly(vinylidene fluoride) (PVDF), polyolefins such as poly(ethylene) or poly(propylene), vinyl- containing polymers or copolymers such as ethylene-vinyl alcohol copolymers and butadiene- containing polymers or copolymers, and (meth)acrylate-containing polymers or copolymers.

[0043] In some embodiments, the porous polymeric substrate can be a macro-porous membrane such as a solvent induced phase separation (SIPS) membrane, for example, a nylon macro-porous film.

[0044] In other embodiments, the porous polymeric substrate can be a nonwoven web, which can include nonwoven webs manufactured by any of the commonly known processes for producing nonwoven webs. As used herein, the term “nonwoven web” refers to a fabric that has a structure of individual fibers or filaments that are randomly and / or unidirectionally interlaid in a mat-like fashion. For example, the fibrous nonwoven web can be made by wet laid, carded, air laid, spunlaced, spunbonding, or melt-blowing techniques, or combinations thereof. Spunbonded fibers are typically small diameter fibers that are formed by extruding molten thermoplastic polymer as filaments from a plurality of fine, usually circular capillaries of a spinneret, with the diameter of the extruded fibers being rapidly reduced. Melt-blown fibers are typically formed by extruding molten thermoplastic material through a plurality of fine, usually circular, die capillaries as molten threads or filaments into a high velocity, usually heated gas (for example, air) stream, which attenuates the filaments of molten thermoplastic material to reduce their diameter. Thereafter, the melt-blown fibers are carried by the high velocity gas stream and are deposited on a collecting surface to form a web of randomly dispersed, melt-blown fibers. Any of thenonwoven webs can be made from a single type of fiber or from two or more fibers that differ in the type of thermoplastic polymer and / or thickness.

[0045] The porosity of nonwoven substrates is typically characterized by properties such as fiber diameter, or basis weight, or solidity, rather than by pore size. The fibers of the nonwoven substrate are typically microfibers having an effective fiber diameter of at least 0.5, 1, 2, or even 4 micrometers and at most 15, 10, 8, or even 6 micrometers as known in the art. The nonwoven substrate preferably has a basis weight in the range of at least 5, 10, 20, or even 50 g / m2; and at most 800, 600, 400, 200, or even 100 g / m2. The minimum tensile strength of the nonwoven web is about 4.0 Newtons. It is generally recognized that the tensile strength of nonwoven substrates is lower in the machine direction than in the cross-web direction due to better fiber bonding and entanglement in the latter.

[0046] In some embodiments, the solid phase is a solid support. The solid support may comprise organic polymers (e.g. plastics) or inorganic materials or combinations of both. Examples of suitable solid support materials include metal oxides such as Al2O3, TiO2, ZrO2, Ta2O3; as well as silica materials such as SiO2and polysilicic acid. The solid supports can be magnetic materials such as iron, cobalt or nickel and oxides, alloys, ceramics or amalgams thereof. Suitable organic polymers include polystyrene, poly(meth)acryl polymers includingpoly(meth)acrylates and poly(meth)acrylamides, polyurethanes, polyamides such as nylon;polyolefins, such as polyethylene, polypropylene, polybutadiene, and copolymers thereof. Other solid support materials include polysaccharides, and in particular hydrogels such as agarose, cellulose, dextran, those available under the trade designation “SEPHADEX” or “SEPHACRYL” from SigmaAldrich, and chitosan. Inorganic supports include, for example, glass or metal surfaces such as gold. In some embodiments, the ligands described herein (e.g. covalently) bond to the solid support material. The solid support may be in the shape of a sphere or spheroid (such as a bead). Such solid supports and their grafting procedures are disclosed in WO 2023 / 084370 (Watts et al.), herein incorporated by reference.

[0047] In some embodiments, the solid phase is provided in a housing to facilitate performance of chromatography.

[0048] Within the meaning of the present invention, the term “multimodal” is understood to mean that the ligands interact with the target nucleic acid species via two or more different chemical and / or physical mechanisms. Examples of these interactions include ionic interactions, hydrogen bonding interactions, hydrophobic interactions, ion-dipole interactions, and cation-π- interactions.

[0049] The multimodal ligand of the present disclosure comprises a nitrogen-containing heterocycle and has a pKa value in the range of 3.5 or 4.0 to 9 or 9.5. In some embodiments, the nitrogen-containing heterocycle has a pKa value of at least 3.5, 4.0, 4.5, 5.0, 5.5. or even 6.0. In some embodiments, the nitrogen-containing heterocycle has a pKa value of at most 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or even 9. The pKa value of the multimodal ligand can, for example, be calculated theoretically.

[0050] The method and software used to calculate the pKa value are provided in the Example section below. The calculations can be used to quickly assess whether the multimodal ligand has a pH within an acceptable range. The estimates of error in calculation of the pKa value based on Hammett-Taft methods are often dependent on the structural similarity of a novel material to those present in the model database. Estimations are useful for biological applications as the error is usually smaller than the range of acceptable pH values for the application. Thus, the calculated pKa values allow for rapid selection of a limited number of potential ligands for evaluation.

[0051] Literature articles that provide information about the calculation of pKa values include, for example, J. R. Greenwood et al., “Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution”, Journal of Computer-Aided Molecular Design, 2010, 24, 591-604 and J. C. Shelley et al., “Epik: a software program for pKaprediction and protonation state generation for drug-like molecules”, Journal of Computer-Aided Molecular Design, 2007, 21, 681-691.

[0052] In some embodiments, the multimodal ligand comprises a nitrogen-containing heterocycle, which is not aromatic. In some embodiments, the nitrogen-containing heterocycle comprises a piperazine or morpholine moiety.

[0053] In some embodiments, the multimodal ligand comprises a unit according to formula (I): Where R4and R5arehaving at least 2 carbon atoms, wherein a sum of ring atoms in a ring group consisting of nitrogen, R4, Q, and R5is either 6 or 7; Q has a single catenated atom and is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to -N(R6)- when N is used to attach the ligand to the solid phase typically through a carbonyl moiety; and R6 is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

[0054] In some embodiments, the multimodal ligand comprises a unit according to formula (IA): (IA) Where R4and R5are each an alkylene having at least 2 carbon atoms, wherein a sum of ring atoms in a ring group consisting of nitrogen, R4, and R5is either 6 or 7; and R6is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

[0055] In some embodiments, the multimodal ligand comprises a unit according to formula (II): Where R2is a (hetero) Z is -NH-(C=O)-, -- , - -NH-R3-, -(C=O)-NH-, and –(C=O)-NH-R3-, where R3is alkylene having at least two catenated carbon atoms; R4and R5are each an alkylene having at least 2 carbon atoms, wherein a sum of ring atoms in a ring group consisting of nitrogen, R4, Q, and R5is either 6 or 7; Q has a single catenated atom and is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to -N(R6)- when Z is -NH-(C=O)-; and R6is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

[0056] In some embodiments, the multimodal ligand is derived from Formula (III)

[0057] In Formula (III),2-O- or -NH-, and R is a (hetero)alkylene. The group Z is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R3-, -(C=O)- NH-, or –(C=O)-NH-R3-, where R3is alkylene having at least 2 catenated carbon atoms. Groups R4and R5are each an alkylene having at least 2 carbon atoms, wherein a sum of ring atoms in a ring group consisting of nitrogen, R4, Q, and R5is either 6 or 7. Group Q has a single catenated atom and is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to -N(R6)-when Z is -NH-(C=O)-. Group R6is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

[0058] The monomers have a (meth)acryloyl group of formula CH2=CR1-(C=O)- where R1is hydrogen or methyl.

[0059] The group X1is either -O- or -NH-. If X1is -O-, the monomer of Formula (III) is a (meth)acrylate but if X is -NH-, the monomer is a (meth)acrylamide.

[0060] Group R2is a (hetero)alkylene. In many embodiments, R2is an alkylene such as one having 1 to 20 carbon atoms. The alkylene R2can have, for example, at least 1, 2, 3, 4, 6, 8, or even 10 carbon atoms and up to 20, 18, 16, 14, 12, 10, 8, 6, or even 4 carbon atoms. In other embodiments, R2is of formula -R-O-R- where each R is an alkylene having 2 to 10 catenated carbon atoms. The alkylene R can have at least 2, 3, or even 4 and up to 10, 8, 6, or even 4 catenated carbon atoms.

[0061] The group Z is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R3-, -(C=O)-NH-, and – (C=O)-NH-R3-, where R3is alkylene having at least 2 catenated carbon atoms. Suitable alkylene groups often have 2 to 10 carbon atoms such as at least 2, at least 3, at least 4 and up to 10, up to 8, up to 6, or up to 4 carbon atoms. As the Z group can form hydrogen bonds, in some embodiments it may be useful for providing an additional mode of interaction (in other words, not ionic bonding interactions), which can be used to provide additional binding selectivity of the ligands to the nucleic acids.

