Methods and apparatus for double-stranded nucleotide purification

Abstract

This specification discloses, for example, a method for purifying oligonucleotides by separating double-stranded oligonucleotides from single-stranded oligonucleotides. An apparatus for carrying out such a method is also provided. Also provided are double-stranded nucleic acid compositions and single-stranded nucleic acid compositions produced using the methods and apparatus disclosed herein. Also provided is a method for generating circular double-stranded nucleic acids.

Description

Technical Field

[0001] Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 368,911, filed Jul. 20, 2022, the entire contents of which are incorporated herein by reference.

Background Art

[0002] Background Oligonucleotides are useful in various biological processes in experimental and industrial settings, as well as in therapeutic applications. Producing oligonucleotides industrially, for example on a large scale, is time-consuming and resource-intensive. Improvements and enhancements are needed in methods for manufacturing oligonucleotides to improve product and / or manufacturing characteristics.

Summary of the Invention

[0003] Summary The present disclosure relates, at least in part, to methods and apparatuses for improving the production of oligonucleotides, methods and apparatuses for improving the purification of oligonucleotides, and / or methods and apparatuses for improving the characteristics (e.g., purity) of products produced using the methods or apparatuses. In some embodiments, the method improves the separation of double-stranded nucleic acids from 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., a nucleic acid comprising a first and a second single-stranded nucleic acid). Without wishing to be bound by theory, in some embodiments, the method and apparatus are thought to allow undesirable mixture components to permeate through the membrane while retaining double-stranded nucleic acids in the retentate.

[0004] Accordingly, in one aspect, the present disclosure is a method for producing a purified double-stranded nucleic acid, comprising: (a) a double-stranded nucleic acid comprising a first single-stranded nucleic acid hybridized to a complementary second single-stranded nucleic acid, and (b) at least one single-stranded nucleic acid, for example, (i) a first single-stranded nucleic acid in single-stranded form (i.e., not hybridized to a second single-stranded nucleic acid), (ii) a second single-stranded nucleic acid in single-stranded form (i.e., not hybridized to the first single-stranded nucleic acid), or (iii) both providing a mixture comprising; subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids comprising hybridized first and second single-stranded nucleic acids but does not retain single-stranded nucleic acids (e.g., the first and / or second single-stranded nucleic acids in single-stranded form); and recovering the retentate comprising thereby producing a purified double-stranded nucleic acid, providing a method.

[0005] In another aspect, the present disclosure is a method for purifying a first and / or second single-stranded nucleic acid, comprising (a) a double-stranded nucleic acid comprising a first single-stranded nucleic acid hybridized to a complementary second single-stranded nucleic acid, and (b) at least one single-stranded nucleic acid, for example, (i) a first single-stranded nucleic acid in single-stranded form (i.e., not hybridized to a second single-stranded nucleic acid), (ii) a second single-stranded nucleic acid in single-stranded form (i.e., not hybridized to the first single-stranded nucleic acid), or (iii) both providing a mixture comprising; subjecting the mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids comprising hybridized first and second single-stranded nucleic acids but does not retain single-stranded nucleic acids (e.g., the first and / or second single-stranded nucleic acids in single-stranded form); and recovering the permeate comprising thereby purifying the first and / or second single-stranded nucleic acid, providing a method.

[0006] In another aspect, the present disclosure is a method for separating double-stranded nucleic acids from single-stranded nucleic acids, comprising Providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid; Subjecting the mixture to a tangential flow filtration step of selectively retaining double-stranded nucleic acid rather than single-stranded nucleic acid; comprising; The tangential flow filtration step includes applying the mixture to a filtration device, such as a cross-flow filtration device, having a membrane with a molecular weight (MW) cut-off that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% smaller 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 smaller than the MW of the double-stranded nucleic acid) and at least 10%, 20%, 30%, 40%, or 50% larger than the MW of the single-stranded nucleic acid (e.g., at least 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 kilodaltons larger than the MW of the single-stranded nucleic acid), thereby separating the double-stranded nucleic acid from the single-stranded nucleic acid; providing a method for separating double-stranded nucleic acid from single-stranded nucleic acid.

[0007] In yet another aspect, the present disclosure provides a method for separating double-stranded nucleic acid from single-stranded nucleic acid, comprising: providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid; subjecting the mixture to a tangential flow filtration step of selectively retaining double-stranded nucleic acid rather than single-stranded nucleic acid; comprising; The tangential flow filtration step includes 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 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 or less, thereby separating the double-stranded nucleic acid from the single-stranded nucleic acid; providing a method for separating double-stranded nucleic acid from single-stranded nucleic acid.

[0008] In another aspect, the present disclosure provides a method for separating double-stranded nucleic acid from single-stranded nucleic acid, comprising: providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and subjecting the mixture to a tangential flow filtration step of selectively retaining double-stranded nucleic acid rather than single-stranded nucleic acid; comprising, The ultrafiltration step comprises applying the mixture to a filtration device, such as a cross-flow filtration device, the filtration device comprising 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), where Z is the numerical average of X and Y, X is the minimum pore size that allows at least 90% of each of the first single-stranded nucleic acid and / or the second single-stranded nucleic acid to pass through the membrane (e.g., at a diafiltration volume (DTV) of 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); and Y is the maximum pore size that allows at least 90% of the double-stranded nucleic acid to be retained by the membrane (e.g., at 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), thereby providing a method for separating single-stranded nucleic acid from double-stranded nucleic acid.

[0009] In another aspect, the present disclosure is a method for separating single-stranded nucleic acid from double-stranded nucleic acid, 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 acid rather than single-stranded nucleic acid comprising, The ultrafiltration step comprises applying the mixture to a filtration device, such as a cross-flow filtration device, the filtration device comprising a membrane having a pore size that allows at least 90% of the single-stranded nucleic acid and at most 10% of the double-stranded nucleic acid to pass through the membrane (e.g., at 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).

[0010] In another aspect, the present disclosure provides a first chamber configured to hold a mixture to be filtered (optionally, to capture a holding solution), a second chamber configured to capture a permeate, and a filtration element disposed between the first chamber and the second chamber wherein the mixture comprises double-stranded nucleic acids (e.g., comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid) and single-stranded nucleic acids (e.g., the first single-stranded nucleic acid in single-stranded form, the second single-stranded nucleic acid in single-stranded form, or both), and the filtration element selectively retains double-stranded nucleic acids (e.g., comprising the first and second single-stranded nucleic acids) rather than single-stranded nucleic acids (e.g., the first and / or second single-stranded nucleic acids in single-stranded form).

[0011] In some embodiments of any of the methods or apparatuses disclosed herein, the ultrafiltration step or the filtration element also does not retain one or more other mixture components, such as salts (e.g., halide salts or mineral salts), buffers, or production reagents or by-products. In certain embodiments, the other mixture components are single-stranded nucleic acids of shorter length (e.g., shorter than the first and / or second single-stranded nucleic acids), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine), thiolation reagents / by-products (e.g., xanthane, hydride, or PADS), capping reagents (e.g., acetic anhydride or NMI), coupling reagents (e.g., phosphoramidite or ETT), detritylation reagents / by-products (e.g., dichloroacetic acid), deprotection reagents / by-products (e.g., diethylamine, methylamine, or ammonia), or conjugation reagents (e.g., carboxylic acids, carboxylic acid esters, carbonates, and carboxylic acid activating reagents).

[0012] ​In some embodiments of any of the methods or apparatuses disclosed herein, the ultrafiltration step or filtration element produces a retentate and a permeate. In certain embodiments, the retentate contains one or more salts, buffers, or production reagents or by-products at a different concentration than the permeate, the mixture, or both. In another embodiment, the retentate has a lower concentration of one or more of salts, buffers, or production reagents or by-products than the permeate, the mixture, or both.

[0013] In certain embodiments of any of the methods disclosed herein, the step of providing a mixture For example, as described in Examples 1-5, providing a first single-stranded nucleic acid and a second single-stranded nucleic acid that include sequences that are sufficiently complementary to each other to hybridize under conditions suitable for hybridization, and 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 in single-stranded form, the second single-stranded nucleic acid in single-stranded form, or both combining the first single-stranded nucleic acid and the second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture comprising is included.

[0014] In some embodiments, the step of providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes a synthesis step, e.g., solid-phase chemical synthesis, liquid-phase chemical synthesis, enzymatic synthesis, hybrid / chemical-enzymatic synthesis, PCR-based synthesis, and cell synthesis of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both. In certain embodiments, the step of providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes a post-synthesis step, e.g., conjugation, chemical or enzymatic cleavage from a solid substrate, removal of one or more chemical moieties (e.g., removal of a protecting group), or combinations thereof. In certain embodiments, the first single-stranded nucleic acid and / or the second single-stranded nucleic acid may be conjugated during synthesis, after synthesis, or after single-strand purification or ultrafiltration. In certain embodiments, the step of providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes a purification step, e.g., a step of concentrating the nucleic acid component and reducing the level of non-nucleic acid components, e.g., a chromatography step, e.g., a step including anion-exchange chromatography. In some embodiments, the step of providing the first single-stranded nucleic acid and / or the second single-stranded nucleic acid includes subjecting the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to an ultrafiltration step that selectively retains the single-stranded nucleic acid (and, e.g., does not retain shortmers (i.e., those shorter than the desired full-length single-stranded nucleic acid) or non-nucleic acid components).

