Method and apparatus for double-stranded nucleotide purification

Abstract

Disclosed herein are methods of purifying oligonucleotides, such as by separating double stranded oligonucleotides from single stranded oligonucleotides. An apparatus for performing such a method is also provided. Also provided are double-stranded nucleic acid compositions and single-stranded nucleic acid compositions made using the methods and devices disclosed herein. Methods of producing the cyclic double-stranded nucleic acids are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 368,911, filed on July 20, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Oligonucleotides can be used in a variety of biological processes and therapeutic applications in laboratory and industrial settings. For example, industrial large-scale production of oligonucleotides is time and resource intensive. There is a need to improve and improve methods for making oligonucleotides to improve product and / or manufacturing characteristics. Summary of the invention

[0004] The present disclosure relates at least in part to methods and apparatus for improving the manufacture of oligonucleotides, improving the purification of oligonucleotides, and / or improving the characteristics (e.g., purity) of products made by the method or using the device. In some embodiments, the method improves the separation of double-stranded nucleic acids and single-stranded nucleic acids. In some embodiments, the method improves the purity of a composition comprising double-stranded nucleic acids. In some embodiments, the method includes an ultrafiltration step that selectively retains double-stranded nucleic acids (e.g., nucleic acids comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid). Without wishing to be bound by theory, it is believed that in some embodiments, the method and apparatus retain double-stranded nucleic acids in the retentate while allowing undesirable mixture components to penetrate the membrane.

[0005] Therefore, in one aspect, the present disclosure provides a method for producing a purified double-stranded nucleic acid, the method comprising:

[0006] A mixture is provided, the mixture comprising:

[0007] (a) a double-stranded nucleic acid comprising a first single-stranded nucleic acid hybridized to a complementary second single-stranded nucleic acid, and

[0008] (b) at least one single-stranded nucleic acid, e.g., (i) the first single-stranded nucleic acid in single-stranded form (i.e., does not hybridize to the second single-stranded nucleic acid), (ii) the second single-stranded nucleic acid in single-stranded form (i.e., does not hybridize to the first single-stranded nucleic acid), or (iii) both;

[0009] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids comprising the hybridized first single-stranded nucleic acid and the second single-stranded nucleic acid, but does not retain the single-stranded nucleic acids (e.g., the first single-stranded nucleic acid and / or the second single-stranded nucleic acid in a single-stranded form); and

[0010] collecting the retentate,

[0011] Thus, purified double-stranded nucleic acid is produced.

[0012] In another aspect, the present disclosure provides a method of purifying a first single-stranded nucleic acid and / or a second single-stranded nucleic acid, the method comprising:

[0013] A mixture is provided, the mixture comprising:

[0014] (a) a double-stranded nucleic acid comprising a first single-stranded nucleic acid hybridized to a complementary second single-stranded nucleic acid, and

[0015] (b) at least one single-stranded nucleic acid, e.g., (i) the first single-stranded nucleic acid in single-stranded form (i.e., does not hybridize to the second single-stranded nucleic acid), (ii) the second single-stranded nucleic acid in single-stranded form (i.e., does not hybridize to the first single-stranded nucleic acid), or (iii) both;

[0016] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids comprising the hybridized first single-stranded nucleic acid and the second single-stranded nucleic acid, but does not retain the single-stranded nucleic acids (e.g., the first single-stranded nucleic acid and / or the second single-stranded nucleic acid in a single-stranded form); and

[0017] Harvest the permeate,

[0018] The first single-stranded nucleic acid and / or the second single-stranded nucleic acid is thereby purified.

[0019] In another aspect, the present disclosure provides a method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, the method comprising:

[0020] Providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid,

[0021] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0022] wherein the ultrafiltration step comprises applying the mixture to a filtration device, such as a cross-flow filtration device, having a membrane having a molecular weight (MW) cutoff that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the MW of the double-stranded nucleic acid (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kilodaltons less than the MW of the double-stranded nucleic acid) and at least 10%, 20%, 30%, 40%, or 50% greater than the MW of a single-stranded nucleic acid (e.g., at least 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 kilodaltons greater than the MW of a single-stranded nucleic acid),

[0023] Thereby, double-stranded nucleic acids are separated from single-stranded nucleic acids.

[0024] In yet another aspect, the present disclosure provides a method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, the method comprising:

[0025] Providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid,

[0026] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0027] wherein the ultrafiltration step comprises applying the mixture to a filtration device, such as a cross-flow filtration device, having an average pore size (diameter) of at least 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8 or 3 nm and no greater than 4, 3.8, 3.6, 3.4, 3.2, 3, 2.8, 2.6, 2.4, 2.2, 2, 1.8 or 1.6 nm,

[0028] Thereby, double-stranded nucleic acids are separated from single-stranded nucleic acids.

[0029] In another aspect, the present disclosure provides a method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, the method comprising:

[0030] providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and

[0031] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0032] wherein the ultrafiltration step comprises applying the mixture to a filtration device, such as a cross-flow filtration device, wherein the filtration device comprises a membrane having an average pore size greater than Z (and optionally less than 0.8Y, 0.85Y, 0.9Y, 0.95Y, 0.99Y, Y or 1.1Y), wherein Z is the numerical average of X and Y,

[0033] wherein X is the minimum pore size that will allow at least 90% of each of the first single stranded nucleic acid and / or the second single stranded nucleic acid to permeate through the membrane (e.g., within less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV)); and

[0034] wherein Y is the maximum pore size that will allow at least 90% of the double stranded nucleic acid to be retained by the membrane (e.g., within less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 DTVs),

[0035] Thus, a method for separating double-stranded nucleic acids from single-stranded nucleic acids is provided.

[0036] In another aspect, the present disclosure provides a method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, the method comprising:

[0037] providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and

[0038] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0039] wherein the ultrafiltration step comprises applying the mixture to a filtration device, such as a cross-flow filtration device, wherein the filtration device comprises a membrane having a pore size (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 DTV) that will allow at least 90% of single-stranded nucleic acids and at most 10% of double-stranded nucleic acids to permeate through the membrane.

[0040] In another aspect, the present disclosure provides an apparatus comprising:

[0041] a first chamber configured to hold a mixture to be filtered (and optionally capture a retentate), a second chamber configured to capture a permeate, and a filter element disposed between the first chamber and the second chamber;

[0042] wherein the mixture comprises a double-stranded nucleic acid (e.g., comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid) and a single-stranded nucleic acid (e.g., the first single-stranded nucleic acid in single-stranded form, the second single-stranded nucleic acid in single-stranded form, or both);

[0043] The filter element selectively retains double-stranded nucleic acids (e.g., comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid) and does not retain single-stranded nucleic acids (e.g., the first single-stranded nucleic acid and / or the second single-stranded nucleic acid in single-stranded form).

[0044] In some embodiments of any of the methods or devices disclosed herein, the ultrafiltration step or filter element also does not retain one or more other mixture components, such as salts (e.g., halides or mineral salts), buffers, or production reagents or byproducts. In certain embodiments, the other mixture components are selected from: single-stranded nucleic acids of shorter length (e.g., shorter than the first single-stranded nucleic acid and / or the second single-stranded nucleic acid), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine), thiolation reagents / byproducts (e.g., xanthane, hydride, or PADS), capping reagents (e.g., acetic anhydride or NMI), coupling reagents (e.g., phosphoramidites or ETT), detritylation reagents / byproducts (e.g., dichloroacetic acid), deprotection reagents / byproducts (e.g., diethylamine, methylamine, or ammonia) or conjugation reagents (e.g., carboxylic acids, carboxylates, carbonates, and carboxylic acid activator reagents).

[0045] In some embodiments of any of the methods or devices disclosed herein, the ultrafiltration step or filter element produces a retentate and a permeate. In certain embodiments, the retentate comprises a different concentration of salt, buffer, or one or more of a production reagent or by-product than the permeate, mixture, or both. In another embodiment, the retentate has a lower concentration of salt, buffer, or one or more of a production reagent or by-product than the permeate, mixture, or both.

[0046] In certain embodiments of any of the methods disclosed herein, providing the mixture comprises:

[0047] providing a first single-stranded nucleic acid and a second single-stranded nucleic acid, wherein the first single-stranded nucleic acid and the second single-stranded nucleic acid comprise sequences sufficiently complementary to each other to hybridize under conditions suitable for hybridization, such as those described in Examples 1 to 5, and

[0048] combining the first single-stranded nucleic acid and the second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture comprising:

[0049] a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and

[0050] The first single-stranded nucleic acid is in single-stranded form, the second single-stranded nucleic acid is in single-stranded form, or both.

[0051] In some embodiments, providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes a synthesis step, such as solid-state chemical synthesis, solution-phase chemical synthesis, enzymatic synthesis, hybridization / chemoenzymatic synthesis, PCR-based synthesis and cell synthesis including the first single-stranded nucleic acid, the second single-stranded nucleic acid or both. In certain embodiments, providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes a post-synthesis step, such as including conjugation, chemical or enzymatic cutting from a solid substrate, removing one or more chemical parts (e.g., removing protective groups), or a combination thereof. In certain embodiments, the first single-stranded nucleic acid and / or the second single-stranded nucleic acid can be conjugated during synthesis, after synthesis, or after single-strand purification or ultrafiltration. In certain embodiments, providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes a purification step, such as enriching nucleic acid components and reducing the level of non-nucleic acid components, such as a chromatography step, such as including anion exchange chromatography. In some embodiments, providing a first single-stranded nucleic acid and / or a second single-stranded nucleic acid comprises subjecting the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to an ultrafiltration step that selectively retains single-stranded nucleic acids (e.g., rather than shortmers (i.e., shorter than the full-length desired single-stranded nucleic acid) or non-nucleic acid components).

[0052] In some embodiments, providing a double-stranded nucleic acid comprises a synthesis step and / or a post-synthesis step, for example, comprising conjugating the double-stranded nucleic acid to another component (e.g., a nucleic acid, a carbohydrate, a peptide, a protein domain, a lipid, a steroid polyethylene glycol, a fluorescent label). In some embodiments, a single-stranded nucleic acid is conjugated to another component before forming a double-stranded nucleic acid. In certain embodiments, providing a double-stranded nucleic acid enriches the nucleic acid component and reduces the level of non-nucleic acid components, such as a chromatography step, for example, comprising anion exchange chromatography. In some embodiments, providing a first double-stranded nucleic acid and / or a conjugated component comprises subjecting the first double-stranded nucleic acid, the conjugated component, or both to an ultrafiltration step, wherein the ultrafiltration step selectively retains the double-stranded nucleic acid (e.g., without retaining the non-nucleic acid component).

[0053] In some embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long (and optionally is no more than 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides long). In some embodiments, the second single-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long (and optionally is no more than 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides long). In certain embodiments, the double stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs long (and optionally no more than 100, 80, 60, 50, 45, 40, 35, or 30 base pairs long).

[0054] In some embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-4 0, 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12 -30, 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0055] In certain embodiments of any of the methods or devices disclosed herein, the second single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-4 0, 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12 -30, 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0056] In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46 -50, 48-50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40 , 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12 -30, 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 base pairs in length.

[0057] In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleic acid sequence of interest (e.g., a sequence of a target gene, a non-coding RNA, a primer, or a component for molecular assembly).

[0058] In certain embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid is at least 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, or 5 kilodaltons (and optionally no more than 10, 9, 8, 7, or 6 kilodaltons). In certain embodiments of any of the methods or devices disclosed herein, the second single-stranded nucleic acid is at least 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, or 5 kilodaltons (and optionally no more than 10, 9, 8, 7, or 6 kilodaltons).

[0059] In some embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid is 3-20, 3-15, 3-10, 3.5-10, 4-10, 4.5-10, 5-10, 5.5-10, 6-10, 6.5-10, 7-10, 7.5-10, 8-10, 8.5-10, 9-10, 9.5-10, 3-9, 3.5-9, 4-9, 4.5-9, 5-9, 5.5-9, 6-9, 6.5-9, 7-9, 7.5-9, 8-9, 8.5-9, 3-8, 3.5-8, 4-8, 4.5-8, 5-8, 5.5-8, 6-8, 6.5-8, 7-8, 7.5-8, 3-7, 3.5-7, 4-7, 4.5-7, 5-7, 5.5-7, 6-7, 6.5-7, 3-6, 3.5-6, 4-6, 4.5-6, 5-6, 5.5-6, 3-5, 3.5-5, 4-5, 4.5-5, 3-4, or 3.5-4 kilodaltons.

[0060] In some embodiments of any of the methods or devices disclosed herein, the second single-stranded nucleic acid is 3-20, 3-15, 3-10, 3.5-10, 4-10, 4.5-10, 5-10, 5.5-10, 6-10, 6.5-10, 7-10, 7.5-10, 8-10, 8.5-10, 9-10, 9.5-10, 3-9, 3.5-9, 4-9, 4.5-9, 5-9, 5.5-9, 6-9, 6.5-9, 7-9, 7.5-9, 8-9, 8.5-9, 3-8, 3.5-8, 4-8, 4.5-8, 5-8, 5.5-8, 6-8, 6.5-8, 7-8, 7.5-8, 3-7, 3.5-7, 4-7, 4.5-7, 5-7, 5.5-7, 6-7, 6.5-7, 3-6, 3.5-6, 4-6, 4.5-6, 5-6, 5.5-6, 3-5, 3.5-5, 4-5, 4.5-5, 3-4, or 3.5-4 kilodaltons.

[0061] In some embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid comprises a sequence capable of hybridizing with itself (e.g., forming a hairpin loop) under conditions suitable for hybridization. In certain embodiments of any of the methods or devices disclosed herein, the second single-stranded nucleic acid comprises a sequence capable of hybridizing with itself (e.g., forming a hairpin loop) under conditions suitable for hybridization.

[0062] In some embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid does not include a sequence that can hybridize with itself (e.g., form a hairpin loop) under conditions suitable for hybridization. In certain embodiments of any of the methods or devices disclosed herein, the second single-stranded nucleic acid does not include a sequence that can hybridize with itself (e.g., form a hairpin loop) under conditions suitable for hybridization.

[0063] In some embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both is or comprises DNA, RNA, UNA, PNA, or LNA.

[0064] In certain embodiments of any of the methods or devices disclosed herein, the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both comprise one or more modified and / or non-classical nucleotides selected from the group consisting of MOE, 2' fluoro, 2'OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl uridine, C5-propynyl cytidine, C5-methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, nucleotides containing modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or any combination thereof.

[0065] In some embodiments of any of the methods or apparatus disclosed herein, the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both comprise one or more non-phosphodiester bonds between nucleotides, such as one or more phosphorothioate or 5'-N-phosphoramidate bonds.

[0066] In certain embodiments of any of the methods disclosed herein, the ultrafiltration step comprises applying the double-stranded nucleic acid product mixture to a filtration device comprising a cross-flow filter. In some embodiments of any of the devices disclosed herein, the filtration element comprises a cross-flow filter.

[0067] In some embodiments of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having a pore size (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 DTVs) that will allow at least 50%, 60%, 70%, 80%, or 90% of single-stranded nucleic acids and at most 10%, 20%, 30%, 40%, or 50% of double-stranded nucleic acids to permeate through the membrane.

[0068] In some embodiments of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having an average pore size greater than Z (and optionally, less than 0.8Y, 0.85Y, 0.9Y, 0.95Y, 0.99Y, Y, or 1.1Y), wherein Z is the numerical average of X and Y,

[0069] wherein X is the minimum pore size that will allow at least 90% of the single stranded nucleic acid, the second single stranded nucleic acid, or both to permeate through the membrane (e.g., within less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV)); and

[0070] wherein Y is the maximum pore size that will allow at least 90% of the double stranded nucleic acid to be retained by the membrane (e.g., within less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 DTVs).

[0071] In certain embodiments of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having an average pore size of 0.05-20, 0.1-20, 0.5-20, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20, 0.05-10, 0.1-10, 0.5-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 0.05-8, 0.1-8, 0.5-8, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 0.05-6, 0.1-6, 0.5-6, 1-6, 2-6, 3-6, 4-6, 5-6, 0.05-4, 0.1-4, 0.5-4, 1-4, 2-4, 3-4, 0.05-2, 0.1-2, 0.5-2, 1-2, 0.05-1, 0.1-1, or 0.5-1 μm.

[0072] In some embodiments of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a molecular weight cutoff of:

[0073] is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons greater than the molecular weight of a single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both), and

[0074] At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons less than the molecular weight of the double-stranded nucleic acid.

[0075] In another embodiment of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having a molecular weight cutoff that is 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 15-30, 20-30, 25-30, 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20, 1-15, 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 kilodaltons.

[0076] In certain embodiments of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having a molecular weight cutoff that is 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 15-30, 20-30, 25-30, 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20 , 1-15, 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7- 9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 kilodaltons.

[0077] In some embodiments of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a molecular weight cutoff of:

[0078] is at least 2 kilodaltons greater than the molecular weight of a single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both), and

[0079] At least 5 kilodaltons smaller than the molecular weight of double-stranded nucleic acid.

[0080] In certain embodiments of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having a molecular weight cutoff of 0.5-100, 0.5-80, 0.5-60, 0.5-50, 0.5-40, 0.5-30, 0.5-20, 0.5-15, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-100, 1-80, 1-60, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-8, 1-6, 1-4, 1-3, 1-2, 2-100, 2-80, 2-60, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-8, 2-6, 2-4, 2-3, 3-100, 3-80, 3-60, 3-50, 3-40, 3-30, 3-20, 3-15, 3-10, 3-8, 3-6, 3-4, 4-100, 4-80, 4-60, 4-50, 4- 40, 4-30, 4-20, 4-15, 4-10, 4-8, 4-6, 6-100, 6-80, 6-60, 6-50, 6-40, 6-30, 6-20, 6-15, 6-10, 6-8, 8-100, 8-80, 8-60, 8-50, 8-40, 8-30, 8-20, 8-15, 8-10, 10-100, 10-80, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15, 15 15-40, 15-30, 15-20, 20-100, 20-80, 20-60, 20-50, 20-40, 20-30, 30-100, 30-80, 30-60, 30-50, 30-40, 40-100, 40-80, 40-60, 40-50, 50-100, 50-80, 50-60, 60-80, 60-100, or 80-100 kilodaltons.