[0062] Although the sum of catenated atoms in R2plus Z can be as low as three for some uses of the monomers of Formula (III), the sum is preferably greater if the monomers of Formula (III) are used to form separation articles. The sum preferably is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. The sum can be, for example, up to 20 or more, up to 18, up to 16, up to 14, up to 12, up to 10, up to 8, up to 6, or up to 5.

[0063] Groups R4and R5are each an alkylene having at least two carbon atoms. The ring group consisting of nitrogen, R4, Q, and R5has 6 or 7 ring members. Each group R4and R5typically has either 2 or 3 carbon atoms included in atoms that form the ring (e.g., catenated carbon atoms) but there can be additional carbon atoms in R4and R5that are not ring atoms (e.g., non-catenated carbon atoms). The sum of ring carbon atoms (i.e., catenated atoms) in R4and R5is either 4 or 5. The ring group consisting of nitrogen, R4, Q, and R5is typically saturated (i.e., there are no carbon-carbon double bonds).

[0064] Group Q is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to - N(R6)- when Z is -NH-(C=O)-. When Q is -O-, -S-, -S(=O)-, or -S(=O)2-, the nitrogen atom that is part of the same ring and that is attached to group Z is selected so that it can be protonated.When Q is -N(R6)-, there can be one or two protonated groups in the monomer of Formula (III). Group Q has a single catenated atom. That is, group Q contributes a single ring atom (i.e., a single catenated atom) to the ring consisting of nitrogen, R4, Q, and R5. For example, in the Q group -N(R6)-, nitrogen is the ring atom; and in the Q groups –S(=O) and -S(=O)2, sulfur is the ring atom.

[0065] If the nitrogen bonded to group Z is part of a urea group, then the ring has a single group that can be protonated arising from the group -N(R6)-. That is, if Z is equal to -NH-(C=O)-, then Q is equal to -N(R6)-. For example, in the following monomer, which is 2-[[4-(2- hydroxyethyl)piperazine-1-carbonyl]amino]ethyl 2-methylprop-2-enoate (also referred to as IEM / N-(2-hydroxyethyl)piperazine), the nitrogenof a urea linkage group and this nitrogen atom is unlikely to be protonated. The other nitrogen atom in the ring, which is in a group of formula -N(R6)- where R6is a hydroxy-substituted alkyl, is more likely to be protonated.

[0066] If the nitrogen bonded to group Z is not part of a urea group (i.e., Z is not equal to -NH- (C=O)-), then the nitrogen atom bonded to group Z plus the nitrogen in the group -N(R6)- can be protonated. For example, in the following monomer, which is 2-[(4-methylpiperazin-1- yl)carbamoylamino]ethyl 2-methylprop-2-enoate (also referred to as IEM / 1-amino-4- methylpiperazine or IEM / N-methylpiperazine), both nitrogenThe nitrogen attached to Z (-NH-(C=O)- NH-) is not part of a urea linkage but adjacent to such a linkage. The other nitrogen is the ring is the group -N(R6)- where R6is methyl (e.g., an alkyl).

[0067] The group R6in the Q group -N(R6)- is hydrogen, alkyl, or (hetero)aryl. When R6is an alkyl, it can optionally be further substituted with hydroxy, alkoxy, or (hetero)aryl. When R6is a (hetero)aryl, it can optionally be further substituted with hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy. Suitable R6alkyl groups can have 1 to 10 carbon atoms such as at least 1, at least 2, at least 3, at least 4 and up to 10, up to 8, up to 6, or up to 4 carbon atoms. Suitable R6aryl groups usually have six carbon atoms while R6heteroaryl groups often haveeither 5 or 6 ring atoms with 1 or 2 of these ring atoms being a heteroatom and with the remainder ring atoms being carbon. The heteroatoms are usually nitrogen.

[0068] When group R6is an alkyl, it can be unsubstituted or substituted with hydroxy (-OH), alkoxy, or (hetero)aryl group. Suitable alkoxy groups for substitution are of formula -ORawhere Rais an alkyl having 1 to 10 carbon atoms such as at least 1, at least 2, at least 3, at least 4 and up to 10, up to 8, up to 6, or up to 4 carbon atoms. Suitable (hetero)aryl groups for substitution can be an aryl group with six carbon atoms or a heteroaryl group having either 5 or 6 ring atoms with 1 or 2 of these being a heteroatom and with the remainder being carbon. The heteroatoms in the heteroaryl group are usually nitrogen.

[0069] When group R6is a (hetero)aryl, it can be substituted or unsubstituted with hydroxy (- OH), halo (e.g., chloro or bromo), nitro (-NO2), cyano (-CN), trifluoromethyl (-CF3), alkyl, or alkoxy. Suitable alkyl groups often have 1 to 10 carbon atoms such as at least 1, at least 2, at least 3, at least 4 and up to 10, up to 8, up to 6, or up to 4 carbon atoms. Suitable alkoxy groups for substitution are of formula -ORawhere Rais an alkyl having 1 to 10 carbon atoms such as at least 1, at least 2, at least 3, at least 4 and up to 10, up to 8, up to 6, or up to 4 carbon atoms.

[0070] The monomers of Formula (III) can be prepared using any suitable method. Two methods are often used. In the first method, a (meth)acrylate monomer having an isocyanato group is reacted with a cyclic compound that has a reactive -NH- or -NH2group. In the second method, a cyclic compound that has a reactive -NH- or -NH2group is reacted with an alkenylazlactone.

[0071] Multimodal ligands as described above may be synthesized and / or grafted onto a solid phase as disclosed in U.S. Prov. Pat. Appl. No.63 / 476431 (Rasmussen et al.), herein incorporated by reference. Additional multimodal ligands can be synthesized by procedures described in the above mentioned Rasmussen et al. application, including, for example, 2-[(4-ethylpiperazine-1- carbonyl)amino]ethyl 2-methylprop-2-enoate (also referred to as IEM / N-ethylpiperazine) and 2- [(4-propylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (also referred to as IEM / N- propylpiperazine).

[0072] In some embodiments, the multimodal ligand is derived from a piperazine-containing compound and reacted onto a support as described in WO 2023 / 084370 (Watts et al.).

[0073] It has been discovered that the ligands as disclosed herein surprisingly are multimodal and by varying the pH and / or the ionic strength of the various buffers, washes, and eluents, an optimum set of conditions can be found to selectively elute nucleic acids when run at ambient conditions without the use of organic solvents.

[0074] Disclosed herein are three different methods for separating nucleic acids (in other words, separating or purifying a first nucleic acid from a second nucleic acid). Such nucleic acids caninclude single stranded ribonucleic acid, double stranded ribonucleic acid, single stranded deoxyribonucleic acid, and double stranded deoxyribonucleic acid.

[0075] Described for each method below, a sample comprising at least a first nucleic acid and a second nucleic acid is loaded onto the solid phase using a loading buffer. The loading buffer may simply be the solution matrix in which the nucleic acid mixture was generated, or it may be a separately prepared buffer used to adjust the nucleic acid mixture to the desired pH and ionic strength for loading. As used herein, ionic strength is used to described various buffers and solutions. However, conductivity is related to ionic strength, wherein the higher the ionic strength of a solution, the higher its conductivity. Thus, buffer and eluting solutions with higher ionic strength will also have higher conductivity.

[0076] Preferably, the loading buffer is a true buffer, wherein the solution can resist changes to pH upon the addition of an acid or a base. After the sample is loaded, optionally, a washing solution can be passed through the sample loaded on the solid phase to rinse away any non-bound components. The washing solution is preferably a buffer, however in some embodiments, it may not be a buffer. A first eluting solution is then passed through the sample loaded on the solid phase and at least one nucleic acid is eluted. In some embodiments, as described below, a second eluting solution is then passed through the solid phase and another nucleic acid is eluted.

[0077] The sample comprising the nucleic acids may vary widely within the scope of the process, depending upon how it was generated. For example, the sample may be the result of an enzymatic in vitro transcription reaction. The sample may be the result of enzymatic in vitro transcription further processed with enzymatic modification including 3’ polyadenylation, or 5’ capping. Alternatively, the sample may be produced by lysis of a cell suspension, cell pellet, or cell paste, or the sample may be from a chemical synthesis or chemical modification. The sample may be used as obtained or may be subjected to a pre-purification process to remove at least some of cells, cell debris, proteins, and other non-nucleic acid molecules. Thus, the sample may comprise one or more single stranded RNA molecules, one or more single stranded DNA molecules, double stranded DNA molecules, double stranded RNA molecules, and plasmid DNAs as the other nucleic acid species. The sample may also contain other non-nucleic acid components, such as proteins, lipids, carbohydrates, nucleotides, etc., which are derived from the cells or media used to generate the mixture. In a further preferred embodiment, at least one of the nucleic acids targeted for purification is a single stranded mRNA or RNA transcript.