[0015] In some embodiments, the step of providing the double-stranded nucleic acid includes a synthesis step and / or a post-synthesis step including, for example, conjugation of the double-stranded nucleic acid with another component (e.g., nucleic acid, carbohydrate, peptide, protein domain, lipid, steroid polyethylene glycol, fluorescent label). In some embodiments, the single-stranded nucleic acid is conjugated with another component before forming the double-stranded nucleic acid. In certain embodiments, the step of providing the double-stranded nucleic acid includes a chromatography step including, for example, anion exchange chromatography, which concentrates the nucleic acid component and reduces the level of non-nucleic acid components. In some embodiments, the step of providing the first double-stranded nucleic acid and / or the conjugation component includes subjecting the first double-stranded nucleic acid, the conjugation component, or both to an ultrafiltration step that selectively retains the double-stranded nucleic acid (and, for example, does not retain non-nucleic acid components).

[0016] In some embodiments of any of the methods or apparatuses 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 in length (and optionally, 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less). 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 in length (and optionally, 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less). 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 in length (and optionally, 100, 80, 60, 50, 45, 40, 35, or 30 base pairs in length or less).

[0017] In some embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid is 10 to 50, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides in length.

[0018] In any particular embodiment of the method or apparatus disclosed herein, the second single-stranded nucleic acid is 10 to 50, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides in length.

[0019] In some embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid is 10 to 50, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 base pairs in length.

[0020] In some embodiments of any of the methods or apparatuses 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 target gene, non-coding RNA, primer, or sequence of a component for a molecular assembly).

[0021] In certain embodiments of any of the methods or apparatuses 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, 10, 9, 8, 7, or 6 kilodaltons or less). In certain embodiments of any of the methods or apparatuses 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, 10, 9, 8, 7, or 6 kilodaltons or less).

[0022] In some embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid is 3 to 20, 3 to 15, 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to 10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10, 9.5 to 10, 3 to 9, 3.5 to 9, 4 to 9, 4.5 to 9, 5 to 9, 5.5 to 9, 6 to 9, 6.5 to 9, 7 to 9, 7.5 to 9, 8 to 9, 8.5 to 9, 3 to 8, 3.5 to 8, 4 to 8, 4.5 to 8, 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, 7.5 to 8, 3 to 7, 3.5 to 7, 4 to 7, 4.5 to 7, 5 to 7, 5.5 to 7, 6 to 7, 6.5 to 7, 3 to 6, 3.5 to 6, 4 to 6, 4.5 to 6, 5 to 6, 5.5 to 6, 3 to 5, 3.5 to 5, 4 to 5, 4.5 to 5, 3 to 4, or 3.5 to 4 kilodaltons.

[0023] In some embodiments of any of the methods or apparatuses disclosed herein, the second single-stranded nucleic acid is 3 to 20, 3 to 15, 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to 10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10, 9.5 to 10, 3 to 9, 3.5 to 9, 4 to 9, 4.5 to 9, 5 to 9, 5.5 to 9, 6 to 9, 6.5 to 9, 7 to 9, 7.5 to 9, 8 to 9, 8.5 to 9, 3 to 8, 3.5 to 8, 4 to 8, 4.5 to 8, 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, 7.5 to 8, 3 to 7, 3.5 to 7, 4 to 7, 4.5 to 7, 5 to 7, 5.5 to 7, 6 to 7, 6.5 to 7, 3 to 6, 3.5 to 6, 4 to 6, 4.5 to 6, 5 to 6, 5.5 to 6, 3 to 5, 3.5 to 5, 4 to 5, 4.5 to 5, 3 to 4, or 3.5 to 4 kilodaltons.

[0024] In some embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid includes a sequence that can hybridize to itself under conditions suitable for hybridization, for example, a sequence that forms a hairpin loop. In certain embodiments of any of the methods or apparatuses disclosed herein, the second single-stranded nucleic acid includes a sequence that can hybridize to itself under conditions suitable for hybridization, for example, a sequence that forms a hairpin loop.

[0025] In some embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid does not include a sequence that can hybridize to itself under conditions suitable for hybridization, for example, a sequence that forms a hairpin loop. In certain embodiments of any of the methods or apparatuses disclosed herein, the second single-stranded nucleic acid does not include a sequence that can hybridize to itself under conditions suitable for hybridization, for example, a sequence that forms a hairpin loop.

[0026] In some embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both are, or include, DNA, RNA, UNA, PNA, or LNA.

[0027] In certain embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both include one or more modifications and / or non-canonical nucleotides selected from MOE, 2'-fluoro, 2'-OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, nucleotides containing modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or any combination thereof.

[0028] In some embodiments of any of the methods or apparatuses disclosed herein, the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both include one or more non-phosphodiester bonds between nucleotides, e.g., one or more phosphorothioate or 5'-N-phosphoramidate bonds.

[0029] In certain embodiments of any of the methods disclosed herein, the ultrafiltration step includes applying a mixture of double-stranded nucleic acid products to a filtration device including a crossflow filter. In some embodiments of any of the apparatuses disclosed herein, the filtration element includes a crossflow filter.

[0030] In some embodiments of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a pore size that allows at least 50%, 60%, 70%, 80%, or 90% of single-stranded nucleic acid and at most 10%, 20%, 30%, 40%, or 50% of double-stranded nucleic acid to pass through the membrane (e.g., at 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).

[0031] In some embodiments of any of the devices disclosed herein, the filtration device or filtration 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), where Z is the numerical average of X and Y, X is the minimum pore size that allows at least 90% of single-stranded nucleic acid, a second single-stranded nucleic acid, or both to pass through the membrane (e.g., at 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, or 65, 70, 75, 80, 81, 85, or 90 diafiltration volume (DTV)); and Y is the maximum pore size that allows at least 90% of double-stranded nucleic acid to be retained by the membrane (e.g., at 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, or 65, 70, 75, 80, 81, 85, or 90 DTV).

[0032] In certain embodiments of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having an average pore size of 0.05 to 20, 0.1 to 20, 0.5 to 20, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 0.05 to 10, 0.1 to 10, 0.5 to 10, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 0.05 to 8, 0.1 to 8, 0.5 to 8, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 0.05 to 6, 0.1 to 6, 0.5 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 0.05 to 4, 0.1 to 4, 0.5 to 4, 1 to 4, 2 to 4, 3 to 4, 0.05 to 2, 0.1 to 2, 0.5 to 2, 1 to 2, 0.05 to 1, 0.1 to 1, or 0.5 to 1 μm.

[0033] In some embodiments of any of the devices disclosed herein, the filtration device or filtration element 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 at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons less than the molecular weight of a double-stranded nucleic acid and comprises a membrane with a molecular weight cut-off.

[0034] In another embodiment of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a molecular weight cut-off that is 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, or 1 to 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).

[0035] In certain embodiments of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a molecular weight cut-off that is 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, or 1 to 2 kilodaltons smaller than the molecular weight of the double-stranded nucleic acid.

[0036] In some embodiments of any of the devices disclosed herein, the filtration device or filtration element is at least 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 5 kilodaltons less than the molecular weight of the double-stranded nucleic acid and comprises a membrane having a molecular weight cut-off.

[0037] In any particular embodiment of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a molecular weight cut-off of 0.5 to 100, 0.5 to 80, 0.5 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, 0.5 to 15, 0.5 to 10, 0.5 to 8, 0.5 to 6, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 100, 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2, 2 to 100, 2 to 80, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 2 to 3, 3 to 100, 3 to 80, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 15, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 4 to 100, 4 to 80, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 10, 4 to 8, 4 to 6, 6 to 100, 6 to 80, 6 to 60, 6 to 50, 6 to 40, 6 to 30, 6 to 20, 6 to 15, 6 to 10, 6 to 8, 8 to 100, 8 to 80, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 20, 8 to 15, 8 to 10, 10 to 100, 10 to 80, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 10 to 15, 15 to 100, 15 to 80, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 100, 30 to 80, 30 to 60, 30 to 50, 30 to 40, 40 to 100, 40 to 80, 40 to 60, 40 to 50, 50 to 100, 50 to 80, 50 to 60, 60 to 80, 60 to 100, or 80 to 100 kilodaltons.

[0038] In some embodiments of any of the devices disclosed herein, the filtration device or filtration element comprises a membrane having a molecular weight cut-off 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 filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 kDa. In some embodiments, the filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 8-15 kDa. In certain embodiments, the filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 10 kDa.