[0081] In some embodiments of any of the devices disclosed herein, the filtration device or filter element comprises a membrane having a molecular weight cutoff of about 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 25-35, 50, 100, 300, or 1,000 kDa. In certain embodiments, the filter device or filter element comprises a membrane having a molecular weight cutoff of about 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 kDa. In some embodiments, the filter device or filter element comprises a membrane having a molecular weight cutoff of about 8-15 kDa. In certain embodiments, the filter device or filter element comprises a membrane having a molecular weight cutoff of about 10 kDa.

[0082] In some embodiments of any of the methods disclosed herein, the ultrafiltration step comprises applying a force of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 psi (transmembrane pressure), and optionally no greater than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 psi to the double-stranded nucleic acid product mixture, e.g., within a filtration device. In certain embodiments, the ultrafiltration step comprises applying a force of 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20, 5-15, 10-15, or 5-10 psi (transmembrane pressure). In another embodiment, the ultrafiltration step comprises applying a force within the pressure operating conditions recommended by the filter device manufacturer. In some embodiments, the ultrafiltration step comprises applying at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90 psi to the filter device. filtration volumes (DTV) (e.g., sequentially), and optionally no greater than 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90 DTVs.In some embodiments, the ultrafiltration step comprises applying 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 55-90, 60-90, 65-90, 70-90, 75-90, 80-90, 85-90, 20-85, 25-85, 30-85, 35-85, 40-85, 45-85, 50-85, 55-85, 60-85, 65-85, 70-85, 75-85, 80-85, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 55-80, 60-80, 65-80, 70-80, 75-80, 20-75, 25-75, 30-75, 35-75, 40-75, 45-75, 50-75, 55-75, 60-75, 65-75, 70-75, 20-70, 25-7 0, 30-70, 35-70, 40-70, 45-70, 50-70, 55-70, 60-70, 65-70, 20-65, 25-65, 30-65, 35-65, 40-65, 45-65, 50-65, 55-65, 60-65, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 20-55, 25-55, 30 -55, 35-55, 40-55, 45-55, 50-55, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 20-45, 25-45, 30-45, 35-45, 40-45, 20-40, 25-40, 30-40, 35-40, 20-35, 25-35, 30-35, 20-30, 25-30, or 20-25 DTVs (e.g., sequentially). In some embodiments, the ultrafiltration step comprises achieving at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m at a pressure. 2 and optionally no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 L / min / m 2In certain embodiments, the ultrafiltration step comprises achieving a recirculation rate of 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20, 5-15, 10-15, or 5-10 L / min / m at a pressure. 2 In some embodiments, ultrafiltration is performed until a stable low conductivity threshold is achieved (e.g., a conductivity threshold of less than or equal to about 30, 40, 50, 60, 70, or 80 μS / cm, such as about 50 μS / cm).

[0083] In certain embodiments of any of the devices disclosed herein, the filter device or filter element membrane comprises polysulfone, polypropylene, cellulose acetate, polylactic acid, nitrocellulose, mixed cellulose ester nitrocellulose, regenerated cellulose, polyethersulfone, polyamide, cellulose derivatives Polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track-etched membrane. In some embodiments, the filtration device or filter element is or includes hollow fiber, tubular, spiral wound, cassette, plate, or frame membrane type.

[0084] In some embodiments of any of the methods disclosed herein, the method produces a purified double stranded nucleic acid that achieves a purity standard, and wherein the purity standard comprises a retentate comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% double stranded nucleic acid (e.g., and comprising less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% single stranded nucleic acid). In certain embodiments, the method produces a purified double stranded nucleic acid achieving a purity standard, and wherein the purity standard comprises a retentate comprising 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100%, 80-99%, 85-99%, 90-99%, 91-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, 97-99% or 98-99% double stranded nucleic acid. In certain embodiments, the method produces a purified double stranded nucleic acid that achieves a purity standard, and wherein the purity standard comprises a retentate comprising 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 10-1, 15-1, or 20-1 of single stranded nucleic acid to double stranded nucleic acid.

[0085] In some embodiments of any of the methods disclosed herein, the method achieves a purity standard of no greater than 25, 24, 23, 22, 21, or 20 DTV.

[0086] In some embodiments of any of the methods or devices disclosed herein, the length of at least one strand of the double-stranded nucleic acid is the same as the length of the single-stranded nucleic acid.

[0087] In some embodiments of any of the methods or devices disclosed herein, the single-stranded nucleic acid is a component of a double-stranded nucleic acid (eg, the double-stranded nucleic acid comprises a copy of the single-stranded nucleic acid).

[0088] In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid comprises a flat end. In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid comprises two flat ends. In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid comprises a single-stranded portion, such as an end protrusion, such as at one end. In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid has a single-stranded portion at both ends, such as an end protrusion. In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid comprises a conjugated group, such as or comprising a PEG (polyethylene glycol), a dye, a label, a peptide, a lipid, a steroid, a carbohydrate (e.g., GalNAc), or an ethylene glycol spacer.

[0089] In some embodiments of any of the devices disclosed herein, the second chamber contains permeate, e.g., contains single-stranded nucleic acids. In other embodiments, the second chamber does not retain the permeate, e.g., the permeate flows out of the second chamber and is discarded.

[0090] In certain embodiments of any of the devices disclosed herein, the first chamber comprises a mixture. In certain embodiments of any of the methods or devices disclosed herein, the first chamber comprises a retentate.

[0091] In some embodiments of any of the methods or devices disclosed herein, the mixture comprises no more than 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl, or 400 μl, or no more than 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml l, 6ml, 7ml, 8ml, 9ml, 10ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml, 100ml, 200ml, 300ml, or 400ml, or no more than 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​2 100L, 200L, 300L, 350L, 400L, 450L or 500L (and optionally at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl or 100 μl, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml, or at least 0.5 L, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, or 500 L).

[0092] In some embodiments of any of the methods or devices disclosed herein, the mixture comprises at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl, or 400 μl, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6ml, 7ml, 8ml, 9ml, 10ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml, 100ml, 200ml, 300ml, or 400ml , or at least 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L , 250L, 300L, 350L, 400L, 450L or 500L (and optionally no more than 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl or 100 μl, or no more than 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml , 10ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml, or 100ml, or no more than 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350L, 400L, 450L or 500L).

[0093] In certain embodiments of any of the methods or apparatus disclosed herein, the mixture comprises:

[0094] 1-100μl, 5-100μl, 10-100μl, 20-100μl, 30-100μl, 40-100μl, 50-100μl, 60-100μl, 70-100μl, 80-100μl l, 90-100μl, 1-50μl, 5-50μl, 10-50μl, 20-50μl, 30-50μl, 40-50μl, 1-20μl, 5-20μl, 10-20μl, or 1-10μl,

[0095] 0.1 - 100 ml, 0.5 - 100 ml, 1 - 100 ml, 5 - 100 ml, 10 - 100 ml, 20 - 100 ml, 30 - 100 ml, 40 - 100 ml, 50 - 100 ml, 60 - 100 ml, 70 - 100 ml, 80 - 100 ml, 90 - 100 ml, 0.1 - 50 ml, 0.5 - 50 ml, 1 - 50 ml, 5 - 50 ml, 10 - 50 ml, 20 - 50 ml, 30 - 50 ml, 40 - 50 ml, 0.1 - 20 ml, 0.5 - 20 ml, 1 - 20 ml, 5 - 20 ml, 10 - 20 ml, 0.1 - 10 ml, 0.5 - 10 ml, 1 - 10 ml, 0.1 - 1 ml, 0.5 - 1 ml, or 0.1 - 0.5 ml, or

[0096] 0.1 - 500 L, 0.5 - 500 L, 1 - 500 L, 5 - 500 L, 10 - 500 L, 20 - 500 L, 30 - 500 L, 40 - 500 L, 50 - 500 L, 60 - 500 L, 70 - 500 L, 80 - 500 L, 90 - 500 L, 100 - 500 L, 150 - 500 L, 200 - 500 L, 250 - 500 L, 300 - 500 L, 350 - 500 L, 400 - 500 L, 450 - 500 L, 0.1 - 250 L, 0.5 - 250 L, 1 - 250 L, 5 - 250 L, 10 - 250 L, 20 - 250 L, 30 - 250 L, 40 - 250 L, 50 - 250 L, 60 - 250 L, 70 - 250 L, 80 - 250 L, 90 - 250 L, 100 - 250 L, 150 - 250 L, 200 - 250 L, 0.1 - 100 L, 0.5 - 100 L, 1 - 100 L, 5 - 100 L, 10 - 100 L, 20 - 100 L, 30 - 100 L, 40 - 100 L, 50 - 100 L, 60 - 100 L, 70 - 100 L, 80 - 100 L, 90 - 100 L, 0.1 - 50 L, 0.5 - 50 L, 1 - 50 L, 5 - 50 L, 10 - 50 L, 20 - 50 L, 30 - 50 L, 40 - 50 L, 0.1 - 20 L, 0.5 - 20 L, 1 - 20 L, 5 - 20 L, 10 - 20 L, 0.1 - 10 L, 0.5 - 10 L, 1 - 10 L, 0.1 - 1 L, 0.5 - 1 L, or 0.1 - 0.5 L.

[0097] In some embodiments of any of the methods or devices disclosed herein, the mixture comprises at least 300 OD / ml, 400 OD / ml, 500 OD / ml, 600 OD / ml, 700 OD / ml, 800 OD / ml, 900 OD / ml, 1000 OD / ml, 1100 OD / ml, 1200 OD / ml, 1300 OD / ml, 1400 OD / ml, 1500 OD / ml, 1600 OD / ml, 1700 OD / ml, 1800 OD / ml, 1900 OD / ml, 2000 OD / ml, 2100 OD / ml, 2200 OD / ml, 2300 OD / ml, 2400 OD / ml, 2500 OD / ml, 2600 OD / ml, 2700 OD / ml, 2800 OD / ml, 2900 OD / ml, 3000 OD / ml, 3100 OD / ml, 3200 In some embodiments, the mixture comprises a nucleic acid concentration (e.g., overall nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least about 900 OD / mL (e.g., about 900 OD / mL). In some embodiments, the mixture comprises a nucleic acid concentration (e.g., overall nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least about 1100 OD / mL (e.g., about 1100 OD / mL). In certain embodiments, the mixture comprises a nucleic acid concentration (eg, total nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least about 400 OD / mL (eg, about 400 OD / mL).

[0098] In certain embodiments of any of the methods or devices disclosed herein, the ssDNA in the permeate consists primarily of the sense strand (e.g., wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the ssDNA in the permeate consists of the sense strand). In some embodiments, the ssDNA in the permeate consists primarily of the antisense strand (e.g., wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the ssDNA in the permeate consists of the antisense strand).

[0099] In some embodiments of any of the methods or devices disclosed herein, the double-stranded nucleic acid comprises a mismatch (e.g., at one of the ends of the double-stranded nucleic acid, within 1-2 nucleotides of one of the ends of the double-stranded nucleic acid, or between the ends of the double-stranded nucleic acid).

[0100] In certain embodiments of any of the methods or devices disclosed herein, the amount of single-stranded nucleic acid contained in the mixture exceeds the amount of double-stranded nucleic acid. In some embodiments, the amount of single-stranded nucleic acid is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% in excess relative to the amount of double-stranded nucleic acid. In certain embodiments, the amount of single-stranded nucleic acid is about 5% in excess relative to the amount of double-stranded nucleic acid.

[0101] In some embodiments of any of the methods disclosed herein, the ultrafiltration step comprises diafiltration. In certain embodiments, the diafiltration is performed for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes, or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, diafiltration is performed for a period of time sufficient to filter at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV).

[0102] In certain embodiments of any of the methods disclosed herein, the ultrafiltration is applied at a pressure of about 5-40 psi, 10-40 psi, 15-40 psi, 20-40 psi, 30-40 psi, 35-40 psi, 5-15 psi, 5-20 psi, 5-25 psi, 5-30 psi, 5-35 psi, 10-30 psi, 15-25 psi, 5-10 psi, 10-15 psi, 15-20 psi, 20-25 psi, 25-30 psi, 30-35 psi, or 35-40 psi. In some embodiments, ultrafiltration is applied at a pressure of about 15-30 psi (e.g., about 15-25 psi, 15-20 psi, 25-30 psi, 20-30 psi, 15-20 psi, 20-25 psi, or 25-30 psi, such as about 15 psi, 16 psi, 17 psi, 18 psi, 19 psi, 20 psi, 21 psi, 22 psi, 23 psi, 24 psi, 25 psi, 26 psi, 27 psi, 28 psi, 29 psi, or 30 psi). In certain embodiments, ultrafiltration is performed at a temperature of about 10-40°C, e.g., about 15-40°C, 20-40°C, 25-40°C, 30-40°C, 35-40°C, 10-35°C, 10-30°C, 10-25°C, 10-20°C, 10-15°C, 15-35°C, 20-30°C, 10-15°C, 15-20°C, 20-25°C, 25-30°C, 30-35°C, or 35-40°C, e.g., about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C.

[0103] In some embodiments of any of the methods disclosed herein, the method further comprises analyzing the retentate by non-denaturing HPLC (e.g., to determine the relative amounts of double-stranded nucleic acids and / or single-stranded nucleic acids in the retentate), e.g., as described in Examples 1 to 5.

[0104] In some embodiments of any of the methods or devices disclosed herein, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate (e.g., as determined by native HPLC) increases over time and / or as the total volume of diafiltration (DTV) increases. In certain embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate (e.g., as determined by native HPLC) is at least 90:5, 90.3:5, 90.4:4.8, 90.9:4.8, 91.1:4.6, 91.2:4.5, 91.1:4.4, 91.2:4.4, 91.3:4.1, or 91.5:3.9. In some embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate (e.g., as determined by native HPLC) is at least 90.1:5.3, 91.1:4.7, 91.2:4.8, 91.3:4.9, 92.0:3.5, 92.1:3.4, or 92.0:3.2.

[0105] In some embodiments of any of the methods or devices disclosed herein, the concentration of salt (e.g., cations, such as sodium ions) in the mixture is less than about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM. In certain embodiments, the mixture does not substantially comprise salt. In certain embodiments, the mixture is substantially free of salt.

[0106] In some embodiments of any of the methods or devices disclosed herein, the concentration of the salt (e.g., cations such as sodium ions) in the mixture is about 50 mM to about 1.5 M, such as about 60 nM to about 1.2 M, or about 69 mM to about 1.02 M.

[0107] In certain embodiments of any of the methods or devices disclosed herein, the melting temperature of the double-stranded nucleic acid in the mixture is between about 20-85°C, such as between about 25-80°C, 30-75°C, 35-70°C, 40-65°C, 45-60°C, 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, 70-75°C, 75-80°C, or 80-85°C.

[0108] In certain embodiments of any of the methods or devices disclosed herein, the relationship between the melting temperature of the double-stranded nucleic acid in the mixture and the concentration of the salt (e.g., cations, such as sodium ions) in the mixture is determined according to the method described in Owczarzy et al. (2004, Biochemistry 43:3537-3554; incorporated herein by reference in its entirety).

[0109] In some embodiments of any one of the methods or devices disclosed herein, the double-stranded nucleic acid of the retentate is used for downstream purposes or applications. In some embodiments, the double-stranded nucleic acid of the retentate is lyophilized. In some embodiments, the double-stranded nucleic acid of the retentate is conjugated with an additional reagent (e.g., an additional nucleic acid molecule). In some embodiments, the double-stranded nucleic acid of the retentate is mixed with other oligonucleotide duplexes. In some embodiments, the double-stranded nucleic acid of the retentate is mixed with a pharmaceutical product formulation buffer (e.g., a buffered phosphate solution, a buffered phosphate saline solution, a saline solution, or water).

[0110] In some embodiments of any of the methods or devices disclosed herein, the double stranded nucleic acid is 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-200, 200-250, 250-300, 300-400, or 400-500 nucleotides in length.

[0111] In certain embodiments of any of the methods disclosed herein, the method reduces the level of low molecular weight impurities by at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the low molecular weight impurities each have a molecular weight of less than about 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 Daltons. In certain embodiments, the low molecular weight impurities include salts and / or trace amounts of solvents.

[0112] In some embodiments of any of the methods or devices disclosed herein, the method reduces the level of double-stranded nucleic acid by no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0113] In some embodiments of any of the methods or apparatuses disclosed herein, the ratio of the double stranded nucleic acid concentration in the retentate to the double stranded nucleic acid concentration in the mixture is at least 0.75, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995, or 0.999.

[0114] In some embodiments of any of the methods or devices disclosed herein, the amount of double stranded nucleic acid in the retentate is at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 mg / mL.

[0115] In another aspect, the present disclosure provides a method for optimizing a technique for making purified double-stranded nucleic acids, the method comprising:

[0116] (i) providing a mixture, the mixture comprising:

[0117] a first concentration of double-stranded nucleic acid, the double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and

[0118] a second concentration of single-stranded nucleic acids, such as the first single-stranded nucleic acid in single-stranded form, the second single-stranded nucleic acid in single-stranded form, or both;

[0119] wherein the mixture has a predetermined concentration of salt (e.g., cations, such as sodium ions);

[0120] (ii) subjecting the mixture to an ultrafiltration step, wherein the ultrafiltration step selectively retains the double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, while not retaining the single-stranded nucleic acid (e.g., the first single-stranded nucleic acid and the second single-stranded nucleic acid in single-stranded form),

[0121] wherein the ultrafiltration step comprises applying the mixture to a filtration device having a predetermined molecular weight cutoff for a predetermined period of time;

[0122] (iii) harvesting the retentate;

[0123] (iv) changing one or more of the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined time period, or the salt concentration;

[0124] (v) repeating steps (i) to (iii) one or more times, each time using one or more of the varying first concentration, second concentration, predetermined molecular weight cutoff, predetermined time period, and / or salt concentration;

[0125] (vi) for each repetition of steps (i) to (iii), determining the ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the harvested retentate; and

[0126] (vii) identifying the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined time period, and / or the salt concentration that produces the highest ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the harvested retentate;

[0127] Thereby optimizing the technology for producing purified double-stranded nucleic acids.