[0078] The size and topology of the target nucleic acid (in other words, the nucleic acid desired to be separated, isolated, or purified) may also vary widely within the scope of the invention. In some embodiments, the nucleic acid may comprise at least 500, 1000, 1500, or even 2000 nucleotide bases or base pairs to at most 25000, 20000, 15000, 10000, or even 8000 nucleotidebases or base pairs. Similarly, the topological structure of the target nucleic acid is not particularly limited and may be present in different conformations, such as linear, circular, hairpin, supercoiled, etc.

[0079] The solutions and buffers used for loading, optional washing, and / or eluting are not particularly limited and conventional buffers used for biological liquid chromatography can be used as long as they satisfy the below described pH and / or ionic strength conditions. Some suitable buffers include for example citrate buffer (sodium citrate and citric acid monohydrate), acetate buffer, and TE buffer as further described in the examples; phosphate-buffered saline (PBS); N-2-acetamido-2-aminoethanesulfonic acid (ACES); N-2-acetamido-2-iminodiacetic acid (ADA); amino methyl propanediol (AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO); N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES); N,N-bis-2-hydroxyethylglycine (BICINE); bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris); 1,3-bistrishydroxymethylmethylaminopropane (BIS-TRIS Propane); 4- cyclohexylamino-1-butane sulfonic acid (CABS); 3-cyclohexylamino-1-propane sulfonic acid (CAPS); 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO); 2-N- cyclohexylaminoethanesulfonic acid (CHES); 3-N,N-bis-2-hydroxyethylamino-2- hydroxypropanesulfonic acid (DIPSO); N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS); N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS); N-2- hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES); N-2-hydroxyethylpiperazine-N-2- propanesulfonic acid (HEPPSO); 2-N-morpholinoethanesulfonic acid (MES); 4-N- morpholinobutanesulfonic acid (MOBS); 3-N-morpholinopropanesulfonic acid (MOPS); 3-N- morpholino-2-hydroxypropanesulfonic acid (MOPSO); piperazine-N-N-bis-2-ethanesulfonic acid (PIPES); piperazine-N-N-bis-2-hydroxypropanesulfonic acid (POPSO); N-trishydroxymethyl- methyl-4-aminobutanesulfonic acid (TABS); N-trishydroxymethyl-methyl-3- aminopropanesulfonic acid (TAPS); 3-N-trishydroxymethyl-methylamino-2- hydroxypropanesulfonic acid (TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES); N-trishydroxymethylmethylglycine (TRICINE); trishydroxymethylaminomethane (TRIS); histidine and polyhistidine; imidazole and derivatives thereof; triethanolamine dimers, oligomers and polymers; and di / tri / oligo amino acids, for example Gly-Gly; and Ser-Ser, Gly-Gly-Gly, and Ser-Gly. Additional additives may be used to adjust for example, the ionic strength. Typically, such additives would include salts that do not impact the pH of the solution. Such salts include NaCl, EDTA, MgCl2, (NH4)2SO4, Na2SO4, KCl and CH3COONH4. Advantageously, the solutions and buffers used for loading, optional washing, and / or eluting are substantially free (e.g., less than 1 volume % or even not added) of organic solvents. Exemplary organic solvents include alcohols (such as isopropanol, or ethanol), acetonitrile, etc.

[0080] In all of the methods described below, a sample comprising a plurality of nucleic acids is loaded onto a solid phase comprising a multimodal ligand as described herein. The plurality of nucleic acids comprise at least a first nucleic acid and a second nucleic acid that is different from the first nucleic acid. The methods described below are various ways to separate the first nucleic acid from the second nucleic acid.

[0081] Method 1

[0082] In the first method, the plurality of nucleic acids is loaded onto a solid phase disclosed herein. The sample is loaded onto the solid phase in a solution that has a first pH that is below or near the pKa of the nitrogen-containing heterocycle and a first ionic strength, which is sufficiently low to bind (or retain) one or more nucleic acids.

[0083] The sample is loaded onto the solid phase and is optionally washed with a wash solution. As the first and second nucleic acids bind (or are retained within) the solid phase, they will become bound to the multimodal ligands, while other molecules may pass through the solid phase interstitial spaces or become bound to the solid phase. Preferably, a washing solution is used to flush any unretained molecules from the interstitial spaces of the solid phase.

[0084] The sample is optionally washed with a washing solution, which also has a pH that is below or near than the pKa and a first ionic strength value, which is sufficiently low to bind one or more nucleic acids. The washing solution may be a buffer and, in some embodiments, the washing solution is the same as the loading buffer.

[0085] The loading and washing solutions are adjusted such that the nucleic acids are bound to the solid phase. The pKa value of the ligand is the pH at which the protonated and the unprotonated form of the ligand are in equal molar ratios. Thus, in the case of the loading buffer and washing solution, the pH of these solutions is kept at or below the pKa, so that a majority of the ligands are in the positively-charged (or protonated) form. In this form, it is believed that the positive charge of the nitrogen-containing heterocycles comprised in the multimodal ligands on the solid phase interact with the backbone of nucleic acids, specifically with the negatively charged phosphate backbone. In some embodiments, the pH of the loading buffer is between 3.5 to 9.5, for example 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or even 5.5; and at most 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5, 8.6, 8.8, 9.0, 9.2, 9.4, or even 9.5.

[0086] In addition to the pH, which controls the charge (positive versus neutral) of the multimodal ligands and thus the ionic interactions of the solid phase, the salt concentration (or ionic strength) can be adjusted to impact both the ionic interactions and the secondary interactions occurring between the nucleic acids and the multimodal ligands. The ionic strength of the loading and washing solutions are kept low in method 1 to enable both the first and second nucleic acidsto bind to the multimodal ligand. In some embodiments, the ionic strength of the loading buffer and optional wash solution is no more than 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.01, 0.005, or even 0.002 mole / liter (M). The loading buffer may comprise no additional additives, which contribute to the ionic strength, therefore, the loading buffer may have a low ionic strength, for example of less than 0.1, 0.05, 0.005, or even 0.002 M, however, if a buffer salt is used, the ionic strength will be that contributed by the buffer salt itself, for example 100, or even 50 mM (millimolar). In some embodiments, the loading buffer has a conductivity of no more than 20, 10, or even 5 mS / cm (milliSeimens / centimeter). Conductivity can be measured using techniques known in the art, for example using a conductivity meter.

[0087] A first eluting solution then is used to selectively release the first nucleic acid from the solid phase, while leaving the second nucleic acid bound. This is accomplished by using a high salt concentration solution. The first eluting solution comprises an ionic strength, which is higher than the ionic strength used for the loading and optional washing step. In some embodiments, the first eluting solution has an ionic strength of at least 0.2, 0.5, 1, or even 1.5 M. In some embodiments, the first eluting buffer has a conductivity of at least 30, 50, 70, or even 100 mS / cm. Optionally, the pH of the first eluting solution may be increased. For example, the pH may be higher than the pKa of the multimodal ligand. For example, the pH of the first elution solution is at least 1, 1.5, 2, 2.5, or even 3 units above the pKa. In some embodiments, the pH is at least 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.8, 7.0, or even 7.2; and at most 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, or even 10.0. In method 1, the first eluting solution should selectively elute the first nucleic acid, while the second nucleic acid remains bound. In one embodiment of the present disclosure, it has surprisingly been found that a low pH, high ionic strength first eluting solution will selectively elute double stranded RNA, leaving the majority of single stranded RNA bound to the solid phase.

[0088] Then, a second eluting solution is used to selectively release the second nucleic acid from the solid phase. The second eluting solution has an ionic strength lower than that of the first eluting solution. In some embodiments, the second eluting solution has an ionic strength of at least 20mM and at most 1, 0.8, 0.6, 0.5, 0.4, 0.2, 0.1, 0.08, 0.06, or even 0.05 M. In some embodiments, second eluting solution has a conductivity of no more than 20, 10, or even 5 mS / cm. The pH of the second eluting solution is increased, such that it is higher than the pKa. In some embodiments, the pH is at least 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.8, 7.0, or even 7.2; and at most 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, or even 10.0. In method 1, the second eluting solution should selectively elute the second nucleic acid.

[0089] In one embodiment of the present disclosure, method 1 may be used to separate the first nucleic acid of double stranded RNA from the second nucleic acid of single stranded RNA.

[0090] Method 2

[0091] In the second method, the plurality of nucleic acids is loaded onto a solid phase disclosed herein. The sample is loaded onto the solid phase with a loading buffer that has a pH that is below or near than the pKa of the nitrogen-containing heterocycle and an ionic strength, which is sufficiently high to enable a first nucleic acid or group of nucleic acids to flow through, while binding a second nucleic acid or group of nucleic acids.