[0039] 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 (intermembrane pressure), optionally 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 or less, to a mixture of double-stranded nucleic acid products, for example, 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 (intermembrane pressure). In another embodiment, the ultrafiltration step comprises applying a force within the pressure operating conditions recommended by the manufacturer of the filtration device. In some embodiments, the ultrafiltration step comprises applying (e.g., sequentially) 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, a diafiltration volume (DTV) of 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 or less, to the filtration device.In some embodiments, the ultrafiltration step includes 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 to the filtration device (e.g., sequentially). In some embodiments, the ultrafiltration step is 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 and optionally 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 2It includes a process of achieving a recirculation rate under the following pressures. In certain embodiments, the ultrafiltration process is carried out at 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 It includes a process of achieving a recirculation rate under the pressure of 2 . In some embodiments, the ultrafiltration is carried out until a stable low conductivity threshold (e.g., about 30, 40, 50, 60, 70, or 80 μS / cm or less, e.g., a conductivity threshold of about 50 μS / cm) is achieved.

[0040] In any particular embodiment of the devices disclosed herein, the membrane of the filtration device or filtration element includes polysulfone, polypropylene, cellulose acetate, polylactic acid, nitrocellulose mixed cellulose ester cellulose nitrate, regenerated cellulose, polyethersulfone, polyamide, cellulose derivative (Hydrosart®), polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track - etched membrane. In some embodiments, the filtration device or filtration element is of, or includes, a hollow fiber, tubular, spiral wound, cassette, plate, or frame - membrane type.

[0041] In some embodiments of any of the methods disclosed herein, the method produces a purified double-stranded nucleic acid that meets a purity criterion, where the purity criterion includes a holding solution that contains at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% double-stranded nucleic acid (and for example, contains 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 that meets a purity criterion, where the purity criterion includes a holding solution that contains 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 meets a purity criterion, where the purity criterion includes a holding solution that contains 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 single-stranded nucleic acid to double-stranded nucleic acid.

[0042] In some embodiments of any of the methods disclosed herein, the method meets a purity criterion at 25, 24, 23, 22, 21, or 20 DTVs or less.

[0043] In some embodiments of any of the methods or apparatuses 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.

[0044] In some embodiments of any of the methods or apparatuses disclosed herein, the single-stranded nucleic acid is a component of the double-stranded nucleic acid (for example, the double-stranded nucleic acid contains a copy of the single-stranded nucleic acid).

[0045] In some embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid comprises blunt ends. In some embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid comprises two blunt ends. In certain embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid comprises, for example, a single-stranded portion, such as a terminal overhang, at one end. In some embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid has single-stranded portions, such as terminal overhangs, at both ends. In certain embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid is or comprises a conjugate group, such as PEG (polyethylene glycol), a dye, a label, a peptide, a lipid, a steroid, a carbohydrate, such as GalNAc, or a glycol spacer.

[0046] In some embodiments of any of the apparatuses disclosed herein, the second chamber contains a permeate, such as a permeate containing single-stranded nucleic acid. In other embodiments, the second chamber does not hold the permeate, for example, the permeate flows out of the second chamber and is discarded.

[0047] In certain embodiments of the apparatuses disclosed herein, the first chamber contains a mixture. In certain embodiments of any of the methods or apparatuses disclosed herein, the first chamber contains a holding solution.

[0048] In some embodiments of any of the methods or apparatuses disclosed herein, the mixture is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μl, 200 μl, 300 μl, or 400 μl or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ml, 200 ml, 300 ml, or 400 ml or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 L or less (and optionally, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 L).

[0049] In some embodiments of any of the methods or apparatuses disclosed herein, the mixture comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μl, 200 μl, 300 μl, or 400 μl, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ml, 200 ml, 300 ml, or 400 ml, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 L (and optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 L or less).

[0050] In certain embodiments of any of the methods or apparatuses disclosed herein, the mixture is 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 1 to 20, 5 to 20, 10 to 20, or 1 to 10 μl, 0.1 to 100, 0.5 to 100, 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 0.1 to 50, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20, 0.1 to 10, 0.5 to 10, 1 to 10, 0.1 to 1, 0.5 to 1, or 0.1 to 0.5 ml, 0.1 to 500, 0.5 to 500, 1 to 500, 5 to 500, 10 to 500, 20 to 500, 30 to 500, 40 to 500, 50 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 0.1 to 250, 0.5 to 250, 1 to 250, 5 to 250, 10 to 250, 20 to 250, 30 to 250, 40 to 250, 50 to 250, 60 to 250, 70 to 250, 80 to 250, 90 to 250, 100 to 250, 150 to 250, 200 to 250, 0.1 to 100, 0.5 to 100, 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 0.1 to 50, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20, 0.1 to 10, 0.5 to 10, 1 to 10, 0.1 to 1, 0.5 to 1, or 0.1 to 0.5 L.

[0051] In some embodiments of any of the methods or apparatuses disclosed herein, 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, or 2000 OD / mL (e.g., OD at 250-260 nm, e.g., 258-260 nm) (and optionally, 2500, 2000, 1500, 1400, 1300, 1200, 1100, or 1000 OD / mL or less). In certain 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 about 900 OD / mL (e.g., about 900 OD / mL). 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 about 1100 OD / mL (e.g., about 1100 OD / mL). In certain 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 about 400 OD / mL (e.g., about 400 OD / mL).

[0052] In certain embodiments of any of the methods or apparatuses disclosed herein, the ssDNA in the permeate is mainly composed of the sense strand (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the ssDNA in the permeate is composed of the sense strand). In some embodiments, the ssDNA in the permeate is mainly composed of the antisense strand (e.g., 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).

[0053] In some embodiments of any of the methods or apparatuses disclosed herein, the double-stranded nucleic acid contains a mismatch (e.g., at one of the ends of the double-stranded nucleic acid, within 1 to 2 nucleotides from one end of the double-stranded nucleic acid, or between the ends of the double-stranded nucleic acid).

[0054] In certain embodiments of any of the methods or apparatuses disclosed herein, the mixture contains a quantity of single-stranded nucleic acid that exceeds the quantity of double-stranded nucleic acid. In some embodiments, the quantity 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 of the quantity of double-stranded nucleic acid. In a particular embodiment, the quantity of single-stranded nucleic acid is about 5% in excess of the quantity of double-stranded nucleic acid.

[0055] In some embodiments of any of the methods disclosed herein, the ultrafiltration step includes diafiltration. In certain embodiments, the diafiltration is carried out for at least 10, 20, 30, 40, or 50 minutes, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the diafiltration is carried out for a 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, or 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV).

[0056] In certain embodiments of any of the methods disclosed herein, ultrafiltration is applied at a pressure of about 5 - 40, 10 - 40, 15 - 40, 20 - 40, 30 - 40, 35 - 40, 5 - 15, 5 - 20, 5 - 25, 5 - 30, 5 - 35, 10 - 30, 15 - 25, 5 - 10, 10 - 15, 15 - 20, 20 - 25, 25 - 30, 30 - 35, or 35 - 40 psi. In some embodiments, ultrafiltration is applied at a pressure of about 15 - 30 psi (e.g., about 15 - 25, 15 - 20, 25 - 30, 20 - 30, 15 - 20, 20 - 25, or 25 - 30 psi, or e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 psi). In certain embodiments, ultrafiltration is carried out at a temperature of about 10 - 40 °C, e.g., about 15 - 40, 20 - 40, 25 - 40, 30 - 40, 35 - 40, 10 - 35, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 35, 20 - 30, 10 - 15, 15 - 20, 20 - 25, 25 - 30, 30 - 35, or 35 - 40 °C, e.g., about 10, 15, 20, 25, 30, 35, 37, or 4 °C.

[0057] In some embodiments of any of the methods disclosed herein, the method further comprises analyzing the retentate by non - denaturing HPLC, e.g., as described in Examples 1 - 5 (e.g., to determine the relative amounts of double - stranded nucleic acid and / or single - stranded nucleic acid in the retentate).

[0058] In some embodiments of any of the methods or apparatuses disclosed herein, the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retention solution (e.g., determined by non-denaturing HPLC) increases over time and / or with the total diafiltration volume (DTV). In certain embodiments, the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retention solution (e.g., determined by non-denaturing 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 acid to single-stranded nucleic acid in the retention solution (e.g., determined by non-denaturing 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.

[0059] In some embodiments of any of the methods or apparatuses disclosed herein, the concentration of salt (e.g., cations, e.g., sodium ions) 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, or 1000 mM. In certain embodiments, the mixture is substantially salt-free. In certain embodiments, the mixture is essentially salt-free.

[0060] In some embodiments of any of the methods or apparatuses disclosed herein, the concentration of salt (e.g., cations, e.g., sodium ions) in the mixture is from about 50 mM to about 1.5 M, e.g., from about 60 nM to about 1.2 M, or from about 69 mM to about 1.02 M.

[0061] In any particular embodiment of the methods or devices disclosed herein, the melting temperature of the double-stranded nucleic acid in the mixture is between about 20 and 85 °C, for example, between about 25 and 80, 30 and 75, 35 and 70, 40 and 65, 45 and 60, 20 and 25, 25 and 30, 30 and 35, 35 and 40, 40 and 45, 45 and 50, 50 and 55, 55 and 60, 60 and 65, 65 and 70, 70 and 75, 75 and 80, or 80 and 85 °C.

[0062] In any particular embodiment 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 salt (e.g., cation, e.g., sodium ion) in the mixture is determined according to the method of Owczarzy et al. (2004, Biochemistry 43:3537-3554; which is incorporated herein by reference in its entirety).