[0128] In another aspect, the present disclosure provides a double-stranded nucleic acid composition comprising a harvested retentate produced according to the methods described herein.

[0129] In another aspect, the present disclosure provides a single-stranded nucleic acid composition comprising a harvested permeate produced according to the methods described herein.

[0130] In another aspect, the present disclosure provides a composition comprising a first double-stranded nucleic acid comprising a first overhang hybridized to a second double-stranded nucleic acid comprising a second overhang (eg, complementary to the first overhang).

[0131] In some embodiments, the first double-stranded nucleic acid is prepared according to the methods described herein. In certain embodiments, the second double-stranded nucleic acid is prepared according to the methods described herein.

[0132] In another aspect, the present disclosure provides a method for producing a circular double-stranded nucleic acid, the method comprising:

[0133] (a) providing a composition as described herein, and

[0134] (b) incubating the composition under conditions suitable for circularization of the first double-stranded nucleic acid that hybridizes to the second double-stranded nucleic acid.

[0135] In some embodiments, the circularizing comprises ligation.

[0136] Various embodiments disclosed herein are represented by the following clauses:

[0137] 1. A method for producing a double-stranded nucleic acid, the method comprising:

[0138] A mixture is provided, the mixture comprising:

[0139] a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and

[0140] Single-stranded nucleic acid;

[0141] subjecting the mixture to an ultrafiltration step that selectively retains a double-stranded nucleic acid comprising a hybridized first single-stranded nucleic acid and a second single-stranded nucleic acid without retaining the single-stranded nucleic acid; and

[0142] collecting the retentate,

[0143] Thus, purified double-stranded nucleic acid is produced.

[0144] 2. A method for purifying a first single-stranded nucleic acid and / or a second single-stranded nucleic acid, the method comprising:

[0145] A mixture is provided, the mixture comprising:

[0146] a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and

[0147] Single-stranded nucleic acid,

[0148] Optionally wherein the mixture has been subjected to preliminary purification / ultrafiltration to remove short polymers and / or organic impurities resulting from any nucleic acid synthesis and / or deprotection steps performed;

[0149] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid without retaining single-stranded nucleic acids; and

[0150] Harvest the permeate,

[0151] The first single-stranded nucleic acid and / or the second single-stranded nucleic acid is thereby purified.

[0152] 3. A method for separating double-stranded nucleic acid from single-stranded nucleic acid, the method comprising:

[0153] Providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid,

[0154] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0155] wherein the ultrafiltration step comprises applying the mixture to a filtration device having a molecular weight (MW) cutoff of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kilodaltons less than the MW of the double stranded nucleic acid and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 kilodaltons less than the MW of the double stranded nucleic acid,

[0156] Thereby, the double-stranded nucleic acid is separated from the single-stranded nucleic acid.

[0157] 4. A method for separating double-stranded nucleic acid from single-stranded nucleic acid, the method comprising:

[0158] Providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid,

[0159] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0160] wherein the ultrafiltration step comprises applying the mixture to a filtration device having an average pore size (diameter) of at least 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8 or 3 nm and no greater than 4, 3.8, 3.6, 3.4, 3.2, 3, 2.8, 2.6, 2.4, 2.2, 2, 1.8 or 1.6 nm,

[0161] Thereby, double-stranded nucleic acids are separated from single-stranded nucleic acids.

[0162] 5. A method for separating double-stranded nucleic acid from single-stranded nucleic acid, the method comprising:

[0163] providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and

[0164] subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids,

[0165] wherein the ultrafiltration step comprises applying the mixture to a filtration device, wherein the filtration device comprises a membrane having an average pore size greater than Z and optionally less than 0.8Y, 0.85Y, 0.9Y, 0.95Y, 0.99Y, Y or 1.1Y, wherein Z is the numerical average of X and Y,

[0166] wherein X is the minimum pore size that will allow at least 90% of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to permeate through the membrane; and

[0167] where Y is the maximum pore size that will allow at least 90% of double-stranded nucleic acids to be retained by the membrane,

[0168] Thereby, the double-stranded nucleic acid is separated from the single-stranded nucleic acid.

[0169] 6. A device, comprising:

[0170] a first chamber configured to hold a mixture to be filtered and optionally capture a retentate, a second chamber configured to capture a permeate, and a filter element disposed between the first chamber and the second chamber;

[0171] wherein the mixture comprises double-stranded nucleic acid and single-stranded nucleic acid;

[0172] wherein the filter element selectively retains double-stranded nucleic acids while not retaining single-stranded nucleic acids.

[0173] 7. A method or apparatus according to any of the preceding clauses, wherein the ultrafiltration step or filter element also does not retain one or more other mixture components.

[0174] 8. A method or apparatus according to claim 7, wherein the other mixture components are selected from: single-stranded nucleic acids of shorter length, organic solvents, thiolating agents / by-products, capping reagents, coupling reagents, detritylation reagents / by-products, conjugation reagents (e.g., carboxylic acids, carboxylates, carbonates, and carboxylic acid activator reagents), and / or deprotection reagents / by-products.

[0175] 9. The process according to any one of clauses 1 to 5, wherein the ultrafiltration step produces a retentate and a permeate.

[0176] 10. The method or apparatus of any one of clauses 6-9, wherein the retentate comprises a different concentration of one or more of a salt, a buffer, a deprotection base, or a production reagent or by-product than the permeate, the mixture, or both.

[0177] 11. The method or apparatus of any of clauses 6-10, wherein the retentate has a lower concentration of one or more of salts, buffers, or production reagents or byproducts than the permeate, the mixture, or both.

[0178] 12. The method according to any of the preceding clauses, wherein providing the mixture comprises:

[0179] providing a first single-stranded nucleic acid and a second single-stranded nucleic acid, wherein the first single-stranded nucleic acid and the second single-stranded nucleic acid comprise sequences sufficiently complementary to each other to hybridize under conditions suitable for hybridization, and

[0180] combining the first single-stranded nucleic acid and the second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture comprising:

[0181] a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and

[0182] The first single-stranded nucleic acid is in single-stranded form, the second single-stranded nucleic acid is in single-stranded form, or both.

[0183] 13. The method according to clause 12, wherein providing the first single-stranded nucleic acid and the second single-stranded nucleic acid comprises a synthesis step.

[0184] 14. The method according to any one of clauses 12 or 13, wherein providing the first single-stranded nucleic acid and the second single-stranded nucleic acid comprises a post-synthetic step.

[0185] 15. The method according to any one of clauses 12 to 14, wherein providing the first single-stranded nucleic acid and the second single-stranded nucleic acid comprises a purification step.

[0186] 16. The method according to any one of clauses 12-15, wherein providing a first single-stranded nucleic acid and a second single-stranded nucleic acid comprises subjecting the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to an ultrafiltration step that selectively retains single-stranded nucleic acids.

[0187] 17. A method or device according to any of the preceding clauses, wherein the first single-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long, and optionally is no more than 100, 80, 60, 50, 45, 40, 35 or 30 nucleotides long.

[0188] 18. A method or device according to any of the preceding clauses, wherein the second single-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long, and optionally is no more than 100, 80, 60, 50, 45, 40, 35 or 30 nucleotides long.

[0189] 19. A method or apparatus according to any of the preceding clauses, wherein the double-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs in length, and optionally is no more than 100, 80, 60, 50, 45, 40, 35 or 30 base pairs in length.

[0190] 20. A method or apparatus according to any of the preceding clauses, wherein the first single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-4 0, 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12 -30, 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0191] 21. A method or apparatus according to any of the preceding clauses, wherein the second single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-4 0, 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12 -30, 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0192] 22. A method or apparatus according to any of the preceding clauses, wherein the double-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46 -50, 48-50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40 , 20-40, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12 -30, 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 base pairs in length.

[0193] 23. A method or apparatus according to any of the preceding clauses, wherein the double-stranded nucleic acid comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleic acid sequence of interest.

[0194] 24. A method or device according to any of the preceding clauses, wherein the first single-stranded nucleic acid is at least 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8 or 10 kilodaltons, and optionally no more than 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 kilodaltons.

[0195] 25. A method or device according to any of the preceding clauses, wherein the second single-stranded nucleic acid is at least 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8 or 10 kilodaltons, and optionally no greater than 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 kilodaltons.

[0196] 26. A method or apparatus according to any of the preceding clauses, wherein the first single-stranded nucleic acid is 3-10, 3.5-10, 4-10, 4.5-10, 5-10, 5.5-10, 6-10, 6.5-10, 7-10, 7.5-10, 8-10, 8.5-10, 9-10, 9.5-10, 3-9, 3.5-9, 4-9, 4.5-9, 5-9, 5.5-9, 6-9, 6.5-9, 7-9, 7.5-10. 9, 8-9, 8.5-9, 3-8, 3.5-8, 4-8, 4.5-8, 5-8, 5.5-8, 6-8, 6.5-8, 7-8, 7.5-8, 3-7, 3.5-7, 4-7, 4.5-7, 5-7, 5.5-7, 6-7, 6.5-7, 3-6, 3.5-6, 4-6, 4.5-6, 5-6, 5.5-6, 3-5, 3.5-5, 4-5, 4.5-5, 3-4, or 3.5-4 kilodaltons.

[0197] 27. A method or apparatus according to any of the preceding clauses, wherein the second single-stranded nucleic acid is 3-10, 3.5-10, 4-10, 4.5-10, 5-10, 5.5-10, 6-10, 6.5-10, 7-10, 7.5-10, 8-10, 8.5-10, 9-10, 9.5-10, 3-9, 3.5-9, 4-9, 4.5-9, 5-9, 5.5-9, 6-9, 6.5-9, 7-9, 7.5-10. 9, 8-9, 8.5-9, 3-8, 3.5-8, 4-8, 4.5-8, 5-8, 5.5-8, 6-8, 6.5-8, 7-8, 7.5-8, 3-7, 3.5-7, 4-7, 4.5-7, 5-7, 5.5-7, 6-7, 6.5-7, 3-6, 3.5-6, 4-6, 4.5-6, 5-6, 5.5-6, 3-5, 3.5-5, 4-5, 4.5-5, 3-4, or 3.5-4 kilodaltons.

[0198] 28. A method or apparatus according to any of the preceding clauses, wherein the first single-stranded nucleic acid comprises a sequence capable of hybridizing to itself under conditions suitable for hybridization.

[0199] 29. A method or apparatus according to any of the preceding clauses, wherein the second single-stranded nucleic acid comprises a sequence capable of hybridizing to itself under conditions suitable for hybridization.

[0200] 30. A method or apparatus according to any one of clauses 1 to 27 or 29, wherein the first single-stranded nucleic acid does not comprise a sequence capable of hybridizing to itself under conditions suitable for hybridization.

[0201] 31. A method or apparatus according to any one of clauses 1 to 28 or 30, wherein the second single-stranded nucleic acid does not comprise a sequence capable of hybridizing to itself under conditions suitable for hybridization.

[0202] 32. A method or apparatus according to any of the preceding clauses, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both is or comprises DNA, RNA, UNA, PNA or LNA.

[0203] 33. A method or device according to any of the preceding clauses, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both comprise one or more modified and / or non-canonical nucleotides selected from the group consisting of MOE, 2' fluorine, 2'OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, inserted bases, nucleotides comprising modified sugars, or any combination thereof.

[0204] 34. A method or apparatus according to any of the preceding clauses, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both comprise one or more non-phosphodiester bonds between nucleotides.

[0205] 35. The method according to any of the preceding clauses, wherein the ultrafiltration step comprises applying the double-stranded nucleic acid product mixture to a filtration device comprising a cross-flow filter.

[0206] 36. The device of any of the preceding clauses, wherein the filter element comprises a cross-flow filter.

[0207] 37. A method or device according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having an average pore size greater than Z and optionally less than 0.8Y, 0.85Y, 0.9Y, 0.95Y, 0.99Y, Y or 1.1Y, wherein Z is the numerical average of X and Y,

[0208] wherein X is the minimum pore size that will allow at least 90% of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to permeate through the membrane; and

[0209] Where Y is the maximum pore size that will allow at least 90% of double-stranded nucleic acids to be retained by the membrane.

[0210] 38. A method or device according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having an average pore size of 0.05-20, 0.1-20, 0.5-20, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20, 0.05-10, 0.1-10, 0.5-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10 , 7-10, 8-10, 9-10, 0.05-8, 0.1-8, 0.5-8, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 0.05-6, 0.1-6, 0.5-6, 1-6, 2-6, 3-6, 4-6, 5-6, 0.05-4, 0.1-4, 0.5-4, 1-4, 2-4, 3-4, 0.05-2, 0.1-2, 0.5-2, 1-2, 0.05-1, 0.1-1, or 0.5-1 μm.

[0211] 39. A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cut-off of:

[0212] is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons greater than the molecular weight of the single-stranded nucleic acid, and

[0213] At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons less than the molecular weight of the double-stranded nucleic acid.

[0214] 40. A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cutoff of 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 15-30, 20-30, 25-30, 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-2 0, 1-15, 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7 -9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 kilodaltons.

[0215] 41. A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cutoff that is 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 15-30, 20-30, 25-30, 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-25, 20-25. 0, 1-15, 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7 -9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 kilodaltons.

[0216] 42. A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cut-off of:

[0217] is larger than the molecular weight of a single-stranded nucleic acid, and

[0218] Smaller than the molecular weight of double-stranded nucleic acid.

[0219] 43. A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cut-off of 0.5-100, 0.5-80, 0.5-60, 0.5-50, 0.5-40, 0.5-30, 0.5-20, 0.5-15, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-100, 1-80, 1-60, 1-50, 1-40, 1- 30, 1-20, 1-15, 1-10, 1-8, 1-6, 1-4, 1-3, 1-2, 2-100, 2-80, 2-60, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-8, 2-6, 2-4, 2-3, 3-100, 3-80, 3-60, 3-50, 3-40, 3-30, 3-20, 3-15, 3-10, 3-8, 3-6, 3-4, 4-100, 4-80, 4-60, 4-50, 4-40, 4-30, 4-20, 4-15, 4-10, 4-8, 4-6, 6-100, 6-80, 6-60, 6-50, 6-40, 6-30, 6-20, 6-15, 6-10, 6-8, 8-100, 8-80, 8-60, 8-50, 8-40, 8-30, 8-20, 8-15, 8-10, 10-100, 10-80, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15, 1 15-40, 15-30, 15-20, 20-100, 20-80, 20-60, 20-50, 20-40, 20-30, 30-100, 30-80, 30-60, 30-50, 30-40, 40-100, 40-80, 40-60, 40-50, 50-100, 50-80, 50-60, 60-80, 60-100, or 80-100 kilodaltons.

[0220] 44. A method or device according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cutoff of approximately 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15 or 15-16 kDa.

[0221] 45. A method or device according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cutoff of about 8-9, 9-10, 10-11, 11-12, 12-13, 13-14 or 14-15 kDa.

[0222] 46. ​​A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cut-off of about 8-15 kDa.

[0223] 47. A method or apparatus according to any of the preceding clauses, wherein the filtration device or filter element comprises a membrane having a molecular weight cut-off of about 10 kDa.

[0224] 48. A method according to any of the preceding clauses, wherein the ultrafiltration step comprises applying a force of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 psi (transmembrane pressure) and optionally not more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 psi to the double-stranded nucleic acid product mixture.

[0225] 49. A method according to any of the preceding clauses, wherein the ultrafiltration step comprises applying a force of 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20, 5-15, 10-15 or 5-10 psi (transmembrane pressure).

[0226] 50. A method according to any of the preceding clauses, wherein the ultrafiltration step comprises applying a force under pressure operating conditions recommended by the manufacturer of the filtration device.

[0227] 51. A method according to any of the preceding clauses, wherein the ultrafiltration step comprises applying at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 , 86, 88 or 90 diafiltration volumes (DTV) and optionally no more than 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 or 90 DTV.

[0228] 52. A method according to any of the preceding clauses, wherein the ultrafiltration step comprises applying 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 55-90, 60-90, 65-90, 70-90, 75-90, 80-90, 85-90, 20-85, 25-85, 30-85, 35-85, 40-85, 45-85, 50-85, 55-85 , 60-85, 65-85, 70-85, 75-85, 80-85, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 55-80, 60-80, 65-80, 70-80, 75-80, 20-75, 25-75, 30-75, 35-75, 40-75, 45-75, 50-75, 55-75, 60-75, 65-75, 70-75 , 20-70, 25-70, 30-70, 35-70, 40-70, 45-70, 50-70, 55-70, 60-70, 65-70, 20-65, 25-65, 30-65, 35-65, 40-65, 45-65, 50-65, 55-65, 60-65, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 20-55 , 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 20-45, 25-45, 30-45, 35-45, 40-45, 20-40, 25-40, 30-40, 35-40, 20-35, 25-35, 30-35, 20-30, 25-30, or 20-25 DTVs.

[0229] 53. The method of any of the preceding clauses, wherein the ultrafiltration step comprises achieving a flow rate of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m at a pressure of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m 2 and optionally no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 L / min / m 2 recirculation rate.

[0230] 54. The method of any of the preceding clauses, wherein the ultrafiltration step comprises achieving a flow rate of 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20, 5-15, 10-15, or 5-10 L / min / m 2 recirculation rate.

[0231] 55. A method according to any of the preceding clauses, wherein the ultrafiltration is performed until a stable low conductivity threshold is achieved.

[0232] 56. A method or device according to any of the preceding clauses, wherein the filter device or filter element membrane comprises polysulfone, polypropylene, cellulose acetate, polylactic acid, nitrocellulose, mixed cellulose ester nitrocellulose, regenerated cellulose, polyethersulfone, polyamide, cellulose derivatives Polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track-etched membranes.