[0092] The sample is optionally washed with a washing solution that also has a pH that is below or near than the pKa of the nitrogen-containing heterocycle and an ionic strength, which is sufficiently high to bind (or retain) a second nucleic acid. The washing solution may be a buffer, and in some embodiments, the washing solution is the same as the loading buffer.

[0093] In this method, the nucleic acid of interest is bound to the solid phase, while other nucleic acids and contaminates are not retained on the solid phase. Thus, the loading and washing solutions are adjusted such that the second nucleic acid is bound to the solid phase. In some embodiments, the pH of the loading buffer and optional washing solution is between 3.5 to 9.5, for example 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or even 5.5; and at most 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5, 8.6, 8.8, 9.0, 9.2, 9.4, or even 9.5. However, unlike Method 1, the ionic strength of the loading buffer is increased so as to not retain a first nucleic acid. In some embodiments, the loading buffer and optional washing solution has an ionic strength of at least 0.05, 0.1, 0.2, 0.5, 1, or even 1.5 M. In some embodiments, the loading buffer has a conductivity of at least 30, 50, 70, or even 100 mS / cm.

[0094] The sample is loaded onto the solid phase and is optionally washed with wash solution. As the first and second nucleic acids pass through the solid phase, the first nucleic acid is not retained and will pass through the solid phase, while the second nucleic acid becomes bound to the multimodal ligands. Preferably, a washing solution is used to flush any unretained molecules from the solid phase.

[0095] An eluting solution then is used to selective release the second nucleic acid from the solid phase. The eluting solution comprises an ionic strength lower than that of the loading buffer or washing solution. In some embodiments, the ionic strength of the eluting solution is no more than 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, or even 0.04 M, or even having no ionic strength. In some embodiments, the eluting solution has a conductivity of no more than 20, 10, or even 5 mS / cm. The pH of the eluting solution may optionally be adjusted to be higher than the pKa. For example, the pH of the eluting solution may be at least 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.8, 7.0, or even 7.2; and at most 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, or even 10.0. The second nucleic acid is then released from the solid phase with the eluting solution.

[0096] In one embodiment of the present disclosure, method 2 may be used to separate the first nucleic acid of double stranded RNA from the second nucleic acid of single stranded RNA.

[0097] Method 3

[0098] In the third method, the plurality of nucleic acids is loaded onto a solid phase disclosed herein. The sample is loaded onto the solid phase with a loading buffer that has a pH that is above the pKa of the nitrogen-containing heterocycle and an ionic strength, which is sufficiently low to bind (or retain) one or more nucleic acids.

[0099] The sample is optionally washed with a washing solution that also has a pH that is above the pKa and an ionic strength value, which is sufficiently low such that the first nucleic acid is not retained on the solid phase while a second nucleic acid is bound to the solid phase. In some embodiments, the ionic strength of the loading buffer and optional washing solution is no more than 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, or even 0.04 M. In some embodiments, the washing solution has a conductivity of no more than 20, 10, or even 5 mS / cm. The pH of the eluting solution is higher than the pKa. For example, the pH of the loading buffer and optional washing solution may be at least 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.8, 7.0, or even 7.2; and at most 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, or even 10.0.

[0100] The washing solution may be a buffer, and in some embodiments, the washing solution is the same as the loading buffer.

[0101] In this method, a nucleic acid of interest is bound to the solid phase, while another nucleic acid and other undesired components are not retained on the solid phase. Thus, the loading and washing solutions are adjusted such that the second nucleic acid is bound to the solid phase. The eluting solution is used to selective release the second nucleic acid from the solid phase. The eluting solution comprises a pH, which is higher that the pH of the loading buffer or washing solution. In some embodiments, the pH of the eluting solution is at least 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, or even 3.5 pH units higher that the pH of the loading and washing solutions, whichever is higher. In some embodiments, the pH of the eluting solution is at least 7, 7.2, 7.3, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or even 9.0 and at most 9.5, 9.8, 10.0, 10.2, 10.4, 10.6, 10.8, 11.0, 11.2, 11.4, 11.6, 11.8, or even 12.0.

[0102] In one embodiment of the present disclosure, method 3 may be used to separate the first nucleic acid of double stranded RNA from the second nucleic acid of single stranded RNA.EXAMPLES

[0103] Unless otherwise noted, all parts, percentages, ratios, etc. in the examples and the rest of the specification are by weight, and all reagents used in the examples were obtained, or are available, from general chemical suppliers such as, for example, Sigma-Aldrich Company, Saint Louis, Missouri, or may be synthesized by conventional methods.

[0104] The following abbreviations are used below: cm = centimeter, g = gram, kg = kilograms, M = molar, min = minute, mL = milliliter, mm = millimeter, mM = millimolar, ng = nanogram, µl = microliter, and µL = microliter. TABLE 1. Materials List Material Description IEM / N-methylpiperazine Example 2 disclosed in U.S. Prov. Pat. Appl. No.63 / 476431 IEM / N-(2- Example 1 disclosed in U.S. Prov. Pat. Appl. No.63 / 476431 hydroxyethyl)piperazine IEM / N-(3- Example 4 disclosed in U.S. Prov. Pat. Appl. No.63 / 476431 aminopropyl)morpholine HEMA 2-hydroxyethylmethacrylate available from Sigma-Aldrich Tris Tris(hydroxymethyl)aminomethane available from Invitrogen Corp., Waltham, MA EDTA 2-({2-[bis(carboxymethyl)amino]ethyl}(carboxymethyl) amino)acetic acid dihydrate tetrasodium (Cat. No. AM9260G) available from Thermo Fisher Nylon membrane reinforced nylon 6,6 membrane, 0.8 micrometer nominal pore size, #080ZN, obtained from the 3M Co., St. Paul, MN Well plate 96-well solid phase extraction plate, available under the trade designation “EMPORE” from 3M Co. Deep well plate 96-well plate, RNase / DNase free, sterile, with 2-mL wells (Cat. No. 260251) used to collect eluate available from ThermoFisher NTPs Nucleoside triphosphates, in vitro transcription reaction components ordered in bulk from New England Biolabs, Ipswich, MA (Cat. Nos. N0451B, N0452B, N0453B, N0454B) T7 RNA polymerase Enzyme present in IVT reaction ordered in bulk (Cat. No. M0251B) available from New England BiolabsInorganic pyrophosphatase Enzyme present in IVT reaction (Cat. No. M2403L) available from New England Biolabs 5X IVT buffer Buffer made in-house comprised of 200 mM Tris-HCl pH 8, 200 mM, 50 mM sodium acetate, 375 mM magnesium acetate, 1 mM spermidine DTT 1,4-dithiothreitol available from Sigma Aldrich. IVT template plasmid pT7CFE1-CGFP-HA-His plasmid used to produce RNA (Cat. No. 88869) available from Thermo Fisher FastDigest BcuI Restriction enzyme (Cat. No. FD1254) available from Thermo Fisher Transcription kit In Vitro transcription kit (Cat. No. E2050SB) available under the trade designation “HiScribe T7 Quick High Yield RNA Synthesis Kit” from New England Biolabs Assay Kit 1 RNA quantification kit (Cat. No. Q10211) available under the trade designation “Qubit RNA BR Assay Kit” from Invitrogen Assay Kit 2 RNA quantification kit (Cat. No. Q10213) available under the trade designation “Quant-iT RNA Assay kit, Broad Range” from Invitrogen LiCl Precipitation Solution 7.5 M, (Cat. No. AM9480) used to precipitate RNA for crude purification after IVT available from Thermo Fisher PCR master mix solution Cat. No. M0492L, available under the trade designation “Q5 High Fidelity 2X Master Mix” from New England Biolabs CIMac PrimaS AEX column Multimodal chromatography column available from Sartorius Stedim Biotech Cleanup kit Kit used for desalting / purification of PCR reactions available under the trade designation “QIAquick PCR Purification Kit” from Qiagen chromatography system Preparative chromatography system available under the tade designation “AKTA Avant chromatography system” from Cytiva, Chicago, IL Beads Magnetic beads with surface carboxylic acid groups obtained under the trade designation “Dynabeads MyOne Carboxylic Acid” Thermo Fisher Scientific Inc., Waltham, MA EDC N-ethyl-N’-(3- dimethylaminopropyl)carbodiimide hydrochloride available from Oakwood Chemical, Estill, SCN-methylpiperazine solution 120 micromole / milliliter stock solution of N-methylpiperazine (available from Sigma-Aldrich Co.) made in MES buffer MES buffer Morpholinoethylsulfonate buffer made in-house from the solid, 25 mM, pH 6 available from EMD Biosciences, Inc., La Jolla, CA PBS Phosphate-buffered saline solution (concentration: 1x), (pH 7.4), Invitrogen cDNA Synthesis Kit a kit available under the trade designation “ProtoScript II First Strand cDNA Synthesis Kit” (Cat. No. E6560L) with an oligo d(T) primer from New England Biolabs RNA Cleanup Kit a kit available under the trade designation “Monarch RNA Cleanup Kit” (Cat. No. T2040L) from New England Biolabs RNase H Ribonuclease H (Cat. No. M0297S) available from New England Biolabs RNase A Ribonuclease available under the trade designation “Monarch RNase A” (Cat. No. T3018L) from New England Biolabs RT-qPCR master mix A one step qPCR master mix available under the trade designation “PrimeTime One-Step RT-qPCR Master Mix” (Cat. No. 1000706) from Integrated DNA Technologies, Coralville, IA Gene Expression Master Mix A pre-formulated hot-start qPCR master mix available under the trade designation “PrimeTime Gene Expression Master Mix” (Cat. No.1055772) from Integrated DNA Technologies,