[0063] In any particular embodiment of the methods or devices disclosed herein, the double-stranded nucleic acid of the holding solution is for use in downstream applications or uses. In some embodiments, the double-stranded nucleic acid of the holding solution is lyophilized. In some embodiments, the double-stranded nucleic acid of the holding solution is conjugated with additional agents (e.g., additional nucleic acid molecules). In certain embodiments, the double-stranded nucleic acid of the holding solution is mixed with other oligonucleotide duplexes. In certain embodiments, the double-stranded nucleic acid of the holding solution is mixed with a pharmaceutical formulation buffer, e.g., buffered phosphate solution, buffered phosphate saline, saline, or water.

[0064] In some embodiments of the methods or devices disclosed herein, the double-stranded nucleic acid has a length of 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, or 400 to 500 nucleotides.

[0065] 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 solvents.

[0066] In some embodiments of any of the methods or apparatuses disclosed herein, the method reduces the level of double-stranded nucleic acid by only 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

[0067] In some embodiments of any of the methods or apparatuses disclosed herein, the ratio of the concentration of double-stranded nucleic acid in the holding solution to the concentration of double-stranded nucleic acid 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.

[0068] In some embodiments of any of the methods or apparatuses disclosed herein, the amount of double-stranded nucleic acid in the holding solution is at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 mg / mL.

[0069] In another aspect, the present disclosure is a method of optimizing a technique for manufacturing a purified double-stranded nucleic acid, (i) a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid at a first concentration, and providing a mixture comprising 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 at a second concentration, or both, wherein the mixture has a salt at a predetermined concentration (e.g., a cation, e.g., a sodium ion), the step; (ii) a step of subjecting the mixture to an ultrafiltration step that selectively retains a double-stranded nucleic acid containing the first and second single-stranded nucleic acids, rather than a single-stranded nucleic acid (for example, the first and second single-stranded nucleic acids in single-stranded form), wherein the ultrafiltration step includes applying the mixture to a filtration device having a predetermined molecular weight cut-off for a predetermined length of time; (iii) a step of recovering the retentate; (iv) a step of changing one or more of the first concentration, the second concentration, the predetermined molecular weight cut-off, the predetermined length of time, or the salt concentration; (v) a step of repeating steps (i) to (iii) one or more times, each time using one or more of the changed first concentration, second concentration, predetermined molecular weight cut-off, predetermined length of time, and / or salt concentration; (vi) in each repetition of steps (i) to (iii), a step of determining the ratio of the double-stranded nucleic acid to the single-stranded nucleic acid in the recovered retentate; and (vii) a step of identifying the first concentration, the second concentration, the predetermined molecular weight cut-off, the predetermined length of time, and / or the salt concentration that results in the highest ratio of double-stranded nucleic acid to single-stranded nucleic acid in the recovered retentate including Thereby, a method is provided for optimizing a technique for producing a purified double-stranded nucleic acid.

[0070] In another aspect, the present disclosure provides a double-stranded nucleic acid composition comprising a retentate generated and recovered according to the method described herein.

[0071] In another aspect, the present disclosure provides a single-stranded nucleic acid composition comprising a permeate generated and recovered according to the method described herein.

[0072] In another aspect, the present disclosure provides a composition comprising a first double-stranded nucleic acid comprising a first overhang that hybridizes to a second double-stranded nucleic acid comprising a second overhang (e.g., complementary to the first overhang).

[0073] In some embodiments, the first double-stranded nucleic acid is made according to the methods described herein. In certain embodiments, the second double-stranded nucleic acid is made according to the methods described herein.

[0074] In another aspect, the disclosure is a method of generating a circular double-stranded nucleic acid, comprising: (a) providing a composition as described herein; and (b) incubating the composition under conditions suitable for circularization of the first double-stranded nucleic acid hybridized to the second double-stranded nucleic acid. A method is provided.

[0075] In some embodiments, circularization comprises ligation.

[0076] The various embodiments disclosed herein are represented by the following items.

[0077] 1. A method of manufacturing a double-stranded nucleic acid, comprising: providing a mixture comprising a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid, and a single-stranded nucleic acid ; subjecting the mixture to an ultrafiltration step that selectively retains the double-stranded nucleic acid comprising hybridized first and second single-stranded nucleic acids rather than the single-stranded nucleic acid; and recovering the retentate thereby producing a purified double-stranded nucleic acid. comprising A method.

[0078] 2. A method of purifying a first and / or second single-stranded nucleic acid, comprising: providing a mixture comprising a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid, and a single-stranded nucleic acid ; Optionally, a step in which the mixture is subjected to pre-purification / ultrafiltration to remove shortmers and / or organic impurities resulting from the nucleic acid synthesis and / or deprotection steps carried out; A step of subjecting the mixture to an ultrafiltration step that selectively retains a double-stranded nucleic acid containing first and second single-stranded nucleic acids rather than a single-stranded nucleic acid; and A step of recovering the permeate comprising thereby purifying the first and / or second single-stranded nucleic acid, a method.

[0079] 3. A method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, comprising a step of providing a mixture of a double-stranded nucleic acid and a single-stranded nucleic acid, a step of subjecting the mixture to an ultrafiltration step that selectively retains a double-stranded nucleic acid rather than a single-stranded nucleic acid comprising the ultrafiltration step comprises applying the mixture to a filtration device having a MW cut-off that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kilodaltons smaller than the molecular weight (MW) of the double-stranded nucleic acid and not more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 kilodaltons smaller than the MW of the double-stranded nucleic acid, thereby separating the double-stranded nucleic acid from the single-stranded nucleic acid, a method.

[0080] 4. A method for separating a double-stranded nucleic acid from a single-stranded nucleic acid, comprising a step of providing a mixture of a double-stranded nucleic acid and a single-stranded nucleic acid, a step of subjecting the mixture to an ultrafiltration step that selectively retains a double-stranded nucleic acid rather than a single-stranded nucleic acid comprising 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 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 or less, thereby separating the double-stranded nucleic acid from the single-stranded nucleic acid, a method.

[0081] 5. A method for separating double-stranded nucleic acid from single-stranded nucleic acid, comprising: providing a mixture of double-stranded nucleic acid and single-stranded nucleic acid, and subjecting the mixture to a cross-flow filtration step that selectively retains double-stranded nucleic acid rather than single-stranded nucleic acid wherein the cross-flow filtration step comprises applying the mixture to a filtration device, the filtration device comprising 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, where Z is the numerical average of X and Y, X is the minimum pore size that allows at least 90% of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to pass through the membrane; and Y is the maximum pore size that allows at least 90% of the double-stranded nucleic acid to be retained by the membrane, thereby separating double-stranded nucleic acid from single-stranded nucleic acid.

[0082] 6. An apparatus comprising 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 filtration element disposed between the first chamber and the second chamber, wherein the mixture comprises double-stranded nucleic acid and single-stranded nucleic acid; and the filtration element selectively retains double-stranded nucleic acid rather than single-stranded nucleic acid.

[0083] 7. The method or apparatus according to any one of the preceding items, wherein the cross-flow filtration step or the filtration element does not retain one or more other mixture components.

[0084] ​​8. The method or apparatus according to item 7, wherein the other mixture components are selected from single-stranded nucleic acids of shorter length, organic solvents, thiolation reagents / by-products, capping reagents, coupling reagents, detritylation reagents / by-products, conjugation reagents (e.g., carboxylic acids, carboxylic acid esters, carbonates, and carboxylic acid activation reagents), and / or deprotection reagents / by-products.

[0085] 9. The method according to any one of items 1 to 5, wherein the ultrafiltration step produces a retentate and a permeate.

[0086] 10. The method or apparatus according to any one of items 6 to 9, wherein the retentate contains one or more of salts, buffers, deprotected bases, or production reagents or by-products at a concentration different from that of the permeate, the mixture, or both.

[0087] 11. The method or apparatus according to any one of items 6 to 10, wherein the retentate has a lower concentration of one or more of salts, buffers, or production reagents or by-products than the permeate, the mixture, or both.

[0088] 12. The step of providing the mixture comprises providing a first single-stranded nucleic acid and a second single-stranded nucleic acid comprising sequences that are sufficiently complementary to each other to hybridize under conditions suitable for hybridization, and 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 in single-stranded form, the second single-stranded nucleic acid in single-stranded form, or both combining the first single-stranded nucleic acid and the second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture comprising The method according to any one of items 1 to 4.

[0089] 13. The method according to item 12, wherein the step of providing the first single-stranded nucleic acid and the second single-stranded nucleic acid comprises a synthesis step.

[0090] 14. The method according to any one of items 12 or 13, wherein the step of providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes a post-synthesis step.

[0091] 15. The method according to any one of items 12 to 14, wherein the step of providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes a purification step.

[0092] 16. The method according to any one of items 12 to 15, wherein the step of 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 both to an ultrafiltration step that selectively retains single-stranded nucleic acids.

[0093] 17. The method or apparatus according to any one of the preceding items, 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 in length, and optionally 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less.

[0094] 18. The method or apparatus according to any one of the preceding items, 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 in length, and optionally 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less.