[0233] 57. A method or apparatus according to any of the preceding clauses, wherein the filter device or filter element is or comprises a hollow fiber, tubular, spiral wound, cassette, plate or frame membrane type.

[0234] 58. A method according to any of the preceding clauses, wherein the method produces a purified double-stranded nucleic acid that achieves a purity standard, and wherein the purity standard includes a retentate comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% double-stranded nucleic acid.

[0235] 59. A method according to any of the preceding clauses, wherein the method produces a purified double-stranded nucleic acid that achieves a purity standard, and wherein the purity standard comprises a retentate comprising 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100%, 80-99%, 85-99%, 90-99%, 91-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, 97-99% or 98-99% double-stranded nucleic acid.

[0236] 60. A method according to any of the preceding clauses, wherein the method produces a purified double-stranded nucleic acid that achieves a purity standard, and wherein the purity standard includes a retentate comprising 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 10-1, 15-1 or 20-1 of single-stranded nucleic acid to double-stranded nucleic acid.

[0237] 61. A method according to any of the preceding clauses, wherein the method achieves a purity standard of no greater than 25, 24, 23, 22, 21 or 20 DTV.

[0238] 62. A method or apparatus according to any of the preceding clauses, wherein the length of at least one strand of the double-stranded nucleic acid is the same as the length of the single-stranded nucleic acid.

[0239] 63. A method or apparatus according to any of the preceding clauses, wherein the single-stranded nucleic acid is a component of the double-stranded nucleic acid.

[0240] 64. A method or device according to any of the preceding clauses, wherein the double-stranded nucleic acid comprises blunt ends.

[0241] 65. A method or apparatus according to any of the preceding clauses, wherein the double-stranded nucleic acid comprises two blunt ends.

[0242] 66. A method or device according to any of the preceding clauses, wherein the double-stranded nucleic acid comprises a single-stranded portion.

[0243] 67. A method or apparatus according to any of the preceding clauses, wherein the double-stranded nucleic acid has a single-stranded portion.

[0244] 68. A method or device according to any of the preceding clauses, wherein the double-stranded nucleic acid comprises a conjugated group.

[0245] 69. A device according to any of the preceding clauses, wherein the second chamber comprises a permeate.

[0246] 70. The device of any of the preceding clauses, wherein the second chamber retains permeate.

[0247] 71. The device of any one of clauses 6-69, wherein the second chamber does not retain permeate.

[0248] 72. An apparatus according to any of the preceding clauses, wherein the first chamber comprises the mixture.

[0249] 73. An apparatus according to any of the preceding clauses, wherein the first chamber comprises a retentate.

[0250] 74. A method or apparatus according to any of the preceding clauses, wherein the mixture comprises no more than 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl, or 400 μl, or no more than 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl, or 400 μl. 1, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400 ml, or no more than 0.5 L, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350L, 400L, 450L or 500L, and optionally at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl or 100 μl, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 0L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350L, 400L, 450L, 500L, 600L, 700L, 800L, 900L or 1000L.

[0251] 75. A method or apparatus according to any of the preceding clauses, wherein the mixture comprises at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 300 μl or 400 μl, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8ml, 9ml, 10ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml, 100ml, 200ml, 300ml or 400ml, or at least 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml or 100 μl, or no more than 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml 1, 2, 3, 4, 5, 6, 7, 8, 9, or 100 ml, or not more than 0.5 L, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, 500 L, 600 L, 700 L, 800 L, 900 L, or 1000 L.

[0252] 76. A method or apparatus according to any preceding clause, wherein the mixture comprises:

[0253] 1-100μl, 5-100μl, 10-100μl, 20-100μl, 30-100μl, 40-100μl, 50-100μl, 60-100μl, 70-100μl, 80-100μl l, 90-100μl, 1-50μl, 5-50μl, 10-50μl, 20-50μl, 30-50μl, 40-50μl, 1-20μl, 5-20μl, 10-20μl, or 1-10μl,

[0254] 0.1 - 100 ml, 0.5 - 100 ml, 1 - 100 ml, 5 - 100 ml, 10 - 100 ml, 20 - 100 ml, 30 - 100 ml, 40 - 100 ml, 50 - 100 ml, 60 - 100 ml, 70 - 100 ml, 80 - 100 ml, 90 - 100 ml, 0.1 - 50 ml, 0.5 - 50 ml, 1 - 50 ml, 5 - 50 ml, 10 - 50 ml, 20 - 50 ml, 30 - 50 ml, 40 - 50 ml, 0.1 - 20 ml, 0.5 - 20 ml, 1 - 20 ml, 5 - 20 ml, 10 - 20 ml, 0.1 - 10 ml, 0.5 - 10 ml, 1 - 10 ml, 0.1 - 1 ml, 0.5 - 1 ml, or 0.1 - 0.5 ml, or

[0255] 0.1 - 500 L, 0.5 - 500 L, 1 - 500 L, 5 - 500 L, 10 - 500 L, 20 - 500 L, 30 - 500 L, 40 - 500 L, 50 - 500 L, 60 - 500 L, 70 - 500 L, 80 - 500 L, 90 - 500 L, 100 - 500 L, 150 - 500 L, 200 - 500 L, 250 - 500 L, 300 - 500 L, 350 - 500 L, 400 - 500 L, 450 - 500 L, 0.1 - 250 L, 0.5 - 250 L, 1 - 250 L, 5 - 250 L, 10 - 250 L, 20 - 250 L, 30 - 250 L, 40 - 250 L, 50 - 250 L, 60 - 250 L, 70 - 250 L, 80 - 250 L, 90 - 250 L, 100 - 250 L, 150 - 250 L, 200 - 250 L, 0.1 - 100 L, 0.5 - 100 L, 1 - 100 L, 5 - 100 L, 10 - 100 L, 20 - 100 L, 30 - 100 L, 40 - 100 L, 50 - 100 L, 60 - 100 L, 70 - 100 L, 80 - 100 L, 90 - 100 L, 0.1 - 50 L, 0.5 - 50 L, 1 - 50 L, 5 - 50 L, 10 - 50 L, 20 - 50 L, 30 - 50 L, 40 - 50 L, 0.1 - 20 L, 0.5 - 20 L, 1 - 20 L, 5 - 20 L, 10 - 20 L, 0.1 - 10 L, 0.5 - 10 L, 1 - 10 L, 0.1 - 1 L, 0.5 - 1 L, or 0.1 - 0.5 L.

[0256] 77. A method or device according to any of the preceding clauses, wherein the mixture comprises a nucleic acid concentration of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 9000 OD / ml.

[0257] 78. A method or device according to any of the preceding clauses, wherein the mixture comprises a nucleic acid concentration of at least about 900 OD / mL.

[0258] 79. A method or device according to any of the preceding clauses, wherein the mixture comprises a nucleic acid concentration of at least about 1100 OD / mL.

[0259] 80. A method or device according to any of the preceding clauses, wherein the mixture comprises a nucleic acid concentration of at least about 400 OD / mL.

[0260] 81. A method or apparatus according to any of the preceding clauses, wherein the ssDNA in the permeate consists primarily of the sense strand.

[0261] 82. A method or apparatus according to any of the preceding clauses, wherein the ssDNA in the permeate consists primarily of the antisense strand.

[0262] 83. A method or device according to any of the preceding clauses, wherein the double-stranded nucleic acid contains mismatches.

[0263] 84. A method or apparatus according to any of the preceding clauses, wherein the mixture comprises an amount of single-stranded nucleic acid that exceeds the amount of double-stranded nucleic acid.

[0264] 85. A method or device according to claim 84, wherein the amount of single-stranded nucleic acid is in excess of the amount of double-stranded nucleic acid by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0265] 86. The method or apparatus of clause 84, wherein the amount of single-stranded nucleic acid is in excess of about 5% relative to the amount of double-stranded nucleic acid.

[0266] 87. A method according to any of the preceding clauses, wherein the ultrafiltration step comprises diafiltration.

[0267] 88. A method according to claim 87, wherein the diafiltration is performed for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes, or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours.

[0268] 89. A method according to clause 87 or 88, wherein the diafiltration is performed for a time period sufficient to filter at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85 or 90 diafiltration volumes (DTV).

[0269] 90. A method according to any of the preceding clauses, wherein the ultrafiltration is applied at a pressure of about 5-40psi, 10-40psi, 15-40psi, 20-40psi, 30-40psi, 35-40psi, 5-15psi, 5-20psi, 5-25psi, 5-30psi, 5-35psi, 10-30psi, 15-25psi, 5-10psi, 10-15psi, 15-20psi, 20-25psi, 25-30psi, 30-35psi, or 35-40psi.

[0270] 91. A method according to any of the preceding clauses, wherein the ultrafiltration is applied at a pressure of about 15-30 psi.

[0271] 92. The method according to any of the preceding clauses, wherein the ultrafiltration is performed at a temperature of about 10-40°C, optionally wherein the ultrafiltration is performed at a temperature at least 0.5°C below the melting point of double-stranded nucleic acids.

[0272] 93. A method according to any of the preceding clauses, further comprising analyzing the retentate by non-denaturing HPLC.

[0273] 94. A method according to any of the preceding clauses, wherein the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate increases over time and / or as the total diafiltration volume (DTV) increases.

[0274] 95. A method according to any of the preceding clauses, wherein the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate is at least 90:5, 90.3:5, 90.4:4.8, 90.9:4.8, 91.1:4.6, 91.2:4.5, 91.1:4.4, 91.2:4.4, 91.3:4.1 or 91.5:3.9.

[0275] 96. A method according to any of the preceding clauses, wherein the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate is at least 90.1:5.3, 91.1:4.7, 91.2:4.8, 91.3:4.9, 92.0:3.5, 92.1:3.4 or 92.0:3.2.

[0276] 97. A method or device according to any of the preceding clauses, wherein the salt concentration in the mixture is less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or 3000 mM.

[0277] 98. A method or apparatus according to clause 97, wherein the mixture contains substantially no salt.

[0278] 99. A method or apparatus according to any of the preceding clauses, wherein the salt concentration in the mixture is about 50mM to about 3M.

[0279] 100. A method or apparatus according to any preceding clause, wherein the melting temperature of the double-stranded nucleic acids in the mixture is between about 20-85°C.

[0280] 101. A method or apparatus according to any of the preceding clauses, wherein the relationship between the melting temperature of the double-stranded nucleic acid in the mixture and the salt concentration in the mixture is determined.

[0281] 102. A method according to any of the preceding clauses, wherein the double-stranded nucleic acid of the retentate is used for downstream uses or applications.

[0282] 103. A method according to any of the preceding clauses, wherein the double-stranded nucleic acid of the retentate is lyophilized.

[0283] 104. A method according to any of the preceding clauses, wherein the double-stranded nucleic acid of the retentate is conjugated to an additional reagent.

[0284] 105. A method according to any of the preceding clauses, wherein the double-stranded nucleic acid of the retentate is mixed with other oligonucleotide duplexes.

[0285] 106. The method according to any of the preceding clauses, wherein the double-stranded nucleic acid of the retentate is mixed with a drug product formulation buffer.

[0286] 107. A method or apparatus according to any of the preceding clauses, wherein the double-stranded nucleic acid has a length of 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-200, 200-250, 250-300, 300-400, or 400-500 nucleotides.

[0287] 108. A method according to any of the preceding clauses, wherein the method reduces the level of low molecular weight impurities by at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0288] 109. The method of clause 108, wherein the low molecular weight impurities each have a molecular weight of less than about 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 Daltons.

[0289] 110. The method of clause 109, wherein the low molecular weight impurities comprise salts and / or trace amounts of solvents.

[0290] 111. A method according to any of the preceding clauses, wherein the method reduces the level of double-stranded nucleic acid by no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0291] 112. A method according to any of the preceding clauses, wherein the ratio of the amount of double-stranded nucleic acids in the retentate to the amount of double-stranded nucleic acids in the mixture is at least 0.75, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995 or 0.999.

[0292] 113. A method according to any of the preceding clauses, wherein the amount of double-stranded nucleic acid in the retentate is at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 mg / mL.

[0293] 114. A method for optimizing a technique for making purified double-stranded nucleic acids, the method comprising:

[0294] (i) providing a mixture, the mixture comprising:

[0295] a first concentration of double-stranded nucleic acid, the double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and

[0296] Single-stranded nucleic acid;

[0297] wherein the mixture has a predetermined concentration of salt;

[0298] (ii) subjecting the mixture to an ultrafiltration step, wherein the ultrafiltration step selectively retains the double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, while not retaining the single-stranded nucleic acid,

[0299] wherein the ultrafiltration step comprises applying the mixture to a filtration device having a predetermined molecular weight cutoff for a predetermined period of time;

[0300] (iii) harvesting the retentate;

[0301] (iv) changing one or more of the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined time period, or the salt concentration;

[0302] (v) repeating steps (i) to (iii) one or more times, each time using one or more of the varying first concentration, second concentration, predetermined molecular weight cutoff, predetermined time period, and / or salt concentration;

[0303] (vi) for each repetition of steps (i) to (iii), determining the ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the harvested retentate; and

[0304] (vii) identifying the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined time period, and / or the salt concentration that produces the highest ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the harvested retentate;

[0305] The technique for producing purified double-stranded nucleic acids is thereby optimized.

[0306] 115. A double-stranded nucleic acid composition comprising a harvested retentate produced according to the method of any one of clauses 1 or 7-113.

[0307] 116. A single-stranded nucleic acid composition comprising a harvested permeate produced according to the method of any one of clauses 2 or 7-113.

[0308] 117. A composition comprising a first double-stranded nucleic acid comprising a first overhang hybridized to a second double-stranded nucleic acid comprising a second overhang.

[0309] 118. The composition according to clause 117, wherein the first double-stranded nucleic acid is prepared according to the method of any of the preceding clauses.

[0310] 119. The composition according to clause 117 or 118, wherein the second double-stranded nucleic acid is prepared according to the method of any of the preceding clauses.

[0311] 120. A method for producing a circular double-stranded nucleic acid, the method comprising:

[0312] (a) providing a composition according to any one of clauses 117 to 119, and

[0313] (b) incubating the composition under conditions suitable for circularization of the first double-stranded nucleic acid that hybridizes to the second double-stranded nucleic acid.

[0314] 121. A method according to clause 120, wherein the cyclization comprises ligation.

[0315] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, they are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0316] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0317] Figure 1 The schematic diagram of the exemplary ultrafiltration device used in the method described herein is shown. The duplex oligonucleotide solution may contain excessive unhybridized single strands, and the duplex oligonucleotide solution is loaded into the retentate storage tank of the ultrafiltration system. The material will be pumped through the ultrafiltration membrane and returned to the retentate storage tank. Pressure will be applied to the flow path, and the flow path then mechanically forces the (permeate) compound with a molecular weight lower than the molecular weight cutoff of the ultrafiltration membrane to arrive at the permeate side of the membrane. Process water is added to the retentate storage tank to maintain a suitable working volume, while low molecular weight substances and water permeate pass through the ultrafiltration membrane. The process is monitored by the conductivity of the permeate stream. Set the conductivity threshold target to promote the end point for the removal of ionic low molecular weight substances to reach an acceptable level.

[0318] FIG. 2A to FIG. 2Bis an image of an ultraviolet (UV) chromatogram, a reverse phase chromatogram representing the composition of duplex and non-hybridized single stranded nucleic acid over time from a percolation time point study. Figure 2A represents the total HPLC run time. Figure 2B 2C represents the time difference between single-stranded nucleic acid and double-stranded nucleic acid (left and right peaks, respectively). This study was performed as described in Example 3. DETAILED DESCRIPTION

[0319] The present disclosure is at least partially related to the improved method of producing (for example, purifying) oligonucleotide (for example, separating double-stranded oligonucleotide from single-stranded oligonucleotide).Not wishing to be bound by theory, it is believed that in some embodiments, the method of producing (for example, purifying) oligonucleotide, particularly in large-scale or industrial manufacturing environment or using the method of producing oligonucleotide of modified, synthetic or non-natural nucleotide, is usually highly sensitive to process parameters, and the process parameters are the concentration of reagents in solution, the ratio of reagents to each other in solution, the duration of step (for example, recycle time), membrane pressure, membrane porosity or composition or production solution in pollutant existence and / or level.Therefore, the present disclosure is at least partially based on the following discovery: the change of production (for example, purifying) parameter can change the effect of method, for example, by changing the amount of purified oligonucleotide, product characteristics of oligonucleotide (for example, length, purity, composition (for example, correct nucleotide, for example, modified nucleotide), sequence or annealing state), or one or more of the amount of consumed resources or time.

[0320] I. Definitions

[0321] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. Thus, the term "about" can be used to indicate that a value includes the inherent error variation of the device or method used to determine the value. For example, the term "about" generally means ±10% (e.g., ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of the amount.

[0322] As used herein, "substantially free" with respect to a specified component means that any specified component has not been intentionally formulated into the composition and / or the specified component is present only as a contaminant or trace amount. Thus, the total amount of the specified ingredient resulting from any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified ingredient cannot be detected by standard analytical methods.

[0323] As used in this specification, "a / an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising", the word "a / an" may mean one or more.

[0324] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, but the present disclosure supports definitions referring only to alternatives and "and / or". As used herein, "another" may mean at least a second or more.

[0325] As used herein, the term "cross-flow filter" refers to a filter in which a feed passes tangentially across the surface of the filter. In some embodiments, the cross-flow filter includes a transmembrane pressure.

[0326] As used herein, the term "diafiltration tank volume (DTV)" refers to the working volume in which the material of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) is suspended and maintained throughout a process, such as prior to a purification step described herein (e.g., including ultrafiltration). In some embodiments, the DTV is a measure of the volume that has flowed through during the diafiltration step based on the ratio of the volume introduced into the unit operation to the retentate volume. For example, if the volume that has flowed through is equal to the working volume contained in the retentate tank, the diafiltration volume is equal to 1; if the volume that has flowed through is equal to twice the entire initial volume, the diafiltration volume is equal to 2; and so on.