[0105] Calculation of pKa

[0106] The pKa values were calculated using the EpiK pKa prediction tool with the Maestro GUI, both obtained from Schrödinger LLC (New York, NY). Computations were performed on the monomeric species with Maestro version 2021-4 set to “Sequential pKa Values” with H2O as the solvent to a pH of 9 and allowed tautomerization. Hammett and Taft relations were used by the EpiK pKa prediction tool to predict protonation states based on functional group structure and sensitivity to perturbations from the rest of the molecule. The model is empirically driven and trained on a combination of publicly reported pKa values and proprietary internal measurements performed by Schrodinger.

[0107] The Hammett and Taft Equation (Equation 1) assumes that each chemical group has a base pKa ( ) that is then modulated by a correction factor ( , Equation 2) as well as aperturbility term ( ) for the target constituent group and influence constants ( ) for othersubstituent groups in the molecule.Equation 1 Equation 2 In the equations, denotes equivalent H atoms from an acidic molecule whileH to the conjugate base. RA is an adjustment term for aliphatic rings. Uncertainty for the pKa value is calculated from the uncertainties of and . Novel molecules with low coverage for and have a default uncertainty of 2.0 pKa units.

[0108] The calculated pKa values for the ligands used in the examples below are provided in Table 1. Table 1. Monomers and Calculated pKa Values pKa Monomer Structure Abbreviated Monomer Name Value + / - IEM / N-methylpiperazine 7.46 2.22 IEM / N-(2- 7.02 2.22 hydroxyethyl)piperazine IEM / N-(3- 7.42 0.67 aminopropyl)morpholine

[0109] Method A. Preparation of Polymer Grafted Membranes:

[0110] Grafting solutions (5 grams each) of monomers were prepared at various monomer concentrations in deionized water, based on the measured % solids of the monomer solution. Each monomer solution also contained 3-carboxybenzophenone, sodium salt (62.5 microliters ofa 0.033 g / mL aqueous solution). For each grafting solution, a nylon membrane was placed on a sheet of polyester film, and sufficient grafting solution was pipetted onto the top surface of the substrate to completely wet the substrate. The coating solution was allowed to soak into the substrate for about 1 minute, and then a second sheet of polyester film was placed on top of the substrate. A 2.28 kg cylindrical weight was rolled over the top of the resulting three-layer sandwich to squeeze out excess coating solution. Ultraviolet (UV)-initiated grafting was conducted by irradiating the sandwich using a UV stand (Classic Manufacturing, Inc., Oakdale, MN) equipped with 18 bulbs (Sylvania RG240W F40 / 350BL / ECO, 10 above and 8 below the substrate, 1.17 meters (46 inches) long, spaced 5.1 cm (2 inches) on center), with an irradiation time of 15 minutes. The polyester sheets were removed and the resulting grafted membrane was placed in a polyethylene bottle. The bottle was filled with 0.9% saline solution, sealed and placed on a laboratory bottle roller for 30 minutes to wash off any residual monomer or ungrafted polymer. The saline solution was poured off and replaced with deionized water for an additional 30 minutes of washing. Washing was repeated with fresh 0.9% saline solution for 30 minutes, followed by 30 minutes of washing with deionized water (2 times). Following the wash steps, the polymer grafted membrane was air dried. Graft density of the polymer grafted membrane was estimated based on mass gain.

[0111] Method B. Preparation of ssRNA:

[0112] In vitro transcription reactions for ssRNA production were assembled beginning from a 5X IVT reaction master mix. DTT, IVT template plasmid, T7 RNA polymerase, Inorganic pyrophosphatase, and NTPs were added fresh immediately prior to initiation of the reaction. Reactions were incubated at 37°C in a water bath for 6 hours. RNA yield was measured using either the Assay Kit 1 or Assay Kit 2. Crude purification of IVT reactions was done by LiCl precipitation prior to experimentation. Briefly, 2.5 reaction volumes of water was added, followed by 1.25 reaction volumes of 7 M LiCl. After thorough mixing the solution was incubated at -20°C for a minimum of 30 minutes. The RNA was pelleted by centrifugation at 17,000xg for 15 min at 4°C. The pellet was washed with 20 reaction volumes of ice cold 70% ethanol and dried overnight before resuspension in pH 8 Tris-EDTA buffer (40mM Tris and 1 mM EDTA).

[0113] Method C. Preparation of dsRNA:

[0114] For generation of dsRNA used in the samples below, the IVT template plasmid was PCR amplified using primer sets to generate PCR products with a T7 promoter for both complementary plasmid DNA strands (at the 5’ end of the original forward strand, and at the 5’ end of the original reverse strand). PCR was performed using the PCR master mix solution according to the manufacturer’s protocol. PCR products were purified using the cleanup kit according to the manufacturer’s protocol. Forward and reverse products were then used as theDNA template to generate RNA using the transcription kit according to the manufacturer’s protocol. The resulting RNA products were purified by LiCl precipitation as described for Method B. Purified Forward and Reverse RNA were mixed in equal molar concentrations in annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA). Mixtures were heated at 95°C for two minutes in a heat block. The heat block was removed from the heating element and allowed to slowly cool to room temperature. Products were analyzed by gel electrophoresis using a non-denaturing 1% agarose gel and Bioanalyzer (Agilent Technologies, Inc. Santa Clara, CA) to confirm the formation of dsRNA.

[0115] Method D. Preparation of single-stranded DNA (ssDNA):

[0116] cDNA (complementary DNA) was prepared using a cDNA Synthesis Kit, following the manufacturer’s instructions in a final volume of 1 mL. The RNA template used for cDNA synthesis was ssRNA (synthesis above) that was LiCl precipitated then further purified using an RNA Cleanup Kit. Synthesized cDNA was treated with RNase H following the manufacturers protocol for 30 min at 37°C to digest RNA bound to DNA. After RNase H treatment, RNase A was added to the mixture at a final concentration of 167 ug / ml to digest any residual RNA. Next, cDNA was precipitated by adding 300 mM sodium acetate, pH 5.2 and 3.2 volumes of ice-cold 100% ethanol. The cDNA mixture vortexed for 10 seconds on high speed then kept on ice for 30 min, after which the cDNA mixture was centrifuged at 20,000xg at 4°C for 15 min. The pellet was washed with 1 mL of 70% ethanol, allowed to air dry and reconstituted in molecular grade water. cDNA was analyzed by reverse transcription quantitative polymerase chain reaction (using the RT-qPCR master mix) to quantify the RNA and qPCR (using the Gene Expression Master Mix to quantify the DNA in the sample with gene specific primers targeting the tGFP ORF sequence from the pT7CFE1-CGFP-HA-His plasmid.

[0117] Method E. Determination of single stranded nucleic acids and double stranded nucleic acids including RNA and DNA:

[0118] A Dionex Ultimate 3000 RS HPLC system (Thermo Scientific) equipped with a binary pump system, diode-array detector, temperature-controlled column compartment, and autosampler was used. Separation of the IVT components, impurities and target RNA was performed on a CIMac PrimaS AEX column at 40 ^C (100 µL, Sartorius Stedim Biotech; Cat. No.110.5118.2) using 50 mM HEPES, pH 7 as buffer A and 50 mM HEPES with 200 mM sodium pyrophosphate, pH 8.5 as buffer B. Gradient elution at a flow rate of 2 mL min-1was performed starting with 0% B for the first 0.5 min and a linear gradient to 50% buffer B for 5.5 min, and to 100% buffer B for 1 min, followed by a constant 100% buffer B for another 2 min. The column was re-equilibrated to 100% buffer A for 2 min before the next sample injection. The total method run time was 12 min. The components in the sample were detected at 260 nm and280 nm wavelengths. Chromatographic peak areas were processed using Chromeleon 7.2 SR5 software (Thermo Scientific) and used to evaluate the presence and relative abundance of the ds nucleic acid and ss nucleic acid in the sample. Alternatively, a calibration curve using known amounts of standard ss nucleic acid was used to determine the amount of the target ss nucleic acid generated during IVT and the amount recovered from the examples.