[0095] 19. The method or apparatus according to any one of the preceding items, 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 100, 80, 60, 50, 45, 40, 35, or 30 base pairs in length or less.

[0096] The method or apparatus according to any one of the above items, wherein the first single-stranded nucleic acid has a length of 10 to 50, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides.

[0097] 21. The second single-stranded nucleic acid has a length of 10 to 50, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides, and the method or apparatus according to any one of the preceding items.

[0098] 22. The method or apparatus according to any one of the above items, wherein the double-stranded nucleic acid has a length of 10 to 50, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 base pairs.

[0099] 23. The method or apparatus according to any one of the above items, 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 the target nucleic acid sequence.

[0100] 24. The method or apparatus according to any one of the above items, 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, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 kilodaltons or less.

[0101] 25. 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, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 kilodaltons or less. The method or apparatus according to any one of the preceding items.

[0102] 26. The first single-stranded nucleic acid is 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to 10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10, 9.5 to 10, 3 to 9, 3.5 to 9, 4 to 9, 4.5 to 9, 5 to 9, 5.5 to 9, 6 to 9, 6.5 to 9, 7 to 9, 7.5 to 9, 8 to 9, 8.5 to 9, 3 to 8, 3.5 to 8, 4 to 8, 4.5 to 8, 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, 7.5 to 8, 3 to 7, 3.5 to 7, 4 to 7, 4.5 to 7, 5 to 7, 5.5 to 7, 6 to 7, 6.5 to 7, 3 to 6, 3.5 to 6, 4 to 6, 4.5 to 6, 5 to 6, 5.5 to 6, 3 to 5, 3.5 to 5, 4 to 5, 4.5 to 5, 3 to 4, or 3.5 to 4 kilodaltons. The method or apparatus according to any one of the preceding items.

[0103] 27. The method or apparatus according to any one of the preceding items, wherein the second single-stranded nucleic acid is 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to 10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10, 9.5 to 10, 3 to 9, 3.5 to 9, 4 to 9, 4.5 to 9, 5 to 9, 5.5 to 9, 6 to 9, 6.5 to 9, 7 to 9, 7.5 to 9, 8 to 9, 8.5 to 9, 3 to 8, 3.5 to 8, 4 to 8, 4.5 to 8, 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, 7.5 to 8, 3 to 7, 3.5 to 7, 4 to 7, 4.5 to 7, 5 to 7, 5.5 to 7, 6 to 7, 6.5 to 7, 3 to 6, 3.5 to 6, 4 to 6, 4.5 to 6, 5 to 6, 5.5 to 6, 3 to 5, 3.5 to 5, 4 to 5, 4.5 to 5, 3 to 4, or 3.5 to 4 kilodaltons.

[0104] 28. The method or apparatus according to any one of the preceding items, wherein the first single-stranded nucleic acid comprises a sequence capable of hybridizing to itself under conditions suitable for hybridization.

[0105] 29. The method or apparatus according to any one of the preceding items, wherein the second single-stranded nucleic acid comprises a sequence capable of hybridizing to itself under conditions suitable for hybridization.

[0106] 30. The method or apparatus according to any one of items 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.

[0107] 31. The method or apparatus according to any one of items 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.

[0108] 32. The method or apparatus according to any one of the preceding items, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both are DNA, RNA, UNA, PNA, or LNA, or comprise them.

[0109] 33. The method or apparatus according to any one of the preceding items, wherein one or both of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both contain one or more modified and / or non-canonical nucleotides selected from MOE, 2'-fluoro, 2'-OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, nucleotides containing modified sugars, or combinations thereof.

[0110] 34. The method or apparatus according to any one of the preceding items, wherein one or both of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both contain one or more non-phosphodiester bonds between nucleotides.

[0111] 35. The method according to any one of the preceding items, wherein the ultrafiltration step comprises applying a mixture of double-stranded nucleic acid products to a filtration device comprising a cross-flow filter.

[0112] 36. The apparatus according to any one of the preceding items, wherein the filtration element comprises a cross-flow filter.

[0113] 37. The filtration device or filtration 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, where Z is the numerical average of X and Y, X is the minimum pore size that allows at least 90% of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to pass through the membrane; and Y is the maximum pore size that allows at least 90% of the double-stranded nucleic acid to be retained by the membrane. The method or apparatus according to any one of the preceding items.

[0114] 38. The filtration device or filtration element includes a membrane having an average pore size of 0.05 to 20, 0.1 to 20, 0.5 to 20, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 0.05 to 10, 0.1 to 10, 0.5 to 10, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 0.05 to 8, 0.1 to 8, 0.5 to 8, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 0.05 to 6, 0.1 to 6, 0.5 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 0.05 to 4, 0.1 to 4, 0.5 to 4, 1 to 4, 2 to 4, 3 to 4, 0.05 to 2, 0.1 to 2, 0.5 to 2, 1 to 2, 0.05 to 1, 0.1 to 1, or 0.5 to 1 μm. The method or apparatus according to any one of the preceding items.

[0115] 39. The filtration device or filtration element is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons larger than the molecular weight of single-stranded nucleic acid, and is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 kilodaltons smaller than the molecular weight of double-stranded nucleic acid and includes a membrane having a molecular weight cut-off. The method or apparatus according to any one of the preceding items.

[0116] 40. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off that is 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 15 to 25, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, or 1 to 2 kilodaltons greater than the molecular weight of the single-stranded nucleic acid.

[0117] 41. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off that is 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, or 1 to 2 kilodaltons smaller than the molecular weight of the double-stranded nucleic acid.

[0118] 42. The filtration device or filtration element is greater than the molecular weight of the single-stranded nucleic acid and less than the molecular weight of the double-stranded nucleic acid and comprises a membrane having a molecular weight cut-off, and the method or apparatus according to any one of the preceding items.

[0119] 43. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off of 0.5 to 100, 0.5 to 80, 0.5 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, 0.5 to 15, 0.5 to 10, 0.5 to 8, 0.5 to 6, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 100, 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2, 2 to 100, 2 to 80, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 2 to 3, 3 to 100, 3 to 80, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 15, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 4 to 100, 4 to 80, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 10, 4 to 8, 4 to 6, 6 to 100, 6 to 80, 6 to 60, 6 to 50, 6 to 40, 6 to 30, 6 to 20, 6 to 15, 6 to 10, 6 to 8, 8 to 100, 8 to 80, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 20, 8 to 15, 8 to 10, 10 to 100, 10 to 80, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 10 to 15, 15 to 100, 15 to 80, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 100, 30 to 80, 30 to 60, 30 to 50, 30 to 40, 40 to 100, 40 to 80, 40 to 60, 40 to 50, 50 to 100, 50 to 80, 50 to 60, 60 to 80, 60 to 100, or 80 to 100 kilodaltons.

[0120] 44. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, or 15 to 16 kDa.

[0121] 45. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 kDa.

[0122] 46. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 8-15 kDa.

[0123] 47. The method or apparatus according to any one of the preceding items, wherein the filtration device or filtration element comprises a membrane having a molecular weight cut-off of about 10 kDa.

[0124] 48. The method according to any one of the preceding items, 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 (inter-membrane pressure), optionally 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 or less to the mixture of double-stranded nucleic acid products.

[0125] 49. The method according to any one of the preceding items, 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 (inter-membrane pressure).

[0126] 50. The method according to any one of the preceding items, wherein the ultrafiltration step comprises applying a force within the pressure operating conditions recommended by the manufacturer of the filtration device.

[0127] 51. The method according to any one of the preceding items, wherein the diafiltration step comprises applying to the filtration device a diafiltration volume (DTV) of 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, and optionally, a diafiltration volume (DTV) of 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 or less.

[0128] 52. The ultrafiltration step includes 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 to the filtration device in the method according to any one of the preceding items.

[0129] 53. The ultrafiltration step is 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 , and optionally 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 2The method according to any one of the preceding items, comprising the step of achieving a recirculation rate under the following pressure.

[0130] 54. The ultrafiltration step is carried out at a recirculation rate under a pressure 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 The method according to any one of the preceding items, comprising the step of achieving a recirculation rate under the following pressure.

[0131] 55. The method according to any one of the preceding items, wherein ultrafiltration is carried out until a stable low conductivity threshold is achieved.

[0132] 56. The membrane of the filtration device or filtration element comprises polysulfone, polypropylene, cellulose acetate, polylactic acid, nitrocellulose, mixed cellulose ester cellulose nitrate, regenerated cellulose, polyethersulfone, polyamide, cellulose derivative (Hydrosart®), polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track-etched membrane, in the method or device according to any one of the preceding items.

[0133] 57. The filtration device or filtration element is of a hollow fiber, tubular, spiral wound, cassette, plate, or frame membrane type, or comprises the same, in the method or device according to any one of the preceding items.

[0134] 58. The method manufactures a purified double-stranded nucleic acid that meets a purity criterion, where the purity criterion comprises at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% double-stranded nucleic acid, in the method according to any one of the preceding items.

[0135] 59. The method manufactures a purified double-stranded nucleic acid that meets a purity standard, where the purity standard is a holding solution containing 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, the method according to any one of the preceding items.