[0327] As used herein, the term "molecular weight cutoff" (MWCO) refers to a molecular weight target that reflects an average pore size. In some embodiments, for example, in an ultrafiltration method as described herein, a substance (e.g., a double-stranded nucleic acid, a single-stranded nucleic acid, or other mixture components) that exceeds the MWCO is substantially retained by the membrane (e.g., wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the original substance is retained). In some embodiments, the MWCO is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 kilodaltons less than the MW of the double-stranded nucleic acid. In some embodiments, the MWCO is no more than about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 kilodaltons less than the MW of the double-stranded nucleic acid. In some embodiments, species can be transported across the membrane with a molecular weight distribution or range of up to + / -1 kDa. For example, a 10 kDa MWCO can permeate species with a molecular weight of 10 + / - 1 kDa.

[0328] As used herein, the term "pore size" refers to the average diameter of pores in a barrier (e.g., a membrane, such as a semipermeable membrane), for example, used to filter out particles of a particular size (e.g., by ultrafiltration). In some embodiments, the average diameter of the pore size is at least about 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or 3 nm. In some embodiments, the average diameter of the pore size is no greater than about 0.001, 0.005, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.5, or 1.6 μm. Additionally, in other embodiments, the pore size is expressed as a MWCO of 1, 2, 3, 5, 10, 20, 30, 50, 100 kDa.

[0329] As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid monomers (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid monomers. In some embodiments, "nucleic acid" is or comprises RNA. In some embodiments, "nucleic acid" is DNA or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid is, comprises, or consists of one or more modified, synthetic, or non-naturally occurring nucleotides. In some embodiments, a nucleic acid analog is different from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered to be within the scope of the present invention. Additionally or alternatively, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, the nucleic acid is, comprises or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2'methoxyethyl (2'MOE), 2'fluoro, 2'OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those sugars in natural nucleic acids. In some embodiments, the nucleic acid comprises N-acetylgalactosamine (GalNAc) modifications, such as GalNAc modifications to sugars, phosphates, phosphodiesters, or bases (e.g., to phosphodiesters) of the nucleic acid. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (such as an RNA or a protein).In some embodiments, the nucleic acid is a non-coding nucleic acid (e.g., a primer (e.g., a DNA primer) or a non-coding RNA (e.g., a functional RNA)). In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is partially or entirely single-stranded. In some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element encoding a polypeptide or a portion thereof, or is the complement of a sequence encoding a polypeptide or a portion thereof. In some embodiments, the nucleic acid has enzymatic activity (e.g., the nucleic acid is a ribozyme).

[0330] As used herein, "oligonucleotide" refers to a nucleic acid that is at least 2 nucleotides long and not more than about 500 nucleotides long. In some embodiments, oligonucleotide is 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, 175-200, 10-150 or 14-100 nucleotides long. Oligonucleotide may be useful in a variety of biological environments, including but not limited to the manufacture and use of therapeutic agents, laboratory applications (e.g., PCR, RNAi, genetic modification (e.g., CRISPR gene editing), sequencing) and biomanufacturing.

[0331] As used herein, the term "other mixture components" refers to components in the mixture that are not molecules of interest (e.g., double-stranded nucleic acids or single-stranded nucleic acids). In some embodiments, other mixture components include salts (e.g., halides or mineral salts). In some embodiments, other mixture components include buffers. In certain embodiments, other mixture components include production reagents or byproducts (e.g., truncated nucleic acid sequences or non-desired nucleic acid sequences). In some embodiments, other mixture components include single-stranded nucleic acids of shorter length (e.g., shorter than the first single-stranded nucleic acid and / or the second single-stranded nucleic acid of interest). In some embodiments, other mixture components include organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine or lutidine). In some embodiments, other mixture components include thiolation reagents / byproducts (e.g., xanthane, hydride or PADS) or capping reagents (e.g., acetic anhydride or NMI). In some embodiments, other mixture components include coupling reagents (e.g., phosphoramidites or ETT). In some embodiments, other mixture components include detritylation reagents / byproducts (e.g., dichloroacetic acid). In some embodiments, other mixture components include deprotection reagents / byproducts (e.g., diethylamine, methylamine, or ammonia). In some embodiments, production reagents or byproducts can be selected from single-stranded nucleic acids of shorter length (e.g., shorter than the first single-stranded nucleic acid and / or the second single-stranded nucleic acid), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine), thiolation reagents (e.g., xanthane, hydride, or PADS), capping reagents (e.g., acetic anhydride or NMI), coupling reagents (e.g., phosphoramidite or ETT), detritylation reagents (e.g., dichloroacetic acid), conjugation reagents (e.g., carboxylic acids, carboxylic acid esters, carbonates, and carboxylic acid activator reagents), and / or deprotection reagents (e.g., diethylamine, methylamine, or ammonia).

[0332] As used herein, the term "permeate" refers to a substance (e.g., a single-stranded nucleic acid) that passes through a barrier (e.g., a membrane, such as a semi-permeable membrane), for example, in an ultrafiltration method described herein. In some embodiments, the permeate comprises single-stranded nucleic acids. In some embodiments, the permeate comprises other mixture components. In some embodiments, the permeate comprises little or no detectable level of double-stranded nucleic acids. In some embodiments, the stoichiometric ratio of single-stranded nucleic acids to double-stranded nucleic acids in the retentate is at least 100: 1, 1,000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1 or higher.

[0333] As used herein, "phosphoramidite nucleotide" refers to a nucleotide comprising a phosphate group, including a monoamide phosphorous diester. In some embodiments, the phosphoramidite nucleotide can be used in the nucleotide addition steps described herein. Without wishing to be bound by theory, the phosphoramidite nucleotide is believed to be reactive in the presence of a weak acid and a nucleophile, e.g., a nucleic acid (e.g., 3'OH) of a loaded solid medium.

[0334] As used herein, the term "post-synthesis step" generally refers to a method step performed after the synthesis stage of a method. In some embodiments, the post-synthesis step includes chemical or enzymatic cleavage from a solid substrate. In some embodiments, the post-synthesis step includes removing one or more chemical moieties (e.g., removing protecting groups). In some embodiments, the post-synthesis step includes chemical or enzymatic cleavage from a solid substrate, and / or removing one or more chemical moieties (e.g., removing protecting groups). In some embodiments, the post-synthesis step includes chemical conjugation with a conjugating agent (e.g., carboxylic acid, carboxylate, carbonate, and carboxylic acid activator reagent).

[0335] As used herein, the term "retentate" refers to a substance (e.g., double-stranded nucleic acid) that does not pass through a barrier (e.g., a membrane, e.g., a semi-permeable membrane, e.g., a filter) in, for example, an ultrafiltration method described herein. In some embodiments, the retentate comprises double-stranded nucleic acids. In some embodiments, the permeate comprises other mixture components. In some embodiments, the retentate comprises little or no detectable level of single-stranded nucleic acids. In some embodiments, the stoichiometric ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate is at least 100: 1, 1,000: 1, 10,000: 1, 100,000: 1, 1,000,000: 1 or higher.

[0336] As used herein, the term "selectively retain" refers to retention of desired material, such as in a retentate, such as by a method and / or system or device as described herein. In some embodiments, the methods, devices and systems described herein selectively retain double-stranded nucleic acids.

[0337] As used herein, the term "single-stranded form" refers to a compound comprising a substantially (e.g., completely) single-stranded nucleic acid or oligonucleotide, which, for example, does not interact (e.g., hybridize) with another nucleic acid or oligonucleotide (e.g., another nucleic acid that is sufficiently complementary or a second portion of the nucleic acid that is sufficiently complementary to the first portion of the nucleic acid).

[0338] As used herein, the term "sufficiently complementary" refers to a first nucleic acid sequence comprising a continuous nucleic acid sequence that allows a second nucleic acid (e.g., a nucleic acid sequence comprising a reverse complementary sequence to a continuous nucleic acid sequence of the first nucleic acid having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to hybridize therewith. Sufficiently complementary sequences may include Watson-Crick base pairs formed by natural and / or modified nucleic acids. Sufficiently complementary sequences may also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine and hypoxanthine-cytosine) and Hoostein base pairs. In some embodiments, the nucleic acid sequence from the first single-stranded nucleic acid is sufficiently complementary to hybridize with the nucleic acid sequence from the second single-stranded nucleic acid to form a double-stranded nucleic acid. In some embodiments, the nucleic acid from the second single-stranded nucleic acid is sufficiently complementary to hybridize with the nucleic acid sequence from the first single-stranded nucleic acid to form a double-stranded nucleic acid. In some embodiments, a first nucleic acid sequence from a first nucleic acid is sufficiently complementary to hybridize with a second nucleic acid sequence from the first nucleic acid, e.g., to form a hairpin loop. In some embodiments, a first nucleic acid sequence from a second nucleic acid is sufficiently complementary to hybridize with a second nucleic acid sequence from the second nucleic acid, e.g., to form a hairpin loop. In some embodiments, a first nucleic acid sequence from a first nucleic acid is not sufficiently complementary to hybridize with a second nucleic acid sequence from the first nucleic acid, e.g., to form a hairpin loop. In some embodiments, a first nucleic acid sequence from a second nucleic acid is not sufficiently complementary to hybridize with a second nucleic acid sequence from the second nucleic acid, e.g., to form a hairpin loop.

[0339] As used herein, the term "synthesis phase" generally refers to one or more steps of a method for producing a product or for one or more additions to a substrate. In some embodiments, the synthesis phase includes one or more "synthesis steps" of the method as described herein, for example, wherein the step adds one or more nucleotide monomers to the nucleic acid of the solid medium loaded. In some embodiments, the one or more steps of the synthesis phase can be repeated, for example, one or more nucleotide monomers are added to the nucleic acid of the solid medium loaded sequentially. In some embodiments, except the step included in the synthesis phase or the synthesis phase, the method for making oligonucleotides also includes additional stages or steps. In some embodiments, the synthesis step includes the solid-state chemical synthesis of the first single-stranded nucleic acid, the second single-stranded nucleic acid or both. In some embodiments, the oligonucleotide synthesis relates to creating an oligonucleotide via polymerase chain reaction or generates an oligonucleotide via a cell organism.

[0340] As used herein, the term "transmembrane pressure" refers to the hydrostatic pressure gradient that allows ultrafiltration or convection across a barrier (e.g., a membrane, such as a semipermeable membrane, e.g., as described herein). In some embodiments, the transmembrane pressure is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 psi. In some embodiments, the transmembrane pressure is no greater than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 psi.

[0341] As used herein, the term "ultrafiltration" refers to a filtration step comprising applying a solution to a pressure-driven barrier (e.g., a membrane, such as a semipermeable membrane). High molecular weight solutes are retained, while water and low molecular weight solutes pass through the barrier (e.g., a membrane, such as a semipermeable membrane). In some embodiments, ultrafiltration comprises diafiltration. In certain embodiments, ultrafiltration comprises tangential flow filtration. In some embodiments, ultrafiltration comprises diafiltration or tangential flow filtration.

[0342] II. Production Method

[0343] The present disclosure relates in part to methods for producing (e.g., manufacturing) purified oligonucleotides (e.g., purified single-stranded oligonucleotides and / or purified double-stranded oligonucleotides). Generally, the method for producing (e.g., manufacturing) the purified oligonucleotides of the present disclosure includes one or more steps of purifying duplexed (e.g., double-stranded) oligonucleotides from a mixture (e.g., a mixture comprising double-stranded oligonucleotides and single-stranded oligonucleotides). In certain embodiments, the method includes or is a manufacturing method, such as a large-scale manufacturing method (e.g., not a research-scale method or not an analytical method). In some embodiments, the method for producing (e.g., manufacturing) the purified oligonucleotides of the present disclosure includes one or more steps of purifying oligonucleotides in double-stranded form (e.g., double-stranded oligonucleotides). In some embodiments, the method for producing (e.g., manufacturing) the purified oligonucleotides of the present disclosure includes one or more steps of purifying oligonucleotides in single-stranded form (e.g., single-stranded oligonucleotides). The method for producing (e.g., manufacturing) purified oligonucleotides (e.g., purified single-stranded nucleic acids or purified double-stranded oligonucleotides) may include, for example, providing a mixture; subjecting the mixture to an ultrafiltration step; and harvesting a retentate (e.g., a retentate comprising double-stranded oligonucleotides). A method of producing (eg, manufacturing) a purified oligonucleotide (eg, a purified single-stranded nucleic acid or double-stranded oligonucleotide) can include, for example, providing a mixture; subjecting the mixture to an ultrafiltration step; and harvesting the permeate.

[0344] In some embodiments, providing a mixture includes: providing a first single-stranded nucleic acid and a second single-stranded nucleic acid, wherein the first single-stranded nucleic acid and the second single-stranded nucleic acid contain sequences that are sufficiently complementary to each other to hybridize under conditions suitable for hybridization, for example, as described in Examples 1 to 5; and combining the first single-stranded nucleic acid and the second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture, wherein the mixture contains: a double-stranded nucleic acid containing the first single-stranded nucleic acid and the second single-stranded nucleic acid, and the first single-stranded nucleic acid in single-stranded form, the second single-stranded nucleic acid in single-stranded form, or the first single-stranded nucleic acid in single-stranded form and the second single-stranded nucleic acid in single-stranded form.

[0345] In some embodiments, providing the first single-stranded nucleic acid and the second single-stranded nucleic acid comprises a synthesis step. In some embodiments, the synthesis step comprises solid-state chemical synthesis of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or the first single-stranded nucleic acid and the second single-stranded nucleic acid.

[0346] In some embodiments, providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes a post-synthetic step. In some embodiments, the post-synthetic step includes chemical or enzymatic cleavage from a solid substrate, removal of one or more chemical moieties (e.g., removal of protecting groups), or chemical or enzymatic cleavage from a solid substrate and removal of one or more chemical moieties (e.g., removal of protecting groups). In some embodiments, the post-synthetic step includes chemical conjugation with a conjugation reagent (e.g., carboxylic acids, carboxylate esters, carbonates, and carboxylic acid activator reagents).

[0347] In some embodiments, providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes a purification step. In some embodiments, the purification step is a step of enriching the nucleic acid component and reducing the level of the non-nucleic acid component. In some embodiments, the purification step includes a chromatography step, such as anion exchange chromatography.

[0348] In some embodiments, providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes subjecting the first single-stranded nucleic acid, the second single-stranded nucleic acid, or the first single-stranded nucleic acid and the second single-stranded nucleic acid to an ultrafiltration step that selectively retains single-stranded nucleic acids. In some embodiments, the ultrafiltration step selectively retains single-stranded nucleic acids without retaining non-nucleic acid components.

[0349] In some embodiments, the first single-stranded nucleic acid is at least 10 nucleotides long, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the first single-stranded nucleic acid is no more than 100 nucleotides long, such as no more than 80, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the first single-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long and optionally no more than 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides long.

[0350] In some embodiments, the first single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 1 0-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 2 2-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12-30, 20, 14-20, 16-20, 18-20, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0351] In some embodiments, the first single stranded nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0352] In some embodiments, the second single-stranded nucleic acid is at least 10 nucleotides long, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the second single-stranded nucleic acid is no more than 100 nucleotides long, such as no more than 80, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the second single-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long and optionally no more than 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides long.

[0353] In some embodiments, the second single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 1 0-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 2 2-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12-30, 20, 14-20, 16-20, 18-20, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0354] In some embodiments, the second single stranded nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0355] In some embodiments, the double-stranded nucleic acid is at least 10 base pairs long, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs long. In some embodiments, the double-stranded nucleic acid is no more than 100 base pairs long, such as no more than 80, 60, 50, 45, 40, 35, or 30 base pairs long. In some embodiments, the double-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs long and optionally no more than 100, 80, 60, 50, 45, 40, 35, or 30 base pairs long.

[0356] In some embodiments, the double-stranded nucleic acid is 10-50 base pairs long, for example, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50. 50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40, 20-4 0, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12-30 , 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 base pairs in length.

[0357] In some embodiments, the double stranded nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0358] In some embodiments, the double-stranded nucleic acid comprises a sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleic acid sequence of interest (e.g., a sequence of a target gene, a non-coding RNA, a primer, or a component for molecular assembly). In some embodiments, the double-stranded nucleic acid comprises a sequence that differs from a nucleic acid sequence of interest (e.g., a sequence of a target gene, a non-coding RNA, a primer, or a component for molecular assembly) by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0359] In some embodiments, the double-stranded nucleic acid comprises a sequence having at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to a nucleic acid sequence of interest (e.g., a target gene, a non-coding RNA, a primer, or a sequence of a component for molecular assembly). In some embodiments, the double-stranded nucleic acid comprises a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from a sequence complementary to a nucleic acid sequence of interest (e.g., a target gene, a non-coding RNA, a primer, or a sequence of a component for molecular assembly).

[0360] In some embodiments, the ultrafiltration step includes diafiltration. Generally, diafiltration includes, for example, using a permeable filter, based on the molecular size (for example, molecular weight) of the component in the solution, separating the component (for example, double-stranded nucleic acid, single-stranded nucleic acid, salt, solvent or other molecules as described herein). In some embodiments, diafiltration includes making the solution to be filtered flow through a permeable filter, for example, as described herein. In some embodiments, diafiltration includes filtering the solution repeatedly through a permeable filter, for example, as described herein, for example, up to a specific duration. In some embodiments, diafiltration is performed for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes or 50 minutes, or at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours. In some embodiments, diafiltration is performed for a period of time sufficient to filter at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV). In some embodiments, the ultrafiltration is applied at a pressure of about 5-40 psi, 10-40 psi, 15-40 psi, 20-40 psi, 30-40 psi, 35-40 psi, 5-15 psi, 5-20 psi, 5-25 psi, 5-30 psi, 5-35 psi, 10-30 psi, 15-25 psi, 5-10 psi, 10-15 psi, 15-20 psi, 20-25 psi, 25-30 psi, 30-35 psi, or 35-40 psi. In some embodiments, ultrafiltration is applied at a pressure of about 15-30 psi (e.g., about 15-25 psi, 15-20 psi, 25-30 psi, 20-30 psi, 15-20 psi, 20-25 psi, or 25-30 psi, such as about 15 psi, 16 psi, 17 psi, 18 psi, 19 psi, 20 psi, 21 psi, 22 psi, 23 psi, 24 psi, 25 psi, 26 psi, 27 psi, 28 psi, 29 psi, or 30 psi).In some embodiments, ultrafiltration is performed at a temperature of about 10-40°C, such as about 15-40°C, 20-40°C, 25-40°C, 30-40°C, 35-40°C, 10-35°C, 10-30°C, 10-25°C, 10-20°C, 10-15°C, 15-35°C, 20-30°C, 10-15°C, 15-20°C, 20-25°C, 25-30°C, 30-35°C, or 35-40°C, such as about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C.