[0119] Method F: Preparation of Grafted Beads:

[0120] Beads (1 mL, 10 mg / mL) were added to a 1.5 mL Eppendorf tube. The beads were isolated using a magnet and the supernatant was discarded. The beads were washed 3 times with 500 µL MES buffer, collecting the beads using a magnet between washes. The washed beads were resuspended in 150 µL total of a N-methylpiperazine solution mixed by rotating end over end for 30 minutes.344 µL of freshly prepared EDC solution at 0 ºC (10 mg / mL of EDC in MES buffer, 18 µmol) and 6.5 µL MES buffer were added and the reaction was mixed at room temperature for 2 hours. The beads were isolated and washed twice with 500 µL Tris buffer (pH = 7.5, 50 mM) and twice with 500 µL PBS. The beads were left as a slurry in 1 mL PBS (10 mg / mL) and stored at 4 ºC until further use.

[0121] Surface modification of the beads was verified by measuring their zeta potential in two different pH buffers as follows. Zeta potential measurements were taken on a Malvern Zetasizer Nano ZSP (Malvern 35 Panalytical, Malvern, United Kingdom).2.5 microliters (µL) of a 10 milligram / milliliter (mg / mL) solution of the functionalized beads was added to 5 mL acetate buffer (pH = 4.5, 10 millimolar (mM)) or Tris buffer (pH = 8.5, 10 mM) and loaded onto a disposable capillary cell for testing. The cell was equilibrated at room temperature for 60 seconds (s) and then analyzed with the Smoluchowski approximation in the Zetasizer instrument.

[0122] Example 1:

[0123] An IEM / N-methylpiperazine grafted nylon membrane was prepared following Method A above. Single layers of this grafted membrane (7.5 mm diameter discs) were loaded into the wells of the well plate. Two hundred microliters (200 µl) of Tris-EDTA buffer (40 mM Tris, 1mM EDTA, pH 7) was pipetted into each membrane-containing well. The well plate was placed atop a deep-well plate and centrifuged with a swing bucket rotor at 2000 xg (times gravity) for 5 minutes after which the flowthrough collected in the deep-well plate was discarded.200 µl of Tris-EDTA buffer (40 mM Tris, 1 mM EDTA, pH 7) containing a mixture of ssRNA and dsRNA at a mass ratio of 80:20 with a total concentration of 200 ng / µl was then pipetted into each membrane-containing well. The well plate was placed atop a clean deep-well plate and centrifuged at 2000xg for 5 minutes with the flowthrough collected to determine the amount of RNA bound in each well. The well plate was then placed atop a clean deep-well plate and 200 µlof wash buffer (40 mM Tris, 1 mM EDTA, pH 7) was added to each well, and the plates were centrifuged at 2000xg for 5 minutes. The flowthrough was collected.

[0124] Elution buffers were prepared with pH values ranging from 6.5 to 10 and NaCl concentrations ranging from 0 mM to 500 mM for a total of 35 different elution conditions. A different elution buffer with varying pH and salt concentrations was added to each well. The well plate was placed atop a clean deep-well plate and centrifuged at 2000 xg for 5 minutes and the flowthrough was collected. The eluate collected in the deep-well plate was analyzed following Method E above to quantify the overall yield and relative amount of ssRNA and dsRNA in each well. Results from selected cells are reported in Table 2 below. Table 2 Sample Buffer content Percent of ssRNA dsRNA Name pH NaCl nucleic acid (%) (%) (mM) input in elution Load buffer 7 0 NA 80 20 Elution buffer 1 8 500 6 3 97 Elution buffer 2 9 0 24 56 44 Elution buffer 3 8 0 1 0 100 Elution buffer 4 9 500 6 23 77 NA=not applicable

[0125] These data suggest a process of dsRNA separation from ssRNA in which RNA mixtures bound to IEM / N-methylpiperazine grafted materials are exposed to a high ionic strength elution buffer (for example, Elution buffer 1) to selectively remove dsRNA, followed by a low ionic strength buffer (for example, Elution buffer 2) with elevated pH to recover ssRNA. This result also indicates that it will be essential for the high ionic strength elution solution to remove dsRNA prior to the high pH, low ionic strength elution solution.

[0126] Example 2:

[0127] The same procedure as described in Example 1 was repeated but using IEM / aminopropylmorpholine grafted membranes made with the IEM / aminopropylmorpholine monomer. Results from selected cells are reported in Table 3 below. Table 3 Sample Buffer content Percent of ssRNA dsRNA NamepH NaClnucleic acid (%)(%)(mM) input in elution Load buffer 7 0 NA 80 20Elution buffer 1 8 500 16 18 82 Elution buffer 2 9 0 48 73 27 Elution buffer 3 8 0 0.5 100 0 Elution buffer 4 9 500 15 19 81

[0128] Similar to Example 1, these data suggest a process of dsRNA separation from ssRNA in which RNA mixtures bound to IEM / aminopropylmorpholine grafted materials are exposed to a high ionic strength elution buffer (for example, Elution buffer 1) to selectively remove dsRNA, followed by a low ionic strength buffer with elevated pH (for example, Elution buffer 2) to recover ssRNA.

[0129] Example 3:

[0130] A plastic filtration capsule was used. The capsule consisted of a sealed, circular housing. The capsule housing was prepared from two halves (upper and lower halves) that were mated and sealed together at the perimeter after the filtration elements were inserted in the internal cavity of the lower housing. Fluid inlet and vent ports were located on the upper portion of the housing and a fluid outlet port was located on the lower portion of the housing. The outlet port was centered in the middle of the lower housing surface. An IEM / N-methylpiperazine grafted nylon membrane was prepared following Method A above with the IEM / N- methylpiperazine monomer. A single disc (25.4 mm diameter) of the grafted nylon membrane was placed in the bottom half of the capsule and the two halves were sealed together. The capsule was installed on a chromatography system and the following sequence of solutions was flowed through membrane containing capsule.

[0131] The capsule was first flushed with 5 mL of 100 mM Tris, 1 mM EDTA, pH 7 buffer. A 4 ml volume of Tris-EDTA buffer (100 mM Tris, 1 mM EDTA, pH 7) containing a mixture of ssRNA and dsRNA at a mass ratio of 80:20 with a total concentration of 200 ng / µl was then flowed through the capsule to bind the RNA onto the membrane. The membrane was then washed by flowing 6 ml of 100 mM Tris, 1 mM EDTA, pH 7 buffer through the capsule. This was followed by flowing 6 ml of 100 mM Tris, 1M NaCl, 1 mM EDTA, pH 7 buffer to selectively elute dsRNA. A 10 ml linear gradient of 100 mM Tris, 1M NaCl 1 mM EDTA, pH 10 to 100 mM Tris, 1 mM EDTA, pH 10 was then applied followed by an additional 10 ml of 100 mM Tris, 1 mM EDTA, pH 10 to elute ssRNA. Fractions were collected every 400 µl and single and double-stranded RNA content in each fraction was quantified as described by Method E above. Shown in Fig.1 is a chromatogram overlaying the quantitation of ssRNA and dsDNA in the eluate.

[0132] The results of this experiment show that the dsRNA was eluting with the high ionic strength eluent, while the elution of the ssRNA coincides with the increased pH eluent.

[0133] Example 4:

[0134] An IEM / N-methylpiperazine grafted nylon membrane was prepared following Method A above with the IEM / N-methylpiperazine monomer. A well plate was prepared by loading 7.5 mm diameter discs of the grafted nylon membrane into 35 wells of the well plate. Buffers were prepared with pH values ranging from 5.5 to 9 and NaCl concentrations ranging from 0 mM to 1000 mM for a total of 35 different conditions. Each well was pre-equilibrated with 200 µl of a different buffer as outlined in Table 4. The well plate was placed atop a deep- well plate and centrifuged at 2000 xg for 5 minutes after which the flowthrough was discarded.

[0135] The well plate was moved to a clean deep-well plate and 200 µl of buffer containing a mixture of ssRNA and dsRNA at a mass ratio of 80:20 with a total concentration of 200 ng / µl, and at differing pH values and NaCl concentrations as indicated in Table 4 was added to the wells already equilibrated with buffers of the same pH and NaCl concentration. The plates were centrifuged at 2000 xg for 5 minutes and the flowthrough was collected. The well plate was then removed to a clean 96-well deep-well plate, 200 microliters of wash buffer (same as equilibration buffer for each well) was added to each well, and the plates were centrifuged at 2000 xg for 5 minutes. The flowthrough was collected. The well plate was then moved to a clean 2 mL 96-well deep-well plate and 200 µl of elution buffer (40 mM Tris, 1 mM EDTA, pH 10) was added to each well. The plates were centrifuged at 2000xg for 5 minutes and the eluate was collected. Single and double-stranded RNA in the elution samples was quantified as described in Method E above.