[0136] 60. The method manufactures a purified double-stranded nucleic acid that meets a purity standard, where the purity standard is a holding solution containing 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 single-stranded nucleic acid to double-stranded nucleic acid, the method according to any one of the preceding items.

[0137] 61. The method achieves a purity standard at 25, 24, 23, 22, 21, or 20 DTVs or less, the method according to any one of the preceding items.

[0138] 62. 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, the method or apparatus according to any one of the preceding items.

[0139] 63. The single-stranded nucleic acid is a component of the double-stranded nucleic acid, the method or apparatus according to any one of the preceding items.

[0140] 64. The double-stranded nucleic acid contains blunt ends, the method or apparatus according to any one of the preceding items.

[0141] 65. The double-stranded nucleic acid contains two blunt ends, the method or apparatus according to any one of the preceding items.

[0142] 66. The double-stranded nucleic acid contains single-stranded portions, the method or apparatus according to any one of the preceding items.

[0143] 67. The method or apparatus according to any one of the preceding items, wherein the double-stranded nucleic acid has a single-stranded portion.

[0144] 68. The method or apparatus according to any one of items 1 to 11, wherein the double-stranded nucleic acid contains a conjugate group.

[0145] 69. The apparatus according to any one of the preceding items, wherein the second chamber contains a permeate.

[0146] 70. The apparatus according to any one of the preceding items, wherein the second chamber holds a permeate.

[0147] 71. The apparatus according to any one of items 6 to 69, wherein the second chamber does not hold a permeate.

[0148] 72. The apparatus according to any one of the preceding items, wherein the first chamber contains a mixture.

[0149] 73. The apparatus according to any one of the preceding items, wherein the first chamber contains a holding solution.

[0150] 74. The mixture is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μl, 200 μl, 300 μl, or 400 μl or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ml, 200 ml, 300 ml, or 400 ml or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 L or less, and optionally contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 L, of the method or apparatus according to any one of the preceding items.

[0151] 75. The method or apparatus according to any one of the preceding items, wherein the mixture comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μl, 200 μl, 300 μl, or 400 μl, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ml, 200 ml, 300 ml, or 400 ml, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 L, and optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 L or less.

[0152] 76. The mixture is 1 - 100, 5 - 100, 10 - 100, 20 - 100, 30 - 100, 40 - 100, 50 - 100, 60 - 100, 70 - 100, 80 - 100, 90 - 100, 1 - 50, 5 - 50, 10 - 50, 20 - 50, 30 - 50, 40 - 50, 1 - 20, 5 - 20, 10 - 20, or 1 - 10 μl, 0.1 - 100, 0.5 - 100, 1 - 100, 5 - 100, 10 - 100, 20 - 100, 30 - 100, 40 - 100, 50 - 100, 60 - 100, 70 - 100, 80 - 100, 90 - 100, 0.1 - 50, 0.5 - 50, 1 - 50, 5 - 50, 10 - 50, 20 - 50, 30 - 50, 40 - 50, 0.1 - 20, 0.5 - 20, 1 - 20, 5 - 20, 10 - 20, 0.1 - 10, 0.5 - 10, 1 - 10, 0.1 - 1, 0.5 - 1, or 0.1 - 0.5 ml, or 0.1 to 500, 0.5 to 500, 1 to 500, 5 to 500, 10 to 500, 20 to 500, 30 to 500, 40 to 500, 50 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 0.1 to 250, 0.5 to 250, 1 to 250, 5 to 250, 10 to 250, 20 to 250, 30 to 250, 40 to 250, 50 to 250, 60 to 250, 70 to 250, 80 to 250, 90 to 250, 100 to 250, 150 to 250, 200 to 250, 0.1 to 100, 0.5 to 100, 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 0.1 to 50, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20, 0.1 to 10, 0.5 to 10, 1 to 10, 0.1 to 1, 0.5 to 1, or 0.1 to 0.5 L The method or apparatus according to any one of the preceding items, comprising

[0153] 77. The method or apparatus according to any one of the preceding items, 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.

[0154] 78. The method or apparatus according to any one of the preceding items, wherein the mixture comprises a nucleic acid concentration of at least about 900 OD / mL.

[0155] 79. The method or apparatus according to any one of the preceding clauses, wherein the mixture comprises a nucleic acid concentration of at least about 1100 OD / mL.

[0156] 80. The method or apparatus according to any one of the preceding items, wherein the mixture comprises a nucleic acid concentration of at least about 400 OD / mL.

[0157] 81. The method or apparatus according to any one of the preceding items, wherein the ssDNA in the permeate is mainly composed of the sense strand.

[0158] 82. The method or apparatus according to any one of the preceding items, wherein the ssDNA in the permeate is mainly composed of the antisense strand.

[0159] 83. The method or apparatus according to any one of the preceding items, wherein the double-stranded nucleic acid contains a mismatch.

[0160] 84. The method or apparatus according to any one of the preceding items, wherein the mixture comprises an amount of single-stranded nucleic acid that exceeds the amount of double-stranded nucleic acid.

[0161] 85. The method or apparatus according to item 84, wherein 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 of the amount of double-stranded nucleic acid.

[0162] 86. The method or apparatus according to item 84, wherein the amount of single-stranded nucleic acid is about 5% in excess of the amount of double-stranded nucleic acid.

[0163] 87. The method according to any one of the preceding items, wherein the ultrafiltration step includes diafiltration.

[0164] 88. The method according to item 87, wherein the diafiltration is performed for at least 10, 20, 30, 40, or 50 minutes, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0165] 89. The method according to item 87 or 88, wherein diafiltration is carried out for a 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, or 65, 70, 75, 80, 81, 85, or 90 diafiltration volume (DTV).

[0166] 90. The method according to any one of the preceding items, wherein ultrafiltration is applied at a pressure of about 5 - 40, 10 - 40, 15 - 40, 20 - 40, 30 - 40, 35 - 40, 5 - 15, 5 - 20, 5 - 25, 5 - 30, 5 - 35, 10 - 30, 15 - 25, 5 - 10, 10 - 15, 15 - 20, 20 - 25, 25 - 30, 30 - 35, or 35 - 40 psi.

[0167] 91. The method according to any one of the preceding items, wherein ultrafiltration is applied at a pressure of about 15 - 30 psi.

[0168] 92. The method according to any one of the preceding items, wherein ultrafiltration is carried out at a temperature of about 10 - 40 °C, and optionally, ultrafiltration is carried out at a temperature at least 0.5 °C lower than the melting point of the double-stranded nucleic acid.

[0169] 93. The method according to any one of the preceding items, further comprising analyzing the retention solution by non-denaturing HPLC.

[0170] 94. The method according to any one of the preceding items, wherein the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retention solution increases over time and / or with the total diafiltration volume (DTV).

[0171] 95. The method according to any one of the preceding items, wherein the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retention solution 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.

[0172] 96. The ratio of double-stranded nucleic acid to single-stranded nucleic acid in the holding solution 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, the method according to any one of the preceding items.

[0173] 97. The concentration of salt 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, the method or apparatus according to any one of the preceding items.

[0174] 98. The mixture is substantially free of salt, the method or apparatus according to item 97.

[0175] 99. The concentration of salt in the mixture is from about 50 mM to about 3 M, the method or apparatus according to any one of the preceding items.

[0176] 100. The melting temperature of the double-stranded nucleic acid in the mixture is from about 20 to 85 °C, the method or apparatus according to any one of the preceding items.

[0177] 101. The relationship between the melting temperature of the double-stranded nucleic acid in the mixture and the concentration of salt in the mixture is determined, the method or apparatus according to any one of the preceding items.

[0178] 102. The double-stranded nucleic acid of the holding solution is for use in downstream applications or uses, the method according to any one of the preceding items.

[0179] 103. The double-stranded nucleic acid of the holding solution is lyophilized, the method according to any one of the preceding items.

[0180] 104. The double-stranded nucleic acid of the holding solution is conjugated with an additional agent, the method according to any one of the preceding items.

[0181] 105. The method according to any one of the preceding items, wherein the double-stranded nucleic acid in the holding solution is mixed with other oligonucleotide duplexes.

[0182] 106. The method according to any one of the preceding items, wherein the double-stranded nucleic acid in the holding solution is mixed with a pharmaceutical formulation buffer.

[0183] 107. The method or apparatus according to any one of the preceding items, wherein the double-stranded nucleic acid has a length of 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, or 400 to 500 nucleotides.

[0184] 108. The method according to any one of the preceding items, 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%.

[0185] 109. The method according to item 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.

[0186] 110. The method according to item 109, wherein the low molecular weight impurities include salts and / or trace solvents.

[0187] 111. The method according to any one of the preceding items, wherein the method reduces the level of the double-stranded nucleic acid by no more than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

[0188] 112. The method according to any one of the preceding items, wherein the ratio of the concentration of the double-stranded nucleic acid in the holding solution to the concentration of the double-stranded nucleic acid 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.

[0189] 113. The method according to any one of the preceding items, wherein the amount of double-stranded nucleic acid in the retention liquid is at least 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 mg / mL.