[0361] In some embodiments, the manufacturing method further comprises analyzing the retentate by non-denaturing HPLC (e.g., to determine the relative amounts of double-stranded nucleic acids and / or single-stranded nucleic acids in the retentate), e.g., as described in Examples 1 to 5. In some embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate (e.g., as determined by non-denaturing HPLC) increases over time and / or as the total diafiltration volume (DTV) increases. In some embodiments, the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retentate (e.g., as determined by native HPLC) is at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1.

[0362] In some embodiments, the method reduces the level of low molecular weight impurities by at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the low molecular weight impurities each have a molecular weight of less than about 500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 Daltons. In some embodiments, the low molecular weight impurities include salts and / or trace amounts of solvents.

[0363] In some embodiments, the method reduces the level of double-stranded nucleic acids by no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50%. In some embodiments, the ratio of the concentration of double-stranded nucleic acids in the retentate to the concentration of double-stranded nucleic acids in the mixture is at least 0.75, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995, or 0.999. In some embodiments, the ratio of the concentration of double-stranded nucleic acids in the retentate to the concentration of double-stranded nucleic acids in the mixture is the ratio of the concentration of double-stranded nucleic acids in the retentate to the concentration of double-stranded nucleic acids in the mixture before a purification step (e.g., before an ultrafiltration step).

[0364] In some embodiments, the method reduces the level of single-stranded nucleic acids by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the ratio of the concentration of single-stranded nucleic acids in the retentate to the concentration of single-stranded nucleic acids in the mixture is at most 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, or 0.01. In some embodiments, the ratio of the concentration of single-stranded nucleic acids in the retentate to the concentration of single-stranded nucleic acids in the mixture is the ratio of the concentration of single-stranded nucleic acids in the retentate to the concentration of single-stranded nucleic acids in the mixture before the purification step (e.g., before the ultrafiltration step).

[0365] In some embodiments, the method produces a purified double-stranded nucleic acid that achieves a purity standard. In some embodiments, the purity standard comprises a retentate comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% double-stranded nucleic acid (e.g., and comprising less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% single-stranded nucleic acid). In some embodiments, the purity standard comprises a retentate comprising 50-100%, 55-100%, 60-100%, 65-100%, 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 91-100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100%, 80-99%, 85-99%, 90-99%, 91-99%, 92-99%, 93-99%, 94-99%, 95-99%, 96-99%, 97-99%, or 98-99% double stranded nucleic acid. In some embodiments, the purity standard comprises a retentate comprising 1-50%, 1-45%, 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, or 1-2% single stranded nucleic acid. In some embodiments, the method achieves a purity standard of no greater than 25, 24, 23, 22, 21, or 20 DTV.

[0366] A. Purification of double-stranded nucleic acids

[0367] The present disclosure relates in part to methods of producing (eg, manufacturing) double-stranded nucleic acids. Generally, the methods of the present disclosure include one or more steps of selectively retaining double-stranded nucleic acids, eg, from a mixture containing other mixture components.

[0368] In some embodiments, a method for producing (e.g., manufacturing) a purified double-stranded nucleic acid comprises one or more steps of selectively retaining the double-stranded nucleic acid from a mixture (e.g., comprising other mixture components (e.g., undesired products (e.g., single-stranded nucleic acids), salts (e.g., halides or mineral salts), buffers, production reagents or byproducts (e.g., truncated nucleic acid sequences or undesired nucleic acid sequences), double-stranded nucleic acids of shorter length (e.g., shorter than the first single-stranded nucleic acid and / or the second single-stranded nucleic acid of interest), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine), thiolation agents (e.g., xanthane, hydride, or PADS), capping agents (e.g., acetic anhydride or NMI), coupling agents (e.g., phosphoramidite or ETT), detritylation agents (e.g., dichloroacetic acid), deprotection agents (e.g., diethylamine, methylamine, or ammonia))) and / or conjugation agents (e.g., a mixture of carboxylic acids, carboxylic acid esters, carbonates, and carboxylic acid activator reagents).

[0369] In some embodiments, the mixture comprises a double-stranded nucleic acid, the double-stranded nucleic acid comprises a first single-stranded nucleic acid and a second single-stranded nucleic acid; and a single-stranded nucleic acid, such as a first single-stranded nucleic acid in single-stranded form, a second single-stranded nucleic acid in single-stranded form, or a first single-stranded nucleic acid in single-stranded form and a second single-stranded nucleic acid in single-stranded form. In some embodiments, the method for producing (e.g., manufacturing) purifying double-stranded nucleic acids includes subjecting the mixture (e.g., a mixture described herein) to an ultrafiltration step, the ultrafiltration step selectively retaining a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid without retaining single-stranded nucleic acids (e.g., the first single-stranded nucleic acid and the second single-stranded nucleic acid in single-stranded form). In some embodiments, the method for producing (e.g., manufacturing) purifying double-stranded nucleic acids includes harvesting a retentate after subjecting the mixture to an ultrafiltration step. In some embodiments, the retentate comprises a double-stranded nucleic acid containing the first single-stranded nucleic acid and the second single-stranded nucleic acid, and does not comprise single-stranded nucleic acids (e.g., the first single-stranded nucleic acid and the second single-stranded nucleic acid in single-stranded form). In some embodiments, a method of producing (e.g., manufacturing) a purified double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid comprises separating the double-stranded nucleic acid from the single-stranded form of the first single-stranded nucleic acid and / or the single-stranded form of the second single-stranded nucleic acid.

[0370] In some embodiments, the double-stranded nucleic acid is at least 10 base pairs long, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 base pairs long. In some embodiments, the double-stranded nucleic acid is no more than 100 base pairs long, such as no more than 80, 60, 50, 45, 40, 35, or 30 base pairs long. In some embodiments, the double-stranded nucleic acid is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 to about 200 base pairs. In some embodiments, the double-stranded nucleic acid is a portion of a single-stranded nucleic acid and is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 to about 200 base pairs. In some embodiments, the double-stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 190 to about 200 base pairs long and optionally no more than 100, 80, 60, 50, 45, 40, 35 or 30 base pairs long.

[0371] In some embodiments, the double-stranded nucleic acid is 10-50 base pairs long, for example, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50. 50, 10-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40, 20-4 0, 22-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12-30 , 14-30, 16-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 base pairs in length.

[0372] In some embodiments, the double stranded nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0373] In some embodiments, the double-stranded nucleic acid is 100-200 base pairs long, such as 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or 190-200 base pairs long.

[0374] In some embodiments, the double-stranded nucleic acid comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleic acid sequence of interest (e.g., a sequence of a target gene, a non-coding RNA, a primer, or a component for molecular assembly). In some embodiments, the double-stranded nucleic acid comprises a sequence that differs from a nucleic acid sequence of interest (e.g., a sequence of a target gene, a non-coding RNA, a primer, or a component for molecular assembly) by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0375] In some embodiments, the double-stranded nucleic acid comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to a nucleic acid sequence of interest (e.g., a target gene, a non-coding RNA, a primer, or a sequence of a component for molecular assembly). In some embodiments, the double-stranded nucleic acid comprises a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from a sequence complementary to a nucleic acid sequence of interest (e.g., a target gene, a non-coding RNA, a primer, or a sequence of a component for molecular assembly).

[0376] In some embodiments, the length of at least one strand of the double-stranded nucleic acid is the same as the length of the single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid is a component of the double-stranded nucleic acid (eg, the double-stranded nucleic acid comprises a copy of the single-stranded nucleic acid).

[0377] In some embodiments, a double-stranded nucleic acid comprises a blunt end (e.g., one blunt end). In some embodiments, a double-stranded nucleic acid comprises two blunt ends. In some embodiments, a double-stranded nucleic acid comprises a single-stranded portion (e.g., a terminal overhang, such as on one end). In some embodiments, a double-stranded nucleic acid comprises a single-stranded portion (e.g., a terminal overhang, such as on both ends).

[0378] In some embodiments, the double-stranded nucleic acid comprises a conjugated group. In some embodiments, the double-stranded nucleic acid comprises a conjugated group, for example, is or comprises PEG (polyethylene glycol), an amino linker, GalNAc, or an ethylene glycol spacer.

[0379] In some embodiments, a double-stranded nucleic acid comprises a mismatch (e.g., at one of the ends of the double-stranded nucleic acid, within 1-2 nucleotides of one of the ends of the double-stranded nucleic acid, or between the ends of the double-stranded nucleic acid).

[0380] In some embodiments, the melting temperature of the double-stranded nucleic acid in the mixture is between about 20-85° C., such as between about 25-80° C., 30-75° C., 35-70° C., 40-65° C., 45-60° C., 20-25° C., 25-30° C., 30-35° C., 35-40° C., 40-45° C., 45-50° C., 50-55° C., 55-60° C., 60-65° C., 65-70° C., 70-75° C., 75-80° C., or 80-85° C. In some embodiments, the melting temperature of the double-stranded nucleic acid in the mixture is related to the salt concentration in the mixture. In some embodiments, the relationship between the melting temperature of double-stranded nucleic acids in a mixture and the concentration of salt (e.g., cations such as sodium ions) in the mixture is determined according to the method described in Owczarzy et al. (2004, Biochemistry 43:3537-3554; incorporated herein by reference in its entirety).

[0381] In some embodiments, the ratio of the concentration of double stranded nucleic acids in the retentate to the concentration of double stranded nucleic acids in the mixture is at least 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995, 0.999, 1.00, 1.01, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, or 1.55. In some embodiments, the amount of double stranded nucleic acid in the retentate is at least 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, or 1200 OD / mL. In some embodiments, the amount of double-stranded nucleic acid in the retentate is between about 500-1200 OD / mL (e.g., between about 500-550 OD / mL, 550-600 OD / mL, 600-650 OD / mL, 650-700 OD / mL, 700-750 OD / mL, 750-800 OD / mL, 800-850 OD / mL, 850-900 OD / mL, 900-950 OD / mL, 950-1000 OD / mL, 1000-1050 OD / mL, 1050-1100 OD / mL, 1100-1150 OD / mL, or 1150-1200 OD / mL). In some embodiments, the amount of double-stranded nucleic acid in the retentate is at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 mg / mL. In some embodiments, the amount of double-stranded nucleic acid in the retentate is between about 20-48 mg / mL (e.g., between about 20-25, 25-30, 30-35, 35-40, 40-45, or 45-48 mg / mL).

[0382] In some embodiments, the ratio of the amount of double-stranded nucleic acids in the retentate to the amount of double-stranded nucleic acids in the mixture is at least 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995, 0.999, or 1.00. In some embodiments, the ratio of the amount of single-stranded nucleic acids in the retentate to the amount of single-stranded nucleic acids in the mixture is at most 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10, 0.05, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0383] In some embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate (e.g., as determined by native HPLC) increases over time and / or as the total diafiltration volume (DTV) increases. In certain embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the retentate (e.g., as determined by native HPLC) is at least 50:50, 70:25, 70:30, 75:20, 75:25, 80:15, 80:20, 85:10, 85:15, 90:5, 90:10, 90.3:5, 90.3, 9.7, 90.4:4.8, 90.4:9.6, 90.9:4.8, 90.9:9. 1. 91.1:4.6, 91.1:8.9, 91.2:4.5, 91.2:8.2, 91.1:4.4, 91.1:8.9, 91.2:4.4, 91.2:8.8, 91.3:4.1, 91.3:8.7, 91.5:3.9, 91.5:8.5, 92:3, 92:8, 94:5, 94:6, 95:2, 95:5, 98:1, 98:2, 99:1 or 99.5:0.5.

[0384] In some embodiments, the ratio of the concentration of double-stranded nucleic acids in the retentate to the concentration of single-stranded nucleic acids in the mixture is at least 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995, 0.999, 1.00, 1.01, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, or 1.55. In some embodiments, the ratio of the amount of single-stranded nucleic acid in the retentate to the amount of double-stranded nucleic acid in the mixture is at least 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, 0.995, 0.999, 1.00, 1.01, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, or 1.55.

[0385] In some embodiments, providing a double-stranded nucleic acid includes annealing a first single-stranded nucleic acid and a second single-stranded nucleic acid. An individual purified aqueous oligonucleotide matrix is ​​introduced into a container equipped with mixing, heating / cooling and nitrogen atmosphere capabilities. The amount of each single-stranded nucleic acid is added in equal parts to achieve a desired molar ratio (e.g., equal amounts or excess). The ratio is verified by non-denaturing analytical chromatography (e.g., HPLC, SEC). The mixture of aqueous single-stranded nucleic acids is then thermally cycled (e.g., annealed) to promote hybridization to form a duplex nucleic acid. Annealing is performed in the following manner: the solution is first cooled to below room temperature (about 5°C-10°C (e.g., about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C)), then heated to an elevated temperature with a controlled heating rate (gradient) to achieve a T close to the hybridized duplex. m of the room temperature and maintained at this elevated temperature for a short amount of time, followed by cooling at a controlled rate (gradient) back to below room temperature (about 5°C-10°C (e.g., (e.g., about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C)) to obtain a concentrated aqueous solution of double-stranded nucleic acid.

[0386] B. Purified single-stranded nucleic acid

[0387] The present disclosure relates in part to a method for purifying single-stranded nucleic acids. Generally speaking, the method of the present disclosure includes, for example, one or more steps of selectively retaining double-stranded nucleic acids from a mixture comprising single-stranded nucleic acids, double-stranded nucleic acids, and / or other mixture components. In some embodiments, the method for purifying single-stranded nucleic acids does not include the synthesis of single-stranded nucleic acids, but still includes one or more steps of removing single-stranded nucleic acids from the mixture.

[0388] In some embodiments, a method of making a purified single-stranded nucleic acid comprises one or more steps of selectively retaining double-stranded nucleic acids from a mixture (e.g., comprising other mixture components (e.g., undesired products (e.g., single-stranded nucleic acids), salts (e.g., halides or mineral salts), buffers, production reagents or byproducts (e.g., truncated nucleic acid sequences or undesired nucleic acid sequences), single-stranded nucleic acids of shorter length (e.g., shorter than the first single-stranded nucleic acid of interest and / or the second single-stranded nucleic acid), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine), thiolation agents (e.g., xanthane, hydride, or PADS), capping agents (e.g., acetic anhydride or NMI), coupling agents (e.g., phosphoramidites or ETT), detritylation agents (e.g., dichloroacetic acid), deprotection agents (e.g., diethylamine, ethylenediamine, methylamine, or ammonia))), and / or conjugation agents (e.g., mixtures of carboxylic acids, carboxylic acid esters, carbonates, and carboxylic acid activator reagents). In some embodiments, the single-stranded nucleic acids are within the permeate. In some embodiments, the single-stranded nucleic acid is within the permeate, and the permeate is collected.

[0389] In some embodiments, the first single-stranded nucleic acid is at least 10 nucleotides long, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides long. In some embodiments, the double-stranded nucleic acid is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 to about 200 nucleotides. In some embodiments, the double stranded nucleic acid is a portion of a single stranded nucleic acid and is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 to about 200 nucleotides. In some embodiments, the first single stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length, and optionally is no more than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the first single-stranded nucleic acid is no more than 100 nucleotides long, such as no more than 80, 60, 50, 45, 40, 35, or 30 nucleotides long.

[0390] In some embodiments, the first single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 1 0-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 2 2-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12-30, 20, 14-20, 16-20, 18-20, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0391] In some embodiments, the first single stranded nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0392] In some embodiments, the second single-stranded nucleic acid is at least 10 nucleotides long, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides long. In some embodiments, the second single-stranded nucleic acid is no more than 100 nucleotides long, such as no more than 80, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the second single stranded nucleic acid is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length, and optionally is no more than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length.

[0393] In some embodiments, the second single-stranded nucleic acid is 10-50, 12-50, 14-50, 16-50, 18-50, 20-50, 22-50, 24-50, 26-50, 28-50, 30-50, 32-50, 34-50, 36-50, 38-50, 40-50, 42-50, 44-50, 46-50, 48-50, 1 0-45, 12-45, 14-45, 16-45, 18-45, 20-45, 22-45, 24-45, 26-45, 28-45, 30-45, 32-45, 34-45, 36-45, 38-45, 40-45, 42-45, 10-40, 12-40, 14-40, 16-40, 18-40, 20-40, 2 2-40, 24-40, 26-40, 28-40, 30-40, 32-40, 34-40, 36-40, 38-40, 10-35, 12-35, 14-35, 16-35, 18-35, 20-35, 22-35, 24-35, 26-35, 28-35, 30-35, 32-35, 10-30, 12-30, 20, 14-20, 16-20, 18-20, 20-30, 22-30, 24-30, 26-30, 28-30, 10-25, 12-25, 14-25, 16-25, 18-25, 20-25, 22-25, 10-20, 12-20, 14-20, 16-20, 18-20, 10-15, or 12-15 nucleotides in length.

[0394] In some embodiments, the second single stranded nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0395] In some embodiments, the length of at least one strand of the double-stranded nucleic acid is the same as the length of the single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid is a component of the double-stranded nucleic acid (eg, the double-stranded nucleic acid comprises a copy of the single-stranded nucleic acid).