[0136] In these experiments we demonstrate that adjusting the pH and ionic strength of RNA containing feed material enables dsRNA to flow through IEM / N-methylpiperazine grafted membranes under conditions where ssRNA is preferentially captured for later collection in an elution step.

[0137] Results from selected cells are reported in Table 4 below. Table 4 Sample Solution Buffer content Percent of ssRNA dsRNA pH NaCl nucleic acid (%) (%) (mM) input in flow through Example 4a Load buffer 8 50 NA 80 20 Flow through 24 54 46Elution 10 0 57 94 6 Comparative Load Buffer 8 0 NA 80 20 Example 4a Flow through 22 86 14 Elution 10 0 41 73 27 Comparative Load Buffer 8 500 NA 80 20 Example 4b Flow through 85 78 22 Elution 10 0 3 100 0

[0138] Example 5:

[0139] The same procedure as described in Example 4 was repeated but using IEM / N-(2- hydroxyethyl)piperazine grafted membranes made with the IEM / N-(2-hydroxyethyl)piperazine monomer. Results from selected cells are reported in Table 5 below. Table 5 Sample Solution Buffer content Percent of ssRNA dsRNA pH NaCl input RNA in (%) (%) (mM) flow through Example 5a Load buffer 7 500 NA 80 20 Flow through 35 58 42 Elution 10 0 43 94 6 Comparative Load Buffer 7 0 NA 80 20 Example 5a Flow through 12 86 14 Elution 10 0 37 62 38 Comparative Load Buffer 7 750 NA 80 20 Example 5b Flow through 77 80 20 Elution 10 0 6 100 0

[0140] Example 6:

[0141] The same procedure as described in Example 4 was repeated but using IEM / N- methylpiperazine co-HEMA grafted membranes made with the 75:25 IEM / N-methylpiperazine: HEMA monomers. Results from selected cells are reported in Table 6 below. Table 6 Sample Solution Buffer content Percent of input ssRNA dsRNARNA in flow (%) (%) through pH NaCl (mM) Example 6a Load buffer 6.5 750 NA 80 20 Flow through 33 64 36 Elution 10 0 44 96 4 Comparative Load Buffer 6.5 0 NA 80 20 Example 6a Flow through 17 87 13 Elution 10 0 39 70 30 Comparative Load Buffer 6.5 1000 NA 80 20 Example 6b Flow through 72 80 20 Elution 10 0 6 100 0

[0142] Example 7:

[0143] The same procedure as described in Example 1 was repeated except that the Tris- EDTA loading buffer contained a mixture of ssDNA and dsDNA (mass ratio of 80:20 with a total concentration of 200 ng / µL) instead of the RNA nucleic acids used in Example 1. The amount of ssDNA and dsDNA was determined using Method E and the results from selected cells are reported in Table 7 below. Table 7 Sample Buffer content Percent of ssDNA dsDNA Name pH NaCl nucleic acid (%) (%) (mM) input in elution Load buffer 7 0 NA 80 20 Elution Buffer 1 7.5 500 47 61 39 Elution Buffer 2 7 500 39 53 47 Elution Buffer 3 9 0 92 76 24 Elution Buffer 4 9 50 92 76 24

[0144] These data suggest a process of dsDNA separation from ssDNA in which DNA mixtures bound to IEM / N-methylpiperazine grafted materials were exposed to a high ionic strength elution buffer to selectively remove dsDNA (as observed by a higher proportion dsDNAas compared to ssDNA), followed by a low ionic strength buffer with elevated pH to recover ssDNA.

[0145] Example 8:

[0146] A plastic filtration capsule was used. The capsule consisted of a sealed, circular housing. The capsule housing was prepared from two halves (upper and lower halves) which were mated and sealed together at the perimeter after the filtration elements were inserted in the internal cavity of the lower housing. Fluid inlet and vent ports were located on the upper portion of the housing and a fluid outlet port was located on the lower portion of the housing. The outlet port was centered in the middle of the lower housing surface. An IEM / N-methylpiperazine grafted nylon membrane was prepared following Method A above with the IEM / N- methylpiperazine monomer. A single disc (25.4 mm diameter) of the grafted nylon membrane was placed in the bottom half of the capsule and the two halves were sealed together. The capsule was installed on the chromatography system and the flow rate was set to 1 mL / min for all stages of the experiment. The eluate (solution that passed through the capsule) was monitored with an ultraviolet-visible spectrophotometer at a wavelength of 260 nm. Since the spectrophotometer does not identify the materials eluted, the eluate was collected in three different fractions, which were subsequently tested by Method E to determine the amount of ssRNA and dsRNA in each fraction.

[0147] The following sequence of solutions was flowed through membrane containing capsule. The capsule was first flushed with 5 mL of 100 mM Tris, 1 mM EDTA, pH 7 buffer. Then, 4 mL of RNA in a load buffer (100 mM Tris, 1 mM EDTA, 650 mM NaCl, pH 7) was added to the capsule and collection of the eluate and monitoring of the eluate by UV-VIS began. The sample contained a mixture of ssRNA and dsRNA at a mass ratio of 80:20 with a total concentration of 200 ng / µL. The membrane was then washed by flowing 6 mL of a solution comprising 100 mM Tris, 1 mM EDTA, 1M NaCl at pH 7 through the capsule to flush unbound dsRNA and / or selectively elute any bound dsRNA. This was followed by flowing 6 ml of a second solution comprising 100 mM Tris and 1 mM EDTA with a pH 7. Then 15 ml of a third solution comprising 100 mM Tris and 1 mM EDTA with a pH 10 was then flowed through to elute ssRNA. Fractions were collected in bulk for each step and single and double-stranded RNA content in each fraction was quantified as described by Method E. Total RNA yield was measured as described under Method B. Shown in Fig.2 is a chromatogram of RNA coming off the membrane at each stage of the experiment. Total RNA and dsRNA in each fraction are expressed as a percent of input.

[0148] Comparative Example 8a

[0149] Example 8 was repeated except that the feed solution contained no additional NaCl, therefore it contained 100 mM Tris and 1 mM EDTA with a pH 7.

[0150] Shown in Table 8 below is the percentage of total amount of RNA detected in each fraction and the percentage of the dsRNA detected in each fraction versus the total amount added. Table 8 Sample Fraction tested First 4 4mL to 18 mL 18 mL to 34 mL mL eluate eluate eluate Example 8 Total RNA 6 % 12 % 88 % detected Amount of 40% 30% 0% dsRNA versus added Comparative 8a Total RNA 3% 13% 80% detected Amount of 0% 45% 46% dsRNA versus added

[0151] As shown in the figure and the table above, when the loading buffer comprised a high ionic strength as described in Method 2, the dsRNA appears to preferentially not bind to the solid phase, with a majority eluting in the first fraction, while the ssRNA appears to bind to the support and elutes with the eluting solution.

[0152] Example 9:

[0153] N-methylpiperazine conjugated magnetic beads were prepared following Method F.400 µL of bead slurry was pipetted into each of 2, 1.5 mL centrifuge tubes. The tubes were placed in a magnetic tube rack and the supernatant from the bead slurry was removed by a pipette. The beads in each tube were then resuspended in 400 µL of a wash solution (10 mM sodium acetate, pH 5.2). The beads were pipetted up and down in the tube to mix, and returned to the magnetic rack where the wash solution was removed. The tubes were removed from the magnetic rack and the beads were resuspended in 400 µL of 10 mM sodium acetate, pH 5.2 buffer containing either 125 ng / µl ssRNA or 125 ng / µl dsRNA and pipetted up and down in the tube to mix. One of the tubes contained the ssRNA and the other tube contained the dsRNA. The grafted beads were incubated with the RNA-containing solutions for 5 minutes at room temperature toallow the RNA to bind on to the grafted beads. Following the incubation, the beads in each tube were pipetted up and down to mix, and returned to the magnetic rack where the supernatant containing any unbound RNA was removed. The beads in each tube were then resuspended in 400 µL of a wash solution (10 mM sodium acetate, pH 5.2). Maintaining a uniform bead suspension by periodic agitation, 10 µL of the ssRNA-bound beads were transferred to each of 35 wells in a 96 well plate. Similarly, 10 µL of dsRNA-bound beads were separately transferred to each of 35 empty wells in the same 96 well plate. The 96 well plate was placed on a magnetic rack and the wash solution was removed from each well. The beads were then resuspended in a range of elution buffers prepared with pH values ranging from 6 to 9 and NaCl concentrations ranging from 0 mM to 500 mM for a total of 35 different elution conditions. The same elution buffers were used to elute from the ssRNA-bound and dsRNA-bound beads. ssRNA and dsRNA were quantified using RT-qPCR (IDT PrimeTime One-Step RT-qPCR Master Mix 10007066) with gene specific primers targeting the tFGP ORF sequence from the pT7CFE1-CGFP-HA-His plasmid. The results are shown in Table 9. Table 9 Sample Buffer content Percent of Name pH NaCl nucleic acid (mM) input in elution ssRNA tube Load buffer 5.2 0 100 Eluting Solution 1 6.5 500 3.5 Eluting Solution 2 8 0 79.4 dsRNA tube Load buffer 5.2 0 100 Eluting Solution 1 6.5 500 76.6 Eluting Solution 2 8 0 84.7

[0154] The above data shows that high ionic strength buffer preferentially elutes dsRNA relative to ssRNA. This indicates that the mechanisms of ssRNA / dsRNA separation from piperazine grafted materials is independent of the type of solid support and grafting chemistry.