[0190] 114. A method for optimizing a technique for producing a purified double-stranded nucleic acid, comprising: (i) providing a mixture comprising a double-stranded nucleic acid containing a first single-stranded nucleic acid and a second single-stranded nucleic acid at a first concentration, and single-stranded nucleic acid wherein the mixture has a salt at a predetermined concentration; a step; (ii) subjecting the mixture to a tangential flow filtration step that selectively retains the double-stranded nucleic acid containing the first and second single-stranded nucleic acids rather than the single-stranded nucleic acid, the tangential flow filtration step comprising applying the mixture to a filtration device having a predetermined molecular weight cut-off for a predetermined length of time; (iii) recovering the retention liquid; (iv) changing one or more of the first concentration, the second concentration, the predetermined molecular weight cut-off, the predetermined length of time, or the salt concentration; (v) repeating steps (i) to (iii) one or more times, each time using one or more of the changed first concentration, second concentration, predetermined molecular weight cut-off, predetermined length of time, and / or salt concentration; (vi) in each repetition of steps (i) to (iii), determining the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the recovered retention liquid; and (vii) identifying the first concentration, second concentration, predetermined molecular weight cut-off, predetermined length of time, and / or salt concentration that results in the highest ratio of double-stranded nucleic acid to single-stranded nucleic acid in the recovered retention liquid comprising, whereby the technique for producing a purified double-stranded nucleic acid is optimized.

[0191] A double-stranded nucleic acid composition generated according to the method described in item 1 or any one of items 7 to 113 and containing the recovered retention liquid.

[0192] A single-stranded nucleic acid composition generated according to the method described in item 2 or any one of items 7 to 113 and containing the recovered permeate.

[0193] A composition comprising a first double-stranded nucleic acid containing a first overhang, which hybridizes to a second double-stranded nucleic acid containing a second overhang.

[0194] The composition according to item 117, wherein the first double-stranded nucleic acid is prepared according to the method described in any one of the preceding claims.

[0195] The composition according to item 117 or 118, wherein the second double-stranded nucleic acid is prepared according to the method described in any one of the preceding claims.

[0196] A method for generating a circular double-stranded nucleic acid, comprising: (a) providing a composition according to any one of items 117 to 119; and (b) incubating the composition under conditions suitable for circularization of the first double-stranded nucleic acid hybridized to the second double-stranded nucleic acid. The method comprising.

[0197] The method according to item 120, wherein the circularization includes ligation.

[0198] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples indicate preferred embodiments of the present invention, it should be understood that various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, and are given by way of illustration only.

[0199] The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present invention. The present 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.

Brief Description of the Drawings

[0200]

Figure 1

Figure 2A

Figure 2B

Modes for Carrying Out the Invention

[0201] Detailed Description The present disclosure is directed, at least in part, to improved methods of generating (e.g., purifying) oligonucleotides (e.g., separating double-stranded oligonucleotides from single-stranded oligonucleotides). Without wishing to be bound by theory, in some embodiments, methods of generating (e.g., purifying) oligonucleotides, particularly in large-scale or industrial manufacturing settings, or methods using modified, synthetic, or non-natural nucleotides, are generally very sensitive to process parameters such as the concentration of agents in solution, the ratio of agents to each other in solution, the duration of the process (e.g., recycle time), the membrane pressure, the porosity or composition of the membrane, or the presence and / or level of contaminants in the generated solution. Accordingly, the present disclosure is based, at least in part, on the discovery that changing the generation (e.g., purification) parameters, by changing one or more of, for example, the amount of purified oligonucleotide, the product characteristics of the oligonucleotide (e.g., length, purity, composition (e.g., correct nucleotides, e.g., modified nucleotides), sequence, or annealing state), or the amount of resources or time consumed, can change the effectiveness of the method.

[0202] I. Definitions As used herein, the term “about” refers to the normal error range of each value readily known to one of ordinary skill in the art in this technical field. Thus, the term “about” can be used to indicate that a value includes variations in error inherent in the device or method specified for determining that value. For example, the term “about” generally means ±10% (e.g., ±1, 2, 3, 4, 5, 6, 7, 8, 9, 10%) of the stated amount.

[0203] As used herein, with respect to a particular component, "essentially free of" means that in the present specification, the particular component is not intentionally formulated in the composition and / or is present only as an impurity or in trace amounts. Thus, the total amount of the particular component due to unintentional contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which the particular component is not detected at all by standard analytical methods.

[0204] As used herein, "a" or "an" can mean one or more. In the claims of this specification, when used with the word "comprising", the words "a" or "an" can mean one or more than one.

[0205] The use of the term "or" in the claims supports a definition that the disclosure refers only to alternatives and "and / or" only if it is explicitly indicated that it refers only to alternatives or if the alternatives are mutually exclusive; otherwise, it is used to mean "and / or". As used herein, "another" can mean at least a second or more.

[0206] As used herein, the term "crossflow filter" refers to a filter in which the feed passes tangentially across the surface of the filter. In some embodiments, the crossflow filter includes an intermembrane pressure.

[0207] As used herein, the term "diafiltration tank volume (DTV)" refers to the working volume in which the substance of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) is suspended and maintained through processing, for example, prior to a purification step (e.g., including ultrafiltration) described herein. In some embodiments, DTV is a measure of the volume passed during a diafiltration process based on a comparison of the volume introduced into the unit operation and the hold-up volume. For example, when a volume equal to the working volume contained in the hold-up tank is passed, the diafiltration volume is equal to 1, and when a volume equal to twice the total initial volume is passed, the diafiltration volume is equal to 2, and so on.

[0208] As used herein, the term "molecular weight cut-off" (MWCO) refers to a molecular weight target that reflects the average pore size. In some embodiments, substances that exceed the MWCO (e.g., double-stranded nucleic acid, single-stranded nucleic acid, or other mixture components) are substantially retained by the membrane (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the original substance is retained) in, for example, the ultrafiltration method described herein. 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 not 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, the substance species can pass through the membrane with a molecular weight distribution or range of about ±1 kDa. As an example, a MWCO of 10 kDa can permit the passage of substance species having a molecular weight of 10 ± 1 kDa.

[0209] As used herein, the term "pore size" refers to, for example, the average diameter of the pores of a barrier (e.g., a membrane, e.g., a semipermeable membrane) for filtering particles of a particular size, by a method such as ultrafiltration. In some embodiments, the pore size has an average diameter of 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 pore size is 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 or less. Further, in other embodiments, the pore size is presented as an MWCO of 1, 2, 3, 5, 10, 20, 30, 50, 100 kDa.

[0210] As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is incorporated into, or can be incorporated into, an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into, 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 RNA or includes RNA. In some embodiments, "nucleic acid" is DNA or includes 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 differs 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" known in the art that have peptide bonds in the backbone instead of phosphodiester bonds and are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, a 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, the 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, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating 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 in natural nucleic acids. In some embodiments, the nucleic acid comprises an N-acetylgalactosamine (GalNAc) modification, e.g., a modification to the sugar, phosphate, phosphodiester, or base of the nucleic acid, e.g., a modification to the phosphodiester. 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 encoding a polypeptide or a portion thereof, or comprising at least one element that is a complement of the encoding sequence. In some embodiments, the nucleic acid has enzymatic activity (e.g., the nucleic acid is a ribozyme).

[0211] As used herein, "oligonucleotide" refers to a nucleic acid having a length of at least 2 nucleotides and up to about 500 nucleotides. In some embodiments, the 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 in length. Oligonucleotides can be useful in a variety of biological contexts, 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 biological manufacturing.

[0212] As used herein, the term "other mixture components" refers to components in a mixture that are not the molecule of interest (e.g., double-stranded or single-stranded nucleic acid). In some embodiments, the other mixture components include salts (e.g., halide salts or mineral salts). In some embodiments, the other mixture components include buffers. In certain embodiments, the other mixture components include production reagents or by-products (e.g., cleaved nucleic acid sequences or undesired nucleic acid sequences). In some embodiments, the other mixture components include single-stranded nucleic acids of shorter length (e.g., shorter than the first and / or second single-stranded nucleic acids of interest). In some embodiments, the other mixture components include organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine). In some embodiments, the other mixture components include thiolation reagents / by-products (e.g., xanthane, hydride, or PADS) or capping reagents (e.g., acetic anhydride or NMI). In some embodiments, the other mixture components include coupling reagents (e.g., phosphoramidite or ETT). In some embodiments, the other mixture components include detritylation reagents / by-products (e.g., dichloroacetic acid). In some embodiments, the other mixture components include deprotection reagents / by-products (e.g., diethylamine, methylamine, or ammonia). In some embodiments, the production reagent or by-product can be selected from single-stranded nucleic acids of shorter length (e.g., shorter than the first and / or second single-stranded nucleic acids), 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 activating reagents), and / or deprotection reagents (e.g., diethylamine, methylamine, or ammonia).

[0213] As used herein, the term "permeate" refers to a substance (e.g., single-stranded nucleic acid) that passes through a barrier (e.g., a membrane, e.g., a semipermeable membrane) in, for example, the ultrafiltration method described herein. In some embodiments, the permeate contains single-stranded nucleic acid. In some embodiments, the permeate contains other mixture components. In some embodiments, the permeate contains little or no detectable level of double-stranded nucleic acid. In some embodiments, the stoichiometric ratio of single-stranded nucleic acid to double-stranded nucleic acid in the retentate is at least 100:1, 1,000:1, 10,000:1, 100,000:1, 1,000,000:1, or more.