[0396] III. Device

[0397] In some embodiments, the ultrafiltration step comprises applying the mixture (e.g., a mixture described herein) to a filtration device (e.g., a device comprising a barrier layer (e.g., a membrane, such as a semi-permeable membrane)). In some embodiments, the device comprises a first chamber, a second chamber, and / or a filter element, such as a barrier layer (e.g., a membrane (e.g., a semi-permeable membrane)), the filter element being disposed between the first chamber and the second chamber, for example, as described herein. In some embodiments, the first chamber is configured to hold a mixture to be filtered (e.g., a mixture described herein) and optionally capture a retentate. In some embodiments, the second chamber is configured to capture a permeate. In some embodiments, the mixture comprises a double-stranded nucleic acid (e.g., comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid) and a single-stranded nucleic acid (e.g., a first single-stranded nucleic acid in single-stranded form, a second single-stranded nucleic acid in single-stranded form, or a first single-stranded nucleic acid in single-stranded form and a second single-stranded nucleic acid in single-stranded form). In some embodiments, the filter element (e.g., a barrier layer (e.g., a membrane, such as a semi-permeable membrane)) selectively retains double-stranded nucleic acids (e.g., comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid), while not retaining single-stranded nucleic acids (e.g., the first single-stranded nucleic acid and / or the second single-stranded nucleic acid in single-stranded form). In some embodiments, the device does not substantially retain one or more other mixture components (e.g., the device does not retain a detectable amount of one or more other mixture components), e.g., as described herein.

[0398] In some embodiments, the device is pressurized (e.g., at a pressure of about 5-40 psi, in some cases in the mid-psi range (about 15-30 psi)) to concentrate the matrix onto the retentate side of the membrane (e.g., a semi-permeable membrane). Then, in some embodiments, process water can be introduced under pressure to facilitate filtration (e.g., diafiltration) of the other mixture components.

[0399] In some embodiments, the ultrafiltration step produces retentate and permeate. In some embodiments, relative to the mixture through ultrafiltration, the retentate is enriched with for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the desired molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid). In some embodiments, relative to the mixture through ultrafiltration, the retentate is enriched with for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the double-stranded nucleic acid. In some embodiments, the retentate is enriched in single-stranded nucleic acid relative to the ultrafiltered mixture, for example by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0400] In some embodiments, the desired molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) is enriched to 2% to at least 90% purity, 5% to at least 90% purity, 10% to at least 90% purity, 15% to at least 90% purity, 20% to at least 90% purity, 25% to at least 90% purity, 30% to at least 90% purity, 35% to at least 90% purity, 40% to at least 90% purity, 45% to at least 90% purity, 90% purity, 50% to at least 90% purity, 55% to at least 90% purity, 60% to at least 90% purity, 65% to at least 90% purity, 70% to at least 90% purity, 75% to at least 90% purity, 80% to at least 90% purity, 85% to at least 90% purity, 90% to at least 92.5% purity, 95% to at least 97.5% purity, or 97.5% to at least 99% purity. In some embodiments, the desired molecule of interest is enriched to a purity of 50% to at least 90% purity, 55% to at least 90% purity, 60% to at least 90% purity, 65% to at least 90% purity, 70% to at least 90% purity, 75% to at least 90% purity, 80% to at least 90% purity, 85% to at least 90% purity, 90% to at least 92.5% purity, 92.5% to at least 95% purity, 95% to at least 97.5% purity, or 97.5% to at least 99% purity.

[0401] In some embodiments, the purity of the molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) is increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, or 5,000-fold after ultrafiltration.

[0402] In some embodiments, the ultrafiltration step produces a retentate and a permeate. In some embodiments, relative to the permeate, the retentate is enriched with, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the desired molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid). In some embodiments, relative to the permeate, the retentate is enriched with, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the double-stranded nucleic acid. In some embodiments, the permeate is enriched in, e.g., at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% single stranded nucleic acid relative to the permeate.

[0403] In some embodiments, the retentate comprises a different concentration of salt, buffer, or one or more of production reagents or by-products than the permeate, mixture, or permeate and mixture. In some embodiments, the retentate has a lower concentration of salt, buffer, or one or more of production reagents or by-products than the permeate, mixture, or permeate and mixture.

[0404] In some embodiments, the second chamber comprises a permeate. In some embodiments, the second chamber comprises a permeate, e.g., comprises a single-stranded nucleic acid. In some embodiments, the second chamber retains the permeate. In some embodiments, the second chamber does not retain the permeate. In some embodiments, the second chamber does not retain the permeate, and the permeate flows out of the second chamber and is discarded.

[0405] In some embodiments, the single-stranded nucleic acids in the permeate consist primarily of the sense strand (e.g., wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the single-stranded nucleic acids in the permeate consist of the sense strand). In some embodiments, the single-stranded nucleic acids in the permeate consist primarily of the antisense strand (e.g., wherein at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the single-stranded nucleic acids in the permeate consist of the antisense strand).

[0406] A. Membrane

[0407] In some embodiments, the filter device or filter element membrane comprises one or more of the following: polysulfone, polypropylene, cellulose acetate, polylactic acid, nitrocellulose, mixed cellulose ester nitrocellulose, regenerated cellulose, polyethersulfone, polyamide, cellulose derivatives Polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track-etched membrane. In some embodiments, the filtration device or filter element is or includes a hollow fiber, tubular, spiral wound, cassette, plate, or frame membrane type filter.

[0408] 1. Molecular weight cut-off (MWCO)

[0409] In some embodiments, the barrier (e.g., a membrane, such as a semi-permeable membrane) comprises a molecular weight cutoff (MWCO). In some embodiments, a barrier having a MWCO selectively retains double-stranded nucleic acids (e.g., in preference to single-stranded nucleic acids). In some embodiments, a barrier having a MWCO selectively allows single-stranded nucleic acids to flow through (e.g., in preference to double-stranded nucleic acids). In some embodiments, the MWCO is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kilodaltons less than the MW of the double-stranded nucleic acid. In some embodiments, the MWCO is no more than about 300, 250, 200, 150, 100, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 kilodaltons less than the MW of the double-stranded nucleic acid. In some embodiments, the MWCO is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kilodaltons less than the MW of a double-stranded nucleic acid, and is no more than about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 kilodaltons less than the MW of a double-stranded nucleic acid. In some embodiments, the MWCO is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300 kilodaltons greater than the molecular weight of a single-stranded nucleic acid (e.g., a first single-stranded nucleic acid, a second single-stranded nucleic acid, or both). In some embodiments, the MWCO is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300 kilodaltons less than the molecular weight of a double-stranded nucleic acid. In some embodiments, the MWCO is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300 kilodaltons greater than the molecular weight of a single stranded nucleic acid (e.g., a first single stranded nucleic acid, a second single stranded nucleic acid, or both) and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300 kilodaltons less than the molecular weight of a double stranded nucleic acid.In some embodiments, the MWCO is about 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 15-30, 20-30, 25-30, 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20, 1-15, 1-2 kilodaltons. In some embodiments, the MWCO is about 1-30, 2-30, 3-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 15-30, 20-30, 25-30, 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 15-25, 20-25, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20, 1-15, 2-15, 3-15, 4 -15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1-9, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, 4-5, 1-4, 2-4, 3-4, 1-3, 2-3, or 1-2 kilodaltons. In certain embodiments, the MWCO is at least about 2 kilodaltons greater than the molecular weight of a single-stranded nucleic acid (eg, a first single-stranded nucleic acid, a second single-stranded nucleic acid, or both).In certain embodiments, the MWCO is at least about 5 kilodaltons less than the molecular weight of the double-stranded nucleic acid. In certain embodiments, the MWCO is at least about 2 kilodaltons greater than the molecular weight of a single-stranded nucleic acid (e.g., a first single-stranded nucleic acid, a second single-stranded nucleic acid, or both) and at least about 5 kilodaltons less than the molecular weight of the double-stranded nucleic acid.

[0410] In some embodiments, the MWCO is about 0.5-100, 0.5-80, 0.5-60, 0.5-50, 0.5-40, 0.5-30, 0.5-20, 0.5-15, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-100, 1-80, 1-60, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-8, 1-6, 1 -4, 1-3, 1-2, 2-100, 2-80, 2-60, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-8, 2-6, 2-4, 2-3, 3-100, 3-80, 3-60, 3-50, 3-40, 3-30, 3-20, 3-15, 3-10, 3-8, 3-6, 3-4, 4-100, 4-80, 4-60, 4-50, 4-40, 4-30, 4-20, 4-1 5, 4-10, 4-8, 4-6, 6-100, 6-80, 6-60, 6-50, 6-40, 6-30, 6-20, 6-15, 6-10, 6-8, 8-100, 8-80, 8-60, 8-50, 8-40, 8-30, 8-20, 8-15, 8-10, 10-100, 10-80, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15, 15-100, 15- In some embodiments, the MWCO is at least 100, 125, 150, 175, 200, 225, 250, 275, or 300 kilodaltons. In some embodiments, the MWCO is about 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, or 275-300 kilodaltons. In some embodiments, the MWCO is about 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, or 15-16 kDa. In certain embodiments, the MWCO is about 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 kDa. In specific embodiments, the MWCO is about 8-15 kDa.In certain embodiments, the MWCO is about 10 kDa.

[0411] In some embodiments, a barrier having a MWCO of about 10 kDa (e.g., about 7, 8, 9, 10, 11, 12, 13, or 14 kDa) selectively retains double-stranded nucleic acids without significant denaturation (e.g., no detectable denaturation of the double-stranded nucleic acids), e.g., at a transmembrane pressure of about 15-50 psi (e.g., about 15-20, 20-25, 23-30, 25-35, 35-45, or 40-50, 55-60). In some embodiments, a barrier having a MWCO of about 10 kDa (e.g., about 7, 8, 9, 10, 11, 12, 13, or 14 kDa) selectively retains double-stranded nucleic acids, wherein less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, 0.0001%, or 0.00001% of the double-stranded nucleic acids in the mixture are denatured.

[0412] 2. Average pore size

[0413] In some embodiments, the barrier layer (eg, a membrane, such as a semi-permeable membrane) comprises pores having a preselected average pore size. In some embodiments, the average pore size is an average pore size that selectively retains double-stranded nucleic acids.

[0414] In some embodiments, the average pore size is at least about 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or 3 nm, and no greater than 4, 3.8, 3.6, 3.4, 3.2, 3, 2.8, 2.6, 2.4, 2.2, 2, 1.8, or 1.6 nm.

[0415] In some embodiments, the average pore size is about 0.05-20, 0.1-20, 0.5-20, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 15-20, 0.05-10, 0.1-10, 0.5-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 0.05-8, 0.1-8, 0.5-8, 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, 0.05-6, 0.1-6, 0.5-6, 1-6, 2-6, 3-6, 4-6, 5-6, 0.05-4, 0.1-4, 0.5-4, 1-4, 2-4, 3-4, 0.05-2, 0.1-2, 0.5-2, 1-2, 0.05-1, 0.1-1, or 0.5-1 μm.

[0416] In some embodiments, the average pore size is greater than Z (and optionally, less than 0.8Y, 0.85Y, 0.9Y, 0.95Y, 0.99Y, Y, or 1.1Y), where Z is the numerical average of X and Y, where X is the minimum pore size (e.g., less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV)); and wherein Y is the minimum pore size that will allow at least 90% of the double-stranded nucleic acids to permeate through the membrane (e.g., within less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85, or 90 DTV).

[0417] B. Parameters

[0418] Do not wish to be bound by theory, it is envisioned that in some embodiments, some process parameters are combined with each other to determine the effectiveness of the method for producing (e.g., manufacturing) oligonucleotides (e.g., purification of double-stranded oligonucleotides and / or purification of single-stranded oligonucleotides) is important. In some embodiments, the combination of process parameters is for example about each other, rather than having an optimal configuration when separated. The exemplary combination of process parameters is described below.

[0419] C. Mixture components

[0420] In some embodiments, the mixture comprises a double-stranded nucleic acid and a single-stranded nucleic acid. In some embodiments, the mixture comprises a first single-stranded nucleic acid, a second single-stranded nucleic acid, and a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid. In some embodiments, the mixture comprises a first single-stranded nucleic acid and a second single-stranded nucleic acid, wherein the first single-stranded nucleic acid and the second single-stranded nucleic acid are fully complementary to form a double-stranded nucleic acid, and the mixture comprises a double-stranded nucleic acid. In some embodiments, the mixture comprises a first single-stranded nucleic acid, a second single-stranded nucleic acid, and a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid and other mixture components.

[0421] In some embodiments, the mixture comprises other mixture components. In some embodiments, other mixture components comprise components that are not desired products (e.g., desired molecules of interest, e.g., present in retentate or permeate). In some embodiments, the product comprises double-stranded nucleic acids. In some embodiments, the product comprises single-stranded nucleic acids. In some embodiments, other mixture components include, but are not limited to, salts (e.g., halides or mineral salts), buffers, production reagents or byproducts (e.g., truncated nucleic acid sequences or undesired nucleic acid sequences), single-stranded nucleic acids of shorter length (e.g., shorter than the first single-stranded nucleic acid and / or the second single-stranded nucleic acid of interest), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine), thiolation reagents (e.g., xanthane, hydride, or PADS), capping reagents (e.g., acetic anhydride or NM), coupling reagents (e.g., phosphoramidite or ETT), detritylation reagents (e.g., dichloroacetic acid), deprotection reagents (e.g., diethylamine, methylamine, or ammonia), conjugation reagents (e.g., carboxylic acids, carboxylates, amines, carbonates, and carboxylic acid activator reagents), or any combination thereof.

[0422] In some embodiments, the mixture comprises no more than 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, or 100 μl, or no more than 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml l, 9ml, 10ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml or 100ml, or no more than 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 25 0L, 300L, 350L, 400L, 450L, 500L, 600L, 700L, 800L, 900L or 1000L (and optionally, at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl or 100 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350L, 400L, 450L, 500L, 600L, 700L, 800L, 900L or 1000L).

[0423] In some embodiments, the mixture comprises at least 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl or 100 μl, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20ml, 30ml, 40ml, 50ml, 60ml, 70ml, 80ml, 90ml or 100ml, or at least 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350L, 400L, 450 500L, 600L, 700L, 800L, 900L or 1000L (and optionally no more than 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl or 100 μl, or no more than 0.5L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L , 150L, ​​200L, 250L, 300L, 350L, 400L, 450L, 500L, 600L, 700L, 800L, 900L or 1000L).

[0424] In some embodiments, the mixture comprises: 1-100 μl, 5-100 μl, 10-100 μl, 20-100 μl, 30-100 μl, 40-100 μl, 50-100 μl, 60-100 μl, 70-100 μl, 80-100 μl, 90-100 μl, 1-50 μl, 5-50 μl, 10-50 μl, 20-50 μl, 30-50 μl, 40-50 μl, 1-20 μl, 5-20 μl, 10-20 μl, or 1-10 μl; 0.1-100 ml, 0.5-100 ml, 1-100 ml, 5-100 ml, 10-100 ml, 20-100 ml, 30-10 0ml, 40-100ml, 50-100ml, 60-100ml, 70-100ml, 80-100ml, 90-100ml, 0.1-50ml, 0.5-50ml, 1-50ml, 5-50ml, 10-50ml, 20-50ml, 30-50ml, 40-50ml, 0.1-20ml, 0.5-20ml, 1-20ml, 5-20ml, 10-20ml, 0.1-10ml, 0.5-10ml, 1-10ml, 0.1-1ml, 0.5-1ml, or 0.1-0.5ml; or 0.1-500L, 0.5-500L, 1-500L, 5-500L, 10 -500L, 20-500L, 30-500L, 40-500L, 50-500L, 60-500L, 70-500L, 80-500L, 90-500L, 100-500L, 150-500L, 200-500L, 250-500L, 300-500L, 350-500L, 400-500L, 450-500L, 0.1-250L, 0.5-250L, 1-250L, 5-250L, 10-250L, 20-2 50L, 30-250L, 40-250L, 50-250L, 60-250L, 70-250L, 80-250L, 90-250L, 100 -250L, 150-250L, 200-250L, 0.1-100L, 0.5-100L, 1-100L, 5-100L, 10-100 L, 20-100L, 30-100L, 40-100L, 50-100L, 60-100L, 70-100L, 80-100L, 90-10 0L, 0.1-50L, 0.5-50L, 1-50L, 5-50L, 10-50L, 20-50L, 30-50L, 40-50L, 0.1 -20L, 0.5-20L, 1-20L, 5-20L, 10-20L, 0.1-10L, 0.5-10L, 1-10L, 0.1-1L, 0.5-1L, or 0.1-0.5L. .

[0425] In some embodiments, the mixture comprises a nucleic acid concentration (e.g., total nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 OD / ml (e.g., at an OD of 250-260 nm, e.g., 258-260 nm) (and optionally no greater than 2500, 2000, 1500, 1400, 1300, 1200, 1100, or 1000 OD / ml).

[0426] In some embodiments, the mixture comprises a nucleic acid concentration (eg, total nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least about 900 OD / mL (eg, about 900 OD / mL).

[0427] In some embodiments, the mixture comprises a nucleic acid concentration (eg, total nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least about 1100 OD / mL (eg, about 1100 OD / mL).

[0428] In some embodiments, the mixture comprises a nucleic acid concentration (eg, total nucleic acid concentration, double-stranded nucleic acid concentration, or single-stranded nucleic acid concentration) of at least about 400 OD / mL (eg, about 400 OD / mL).

[0429] In some embodiments, the amount of single-stranded nucleic acid contained in the mixture exceeds the amount of double-stranded nucleic acid. In some embodiments, the amount of single-stranded nucleic acid is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% in excess relative to the amount of double-stranded nucleic acid. In a specific embodiment, the amount of single-stranded nucleic acid is about 5% in excess relative to the amount of double-stranded nucleic acid.