[0155] Foreseeable modifications and alterations of this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. This invention should not be restricted to the embodiments that are set forth in this application for illustrative purposes. To the extent that there is any conflict or discrepancy between this specification as written and the disclosure in any document mentioned or incorporated by reference herein, this specification as written will prevail.

Claims

What is claimed is:

1. A method of separating a first nucleic acid and a second nucleic acid, the method comprising the following steps in order: (a) providing a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5; (b) loading a sample matrix onto the solid phase, wherein the sample matrix comprises the first and second nucleic acids and a loading buffer that has a pH that is below or near the pKa and an ionic strength, which is sufficiently low to bind the first and second nucleic acids; (c) optionally washing the solid phase with a washing solution, wherein the washing solution has a pH that is below or near the pKa and an ionic strength, which is sufficiently low to bind the first and second nucleic acids: (d) passing a first eluting solution through the solid phase, wherein the first eluting solution comprises a ionic strength having a higher ionic strength than the first ionic strength, and optionally having a pH, which is higher than the pKa such that first nucleic acid is selectively eluted; and (e) passing a second eluting solution through the solid phase, wherein the second eluting solution comprises a ionic strength lower than the ionic strength of the first eluting solution and a pH, which is higher than the pKa, such that a second nucleic acid is eluted.

2. The method of claim 1, wherein the pH of the loading buffer is in a range of 4.0 to 8 and the ionic strength value is no more than 0.15 mole / liter.

3. The method of any one of the previous claims, wherein the pH of the washing solution is in a range of 3.5 to 8 and the ionic strength value is no more than 0.5 mole / liter.

4. The method of any one of the previous claims, wherein the ionic strength of the first eluting solution is at least 0.2 molar.

5. The method of any one of the previous claims, wherein the pH of the first eluting solution is in a range of 5 to 10.

6. The method of any one of the previous claims, wherein the ionic strength of the second eluting solution is less than 1 molar.

7. The method of any one of the previous claims, wherein the first nucleic acid is double stranded RNA and the second nucleic acid is single stranded RNA.

8. A method of separating a first nucleic acid from a second nucleic acid, the method comprising the following steps in order: (a) providing a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5; (b) loading a sample matrix onto the solid phase, wherein the sample matrix comprises the first and second nucleic acids and a loading buffer has a pH that is lower than the pKa and an ionic strength, which is sufficiently high to allow the first nucleic acid to flow through, while binding the second nucleic acid; (c) optionally washing the solid phase with a washing solution, wherein the washing solution has a pH that is lower the pKa and an ionic strength, which is sufficiently high to preserve binding of the second nucleic acid; (d) passing an eluting solution through the solid phase, wherein the eluting solution comprises a lower ionic strength than the ionic strength of the loading buffer and having a pH, which is higher than the pKa such that the second nucleic acid is selectively eluted.

9. The method of claim 8, wherein the pH of the loading buffer is in a range of 3.5 to 8 and the ionic strength value is more than 0.2 mole / liter.

10. The method of any one of claims 8-9, wherein in the pH of the washing solution is in a range of 3.5 to 8 and the ionic strength value is more than 0.2 mole / liter.

11. The method of any one of claims 8-10, wherein the ionic strength of the eluting solution is no more than 0.5 molar.

12. The method of any one of claims 8-11, wherein the pH of the eluting solution is in a range of 5 to 10.

13. The method of any one of the previous claims, wherein the first nucleic acid is double stranded RNA and the second nucleic acid is single stranded RNA.

14. A method of separating a first nucleic acid from a second nucleic acid, the method comprising the following steps in order:(a) providing a solid phase comprising a multimodal ligand comprising a nitrogen-containing heterocycle, wherein the nitrogen-containing heterocycle has a pKa value of 3.5 to 9.5; (b) loading a sample matrix onto the solid phase, wherein the sample matrix comprises a plurality of nucleic acids and a loading buffer that has a pH that is higher than the pKa and an ionic strength, which is sufficiently low to bind the second nucleic acid and allow the first nucleic acid to flow through; (c) optionally washing the solid phase with a washing solution, wherein the washing solution has a pH that is higher than the pKa and an ionic strength, which is sufficiently low to bind the second nucleic acid and allow the first nucleic acid to flow through: (d) passing an eluting solution through the solid phase, wherein the eluting solution comprises a pH, which is higher than the pH of the loading buffer such that the second nucleic acid is selectively eluted.

15. The method of claim 14, wherein the pH of the loading buffer is in a range of 5 to 10 and the ionic strength value is no more than 0.5 mole / liter.

16. The method of any one of claims 14-15, wherein in the pH of the washing solution is in a range of 5 to 10 and the ionic strength value is no more than 0.5 mole / liter.

17. The method of any one of claims 14-16, wherein the ionic strength of the eluting solution is no more than 0.5 molar.

18. The method of any one of claims 8-11, wherein the pH of the eluting solution is in a range of 7 to 12.

19. The method of any one of the previous claims, wherein the first nucleic acid is double stranded RNA and the second nucleic acid is single stranded RNA.

20. The method of any one of the previous claims, wherein the pKa value is 5.0 to 8.

0.

21. The method of any one of the previous claims, wherein the nitrogen-containing heterocycle comprises a piperazine or morpholine moiety.

22. The method of any one of the previous claims, wherein the nitrogen-containing heterocycle is of the formulaI) Where R4and lkylene having at least 2 carbon atoms, wherein a sum of ring atoms in a ring group trogen, R4, Q, and R5is either 6 or 7; Q has a single catenated atom and is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to -N(R6)- when N is used to attach the ligand to the solid phase typically through a carbonyl moiety; andR6 is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy 23. The method of any one of the previous claims, wherein the nitrogen-containing heterocycle is of the formula Where R4and R5are least 2 carbon atoms, wherein a sum of ring atomsin a ring group is either 6 or 7; and R6is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

24. The method of any one of the previous claims, wherein the nitrogen-containing heterocycle is of the formula: R2is a (hetero)Z is -NH-(C=O)-, -NH-(C=O)-NH-, or -NH-(C=O)-NH-R3-, -(C=O)-NH-, and –(C=O)-NH-R3-, where R3is alkylene having at least two catenated carbon atoms; R4and R5are each an alkylene having at least 2 carbon atoms, wherein a sum of ring atoms in a ring group consisting of nitrogen, R4, Q, and R5is either 6 or 7; Q has a single catenated atom and is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to -N(R6)- when Z is -NH-(C=O)-; andR6is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

25. The method of any one of the previous claims, wherein multimodal ligand is derived from Formula (III) wherein R1is hydrogen orX1is -O- or -NH-; R2is a (hetero)alkylene; Z is -NH-(C=O)-, -NH-(C=O)-NH-, or -NH-(C=O)-NH-R3-, -(C=O)-NH-, and –(C=O)- NH-R3-, where R3is alkylene having at least two catenated carbon atoms; R4and R5are each an alkylene having at least 2 carbon atoms, wherein a sum of ring atoms in a ring group consisting of nitrogen, R4, Q, and R5is either 6 or 7; Q has a single catenated atom and is -O-, -N(R6)-, -S-, -S(=O)-, or -S(=O)2- with the proviso that Q is equal to -N(R6)- when Z is -NH-(C=O)-; and R6is hydrogen, alkyl, or (hetero)aryl, wherein the alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl and wherein the (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

26. The method of any one of claims 22 to 25, wherein a sum of catenated atoms in R2plus Z is equal to at least 4.

27. The method of any one of the previous claims, wherein the solid phase is a stationary phase in a chromatographic column.

28. The method of any one of claims 1-26, wherein the solid phase is a nonwoven, a microporous web, or a bead.

29. The method of any one of the previous claims, wherein the method is conducted at ambient conditions.

30. The method of any one of the previous claims, wherein the first eluting solution is substantially free of organic solvent.

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