[0430] In some embodiments, the concentration of the salt (e.g., cation, such as sodium ion) in the mixture is less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or 3000 mM. In some embodiments, the mixture does not substantially contain salt. In some embodiments, the concentration of the salt (e.g., cation, such as sodium ion) in the mixture is about 50 mM to about 1.5 M, such as about 60 nM to about 1.2 M or about 69 mM to about 1.02 M. In some embodiments, the single-stranded oligonucleotide is in a deprotected sample matrix. In some embodiments, the melting temperature of the double-stranded nucleic acid in the mixture is between about 20-85°C, such as between about 25-80°C, 30-75°C, 35-70°C, 40-65°C, 45-60°C, 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, 70-75°C, 75-80°C, or 80-85°C.

[0431] D. Total diafiltration volume and time

[0432] Without wishing to be bound by theory, it is envisioned that the total diafiltration volume (DTV) and / or time of the device, the pressure at which the mixture is ultrafiltered, and the temperature at which the mixture is ultrafiltered are all process parameters that may be important for determining the efficacy of the duplex oligonucleotide purification method. In general, these process parameters include combinations of process parameters whose optimal configurations are associated with (e.g., dependent on) each other.

[0433] In some embodiments, the ultrafiltration step comprises applying at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90 diafiltration volumes (DTV) to the filtration device (e.g., sequentially). In some embodiments, the ultrafiltration step comprises no more than 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90 DTVs. In some embodiments, the ultrafiltration step comprises applying at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90 DTVs to the filtration device. filtration volumes (DTV) (e.g., sequentially), and optionally no greater than 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90 DTVs.In some embodiments, the ultrafiltration step comprises applying 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 55-90, 60-90, 65-90, 70-90, 75-90, 80-90, 85-90, 20-85, 25-85, 30-85, 35-85, 40-85, 45-85, 50-85, 55-85, 60-85, 65-85, 70-85, 75-85, 80-85, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 55-80, 60-80, 65-80, 70-80, 75-80, 20-75, 25-75, 30-75, 35-75, 40-75, 45-75, 50-75, 55-75, 60-75, 65-75, 70-75, 20-70, 25-7 0, 30-70, 35-70, 40-70, 45-70, 50-70, 55-70, 60-70, 65-70, 20-65, 25-65, 30-65, 35-65, 40-65, 45-65, 50-65, 55-65, 60-65, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 20-55, 25-55, 30 -55, 35-55, 40-55, 45-55, 50-55, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 20-45, 25-45, 30-45, 35-45, 40-45, 20-40, 25-40, 30-40, 35-40, 20-35, 25-35, 30-35, 20-30, 25-30, or 20-25 DTVs (e.g., sequentially).In some embodiments, the ultrafiltration step comprises applying 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 55-90, 60-90, 65-90, 70-90, 75-90, 80-90, 85-90, 20-85, 25-85, 30-85, 35-85, 40-85, 45-85, 50-85, 55-85, 60-85, 65-85, 70-85, 75-85, 80-85, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 55-80, 60-80, 65-80, 70-80, 75-80, 20-75, 25-75, 30-75, 35-75, 40-75, 45-75, 50-75, 55-75, 60-75, 65-75, 70-75, 20-70, 25-7 0, 30-70, 35-70, 40-70, 45-70, 50-70, 55-70, 60-70, 65-70, 20-65, 25-65, 30-65, 35-65, 40-65, 45-65, 50-65, 55-65, 60-65, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 20-55, 25-55, 30 -55, 35-55, 40-55, 45-55, 50-55, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 20-45, 25-45, 30-45, 35-45, 40-45, 20-40, 25-40, 30-40, 35-40, 20-35, 25-35, 30-35, 20-30, 25-30, or 20-25 DTVs (e.g., sequentially).

[0434] In some embodiments, the ultrafiltration step comprises applying DTV to the filtration device (e.g., sequentially) until a stable low conductivity threshold is reached. In some embodiments, ultrafiltration is performed until a stable low conductivity threshold is reached (e.g., a conductivity threshold of less than or equal to about 30, 40, 50, 60, 70, or 80 μS / cm, such as about 50 μS / cm), for example, DTV is applied until a stable low conductivity threshold is reached.

[0435] E. Pressure

[0436] Without being bound by theory, it is contemplated that the pressure at which the mixture is ultrafiltered, the total diafiltration volume (DTV) of the device and / or the time, and the temperature at which the mixture is ultrafiltered are all process parameters that are important in determining the efficacy of the duplex oligonucleotide purification method. In general, these process parameters include a combination of process parameters whose optimal configurations are associated with (e.g., dependent on) each other.

[0437] In some embodiments, the ultrafiltration step comprises applying a force of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 psi (transmembrane pressure) to the double-stranded nucleic acid product mixture, e.g., within a filtration device. In some embodiments, the ultrafiltration step comprises applying a force of no greater than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 psi to the double-stranded nucleic acid product mixture, e.g., within a filtration device. In some embodiments, the ultrafiltration step comprises applying a force of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 psi (transmembrane pressure), and optionally no greater than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 psi to the double-stranded nucleic acid product mixture, e.g., within a filtration device. In some embodiments, the ultrafiltration step comprises applying a force of 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20, 5-15, 10-15 or 5-10 psi (transmembrane pressure). In some embodiments, the ultrafiltration step comprises applying a force under the pressure operating conditions recommended by the filtration device manufacturer.

[0438] In some embodiments, the ultrafiltration step comprises achieving a pressure of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m 2In some embodiments, the ultrafiltration step comprises achieving a recirculation rate of no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 L / min / m at a pressure. 2 In some embodiments, the ultrafiltration step comprises achieving a recirculation rate of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m at a pressure. 2 and optionally no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 L / min / m 2 Membrane recirculation rate.

[0439] In some embodiments, the ultrafiltration step comprises achieving a pressure of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m 2 In some embodiments, the ultrafiltration step comprises achieving a recirculation rate of no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 L / min / m at a pressure. 2 In some embodiments, the ultrafiltration step comprises achieving a recirculation rate of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 L / min / m at a pressure. 2and optionally no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 L / min / m 2 In some embodiments, the ultrafiltration step comprises achieving a recirculation rate of at least 5, 6, 7, 8, 9, 10, 11, or 12 L / min / m 2 Membrane recirculation rate.

[0440] In some embodiments, the ultrafiltration step comprises achieving a flow rate of 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 5-35, 10-35, 15-35, 20-35, 25-35, 30-35, 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20, 5-15, 10-15, or 5-10 L / min / m 2 recirculation rate.

[0441] F. Temperature

[0442] Without wishing to be bound by theory, it is believed that the temperature at which the mixture is ultrafiltered, the pressure at which the mixture is ultrafiltered, and the total diafiltration volume (DTV) and / or time of the device are all process parameters that are important for determining the efficacy of the duplex oligonucleotide purification method. In general, these process parameters include a combination of process parameters whose optimal configurations are associated with (e.g., dependent on) each other.

[0443] In some embodiments, ultrafiltration is performed at a temperature of about 10-40°C, such as about 15-40°C, 20-40°C, 25-40°C, 30-40°C, 35-40°C, 10-35°C, 10-30°C, 10-25°C, 10-20°C, 10-15°C, 15-35°C, 20-30°C, 10-15°C, 15-20°C, 20-25°C, 25-30°C, 30-35°C, or 35-40°C, such as about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, or 40°C. In some embodiments, ultrafiltration is performed at a temperature that is at least 0.5°C, 1°C, 1.5°C, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or 45°C less than the denaturation point of the oligonucleotide. In some embodiments, ultrafiltration is performed at a temperature that does not reach or exceed (e.g., fall below) the denaturation point of the oligonucleotide anywhere in the system. In some embodiments, the temperature at which ultrafiltration is performed does not vary by more than about 0°C, 1°C, 2°C, 3°C, 4°C, or 5°C during the filtration.

[0444] IV. Examples

[0445] The present invention is further illustrated by the following examples. These examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way.

[0446] Example 1 - Exemplary Purification Methods

[0447] In this embodiment, a double-stranded oligonucleotide consisting of two sufficiently complementary chains is synthetically formed by annealing equimolar amounts of individual chains together. The content of duplexes and unreacted single strands is measured using analysis by non-denaturing HPLC. The analysis of the duplex oligonucleotide sample is performed using an Agilent 1290UHPLC equipped with a Waters XBridge BEH C4 300A, 2.1×50mm, 3.5μM analytical column running at 35°C. The peak is eluted with mobile phase A: 95mM 1,1,1,3,3,3-hexafluoro-2-propanol, 16mM triethylamine, 24mM tert-butylamine and 1μM ethylenediaminetetraacetic acid; and mobile phase B: acetonitrile in 26.25 minutes to compare the excess single-stranded and double-stranded abundance.

[0448] Example 2 - Exemplary Ultrafiltration or Cross-Flow Filtration

[0449] The oligonucleotide matrix is ​​introduced into an ultrafiltration or cross-flow ultrafiltration device. A standard ultrafiltration facility is used, which consists of a retentate storage tank, a pump, an ultrafiltration membrane with a specific molecular weight retention aspect (e.g., 2, 5, 10 kD), a conductivity meter, and a waste container ( Figure 1). The system is pressurized in the mid-psi range (approximately 15-30 psi) to concentrate the matrix to the retentate side of the permeable membrane. Process water is then introduced under pressure to facilitate diafiltration of low molecular weight contaminants.

[0450] Diafiltration is continued until a stable low conductivity threshold is achieved (typically ≤ 50 μS / cm) and the minimum target volume exchange of the system is reached.

[0451] Example 3 - Duplex Nucleic Acids with a Molecular Weight > 10 kDa at a Concentration of About 900 OD / mL

[0452] The ultrafiltration device was equipped with a membrane with a molecular weight cut-off of 10 kDa ( Ultrafilter, Sartorius). As described in Example 1 and Example 2, duplex oligonucleotides with a molecular weight > 10 kDa, an excess of about 5% single strands, and a total oligonucleotide concentration of about 900 OD / mL were ultrafiltered. Diafiltration was performed based on total diafiltration volume (DTV) and time. Samples of the retentate matrix were analyzed over time by ultraviolet (UV) and non-denaturing HPLC to obtain the composition of duplexes and single strands. As shown in Tables 1 and Figure 2A As shown in Figure 2C, the percentage of lower molecular weight single strands relative to the percentage of duplexes decreases over time.

[0453] Table 1: HPLC data of Example 2

[0454]

[0455]

[0456] Example 4 - Duplex Nucleic Acids with a Molecular Weight > 10 kDa at a Concentration of About 400 OD / mL

[0457] The ultrafiltration device was equipped with a membrane with a molecular weight cutoff of 10 kDa. As described in Example 1 and Example 2, ultrafiltration was performed on duplex oligonucleotides with a molecular weight>10 kDa, containing an excess of about 5% single strands and about 400 OD / mL oligonucleotide concentration. Diafiltration was performed according to the total diafiltration volume (DTV) and time. Samples of the retentate matrix were analyzed over time by ultraviolet (UV) and non-denaturing HPLC to obtain the composition of duplexes and single strands. The percentage of lower molecular weight single strands relative to the percentage of duplexes did not decrease over time, as shown in Table 2 below.

[0458] Table 2: HPLC data of Example 3

[0459] sample Single Chain Duplex T＝0 5.1 90.2 DTV3 5.1 90.4 DTV5 5.0 90.5 DTV8 5.0 90.5 DTV11 5.0 90.5 DTV14 4.9 90.7

[0460] Example 5 - Duplex Nucleic Acids with a Molecular Weight > 10 kDa at a Concentration of About 1100 OD / mL

[0461] The ultrafiltration device is equipped with a membrane with a molecular weight cutoff of 10 kDa. As described in Example 1 and Example 2, ultrafiltration is performed on duplex oligonucleotides with a molecular weight>10 kDa, containing an excess of about 5% single strands and a concentration of 1,100-1,400 OD / mL oligonucleotides. Diafiltration is performed according to the total diafiltration volume (DTV) and time. Samples of the retentate matrix are analyzed over time by ultraviolet (UV) and non-denaturing HPLC to obtain the composition of duplexes and single strands. The percentage of lower molecular weight single strands relative to the percentage of duplexes decreases over time, as shown in Table 3 below.

[0462] Table 3: HPLC data of Example 3

[0463] sample Single Chain Duplex T＝0 5.3 90.1 DTV1 4.7 91.1 DTV2 4.8 91.2 DTV3 4.9 91.3 DTV7 3.5 92.0 DTV11 3.4 92.1 DTV27 3.2 92.0 DTV45 3.0 92.3 DTV63 2.8 92.8 DTV81 2.7 93.0

[0464] ***

[0465] In light of the present disclosure, all methods disclosed and claimed herein can be made and performed without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be applied to the methods described herein and the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can be substituted for the reagents described herein while achieving the same or similar results. For those skilled in the art, all such similar substitutions and modifications are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, the method comprising: providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids and not single-stranded nucleic acids, wherein the ultrafiltration step comprises applying the mixture to a filtration device, wherein the filtration device comprises a membrane having (i) a molecular weight (MW) cutoff that is at least 10% less than the MW of the double-stranded nucleic acid and at least 10% greater than the MW of the single-stranded nucleic acid, and / or (ii) a pore size that will allow at least 90% of the single-stranded nucleic acid and at most 10% of the double-stranded nucleic acid to permeate the membrane, Thereby, the double-stranded nucleic acid is separated from the single-stranded nucleic acid.

2. A device, comprising: a first chamber configured to hold a mixture to be filtered and optionally capture a retentate; a second chamber configured to capture permeate; and a filter element disposed between the first chamber and the second chamber; wherein the mixture comprises double-stranded nucleic acid and single-stranded nucleic acid; wherein the filter element selectively retains the double-stranded nucleic acids and does not retain the single-stranded nucleic acids, optionally wherein the filter element comprises a membrane having (i) a molecular weight (MW) cutoff that is at least 10% less than the MW of the double-stranded nucleic acids and at least 10% greater than the MW of the single-stranded nucleic acids, and / or (ii) a pore size that will allow at least 90% of the single-stranded nucleic acids and at most 10% of the double-stranded nucleic acids to permeate the membrane.

3. The method or apparatus of any of the preceding claims, wherein the ultrafiltration step or filter element also does not retain one or more other mixture components, wherein the other mixture components are selected from the group consisting of: shorter length single-stranded nucleic acids, organic solvents, thiolation agents / byproducts, capping agents, coupling agents, conjugation agents, detritylation agents / byproducts, deprotection agents / byproducts, carbohydrates, peptides, lipids, polyethylene glycol, or fluorescent labels.

4. The method of any one of the preceding claims, wherein the ultrafiltration step produces a retentate and a permeate, and wherein the retentate comprises a different concentration of salt, buffer, deprotection base, or one or more of production reagents or byproducts than the permeate, the mixture, or both.

5. The method of any one of the preceding claims, wherein providing the mixture comprises: providing a first single-stranded nucleic acid and a second single-stranded nucleic acid, wherein the first single-stranded nucleic acid and the second single-stranded nucleic acid comprise sequences sufficiently complementary to each other to hybridize under conditions suitable for hybridization, and combining the first single-stranded nucleic acid and the second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture comprising: a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and The first single-stranded nucleic acid is in single-stranded form, the second single-stranded nucleic acid is in single-stranded form, or both.

6. The method or device of any one of the preceding claims, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both are or comprise DNA, RNA, UNA, PNA, or LNA.

7. The method or apparatus of any of the preceding claims, wherein the first single stranded nucleic acid, the second single stranded nucleic acid, or both comprise one or more modified and / or non-canonical nucleotides selected from the group consisting of MOE, 2' fluoro, 2'OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, nucleotides comprising modified sugars, or any combination thereof.

8. The device of any one of the preceding claims, wherein the filter element comprises a cross-flow filter.

9. A method according to any one of the preceding claims, wherein the ultrafiltration step comprises applying a force (transmembrane pressure) of 5-30 psi.

10. The method according to any one of the preceding claims, wherein the ultrafiltration step comprises applying 20-90 DTV to the filtration device.

11. The method according to any one of the preceding claims, wherein the ultrafiltration is performed until a stable low conductivity threshold is achieved.

12. The method or device of any preceding claim, wherein the double-stranded nucleic acid comprises a conjugated group.

13. A method according to any one of the preceding claims, wherein the ultrafiltration step comprises diafiltration.

14. The method of claim 13, wherein the diafiltration is performed for a period of time sufficient to filter at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 27, 30, 35, 40, 45, 50, 55, 60, 63, 65, 70, 75, 80, 81, 85 or 90 diafiltration volumes (DTV).

15. The method of any one of the preceding claims, wherein the ultrafiltration is applied at a pressure of about 5-40 psi.

16. The method according to any one of the preceding claims, wherein the ultrafiltration is performed at a temperature of about 10-40°C.

17. The method of any one of the preceding claims, wherein the mixture comprises a nucleic acid concentration of at least 100 OD / mL.

18. The method according to any one of the preceding claims, wherein the ratio of the double-stranded nucleic acid relative to the single-stranded nucleic acid in the retentate increases over time and / or as the total diafiltration volume (DTV) increases.

19. The method of any of the preceding claims, wherein the method reduces the level of double-stranded nucleic acid in the retentate by no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% compared to the level in the mixture.

20. A method for optimizing a technique for making purified double-stranded nucleic acids, the method comprising: (i) providing a mixture, the mixture comprising: a first concentration of double-stranded nucleic acid, the double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and Single-stranded nucleic acid; wherein the mixture has a predetermined concentration of salt; (ii) subjecting the mixture to an ultrafiltration step, the ultrafiltration step selectively retaining a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid without retaining the single-stranded nucleic acid, wherein the ultrafiltration step comprises applying the mixture to a filtration device having a predetermined molecular weight cutoff for a predetermined period of time; (iii) harvesting the retentate; (iv) changing one or more of the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined time period, or the salt concentration; (v) repeating steps (i) to (iii) one or more times, each time using one or more of the varying first concentration, second concentration, predetermined molecular weight cutoff, predetermined time period, and / or salt concentration; (vi) for each repetition of steps (i) to (iii), determining the ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the harvested retentate; as well as (vii) identifying the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined time period, and / or the salt concentration that produces the highest ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the harvested retentate; thereby optimizing the technique for producing purified double-stranded nucleic acid.

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