[0214] As used herein, "phosphoramidite nucleotide" refers to a nucleotide containing a phosphate group that includes a monoamidophosphite diester. In some embodiments, the phosphoramidite nucleotide is useful in the nucleotide addition step described herein. Without wishing to be bound by theory, phosphoramidite nucleotides are thought to be reactive in the presence of a weak acid and a nucleophile (e.g., the nucleic acid of a loaded solid support (e.g., 3'OH)).

[0215] As used herein, the term "post-synthesis step" generally refers to a step of a method that is carried out after the synthesis stage of the 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 removal of one or more chemical moieties (e.g., removal of a protecting group). In some embodiments, the post-synthesis step includes chemical or enzymatic cleavage from a solid substrate and / or removal of one or more chemical moieties (e.g., removal of a protecting group). In some embodiments, the post-synthesis step includes chemical conjugation with a conjugation reagent (e.g., a carboxylic acid, a carboxylic acid ester, a carbonate, and a carboxylic acid activating reagent).

[0216] As used herein, the term "retentate" refers to substances (e.g., double-stranded nucleic acids) that do not pass through a barrier (e.g., a membrane, e.g., a semipermeable membrane, e.g., a filter) in, for example, the ultrafiltration method described herein. In some embodiments, the retentate contains double-stranded nucleic acids. In some embodiments, the permeate contains other mixture components. In some embodiments, the retentate contains little or no single-stranded nucleic acids at detectable levels. 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 more.

[0217] As used herein, the term "selectively retain" refers to, for example, retaining a desired substance in a retentate by the methods described herein and / or in a system or apparatus. In some embodiments, the methods, apparatuses, and systems described herein selectively retain double-stranded nucleic acids.

[0218] As used herein, the term "single-stranded form" refers to a component that is substantially (e.g., completely) single-stranded and, 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 a nucleic acid that is sufficiently complementary to a first portion of the nucleic acid).

[0219] The term "sufficiently complementary", as used herein, refers to a first nucleic acid sequence comprising a continuous nucleic acid sequence that enables a second nucleic acid (e.g., comprising a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse complement of a continuous nucleic acid sequence of the first nucleic acid) to hybridize thereto. Sufficiently complementary sequences can include Watson-Crick base pairs formed from natural and / or modified nucleic acids. Sufficiently complementary sequences can also include non-Watson-Crick base pairs such as wobble base pairs (guanine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, hypoxanthine-cytosine) and Hoogsteen base pairs. In some embodiments, a nucleic acid sequence from a first single-stranded nucleic acid is sufficiently complementary to a nucleic acid sequence from a second single-stranded nucleic acid to hybridize and form a double-stranded nucleic acid. In some embodiments, a nucleic acid from a second single-stranded nucleic acid is sufficiently complementary to a nucleic acid sequence from a first single-stranded nucleic acid to hybridize and form a double-stranded nucleic acid. In some embodiments, a first nucleic acid sequence from a first nucleic acid is sufficiently complementary to a second nucleic acid sequence from the first nucleic acid to hybridize and form, for example, a hairpin loop. In some embodiments, a first nucleic acid sequence from a second nucleic acid is sufficiently complementary to a second nucleic acid sequence from the second nucleic acid to hybridize and form, for example, a hairpin loop. In some embodiments, a first nucleic acid sequence from a first nucleic acid is not sufficiently complementary to a second nucleic acid sequence from the first nucleic acid to hybridize and form, for example, a hairpin loop. In some embodiments, a first nucleic acid sequence from a second nucleic acid is not sufficiently complementary to a second nucleic acid sequence from the second nucleic acid to hybridize and form, for example, a hairpin loop.

[0220] As used herein, the term "synthesis step" generally refers to one or more steps of a method that produces a product or results in one or more additions to a substrate. In some embodiments, the synthesis stage includes one or more "synthesis steps" of the methods described herein. For example, the step is adding one or more nucleotide monomers to a nucleic acid on a solid support. In some embodiments, for example, one or more steps of the synthesis step may be repeated to sequentially add one or more nucleotide monomers to the nucleic acid on the loaded solid support. In some embodiments, the method for making an oligonucleotide includes additional steps or processes in addition to the synthesis stage or the steps included in the synthesis stage. In some embodiments, the synthesis step includes solid-phase chemical synthesis of a first single-stranded nucleic acid, a second single-stranded nucleic acid, or both. In some embodiments, oligonucleotide synthesis is related to the production of oligonucleotides by polymerase chain reaction or production via cell organisms.

[0221] As used herein, the term "transmembrane pressure" refers to a hydrostatic pressure gradient that allows ultrafiltration or convection across a barrier (e.g., a membrane, e.g., the semipermeable membrane 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 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 or less.

[0222] As used herein, the term "ultrafiltration" refers to a filtration process that includes applying a solution to a pressure-driven barrier (e.g., a membrane, e.g., a semipermeable membrane). High molecular weight solutes are retained, and water and low molecular weight solutes pass through the barrier (e.g., a membrane, e.g., a semipermeable membrane). In some embodiments, ultrafiltration includes diafiltration. In certain embodiments, ultrafiltration includes tangential flow filtration. In some embodiments, ultrafiltration includes diafiltration or tangential flow filtration.

[0223] II. Production method The present disclosure is directed, in part, to methods of generating (e.g., manufacturing) purified oligonucleotides (e.g., purified single-stranded oligonucleotides and / or purified double-stranded oligonucleotides). Generally, the methods of generating (e.g., manufacturing) the purified oligonucleotides of the present disclosure include one or more steps of purifying double-stranded (e.g., duplex) oligonucleotides from a mixture (e.g., a mixture containing double-stranded oligonucleotides and single-stranded oligonucleotides). In certain embodiments, the method is a manufacturing method, e.g., a large-scale manufacturing method (e.g., not a research-scale method or an analytical method), or includes such. In some embodiments, the methods of generating (e.g., manufacturing) the purified oligonucleotides of the present disclosure include one or more steps of purifying oligonucleotides in duplex form (e.g., double-stranded oligonucleotides). In some embodiments, the methods of generating (e.g., manufacturing) the purified oligonucleotides of the present disclosure include one or more steps of purifying oligonucleotides in single-stranded form (e.g., single-stranded oligonucleotides). Methods of generating (e.g., manufacturing) purified oligonucleotides (e.g., purified single-stranded nucleic acids or purified double-stranded oligonucleotides) can include, for example, providing a mixture, subjecting the mixture to a crossflow filtration step, and recovering a retentate (e.g., a retentate containing double-stranded oligonucleotides). Methods of generating (e.g., manufacturing) purified oligonucleotides (e.g., purified single-stranded nucleic acids or double-stranded oligonucleotides) can include, for example, providing a mixture, subjecting the mixture to a crossflow filtration step, and recovering a permeate.

[0224] In some embodiments, the step of providing the mixture includes, for example, as described in Examples 1-5, providing a first single-stranded nucleic acid and a second single-stranded nucleic acid that contain sequences that are 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 double-stranded nucleic acid containing the first single-stranded nucleic acid and the second single-stranded nucleic acid, and a mixture containing 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.

[0225] In some embodiments, the step of providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes a synthesis step. In some embodiments, the synthesis step includes solid-phase 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.

[0226] In some embodiments, the step of providing the first single-stranded nucleic acid and the second single-stranded nucleic acid includes a post-synthesis step. In some embodiments, the post-synthesis step includes chemical or enzymatic cleavage from a solid substrate, removal of one or more chemical moieties (e.g., removal of a protecting group), or chemical or enzymatic cleavage from a solid substrate and removal of one or more chemical moieties (e.g., removal of a protecting group). In some embodiments, the post-synthesis step includes chemical conjugation with a conjugation reagent (e.g., a carboxylic acid, a carboxylic acid ester, a carbonate, and a carboxylic acid activating reagent).

[0227] In some embodiments, the step of 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 concentrating nucleic acid components and reducing the level of non-nucleic acid components. In some embodiments, the purification step includes a chromatography step, for example, anion exchange chromatography.

[0228] In some embodiments, the step of 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 and does not retain non-nucleic acid components.

[0229] In some embodiments, the first single-stranded nucleic acid is at least 10 nucleotides in length, for example, at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the first single-stranded nucleic acid is 100 nucleotides in length or less, for example, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less. 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 in length and optionally 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less.

[0230] In some embodiments, the first single-stranded nucleic acid is 10 to 50 nucleotides in length, for example, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides in length.

[0231] 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.

[0232] In some embodiments, the second single-stranded nucleic acid is at least 10 nucleotides in length, for example, at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the second single-stranded nucleic acid is 100 nucleotides in length or less, for example, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less. 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 in length and optionally 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less.

[0233] In some embodiments, the second single-stranded nucleic acid is 10 - 50 nucleotides in length, 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, 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 nucleotides in length.

[0234] 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.

[0235] In some embodiments, the double-stranded nucleic acid is at least 10 base pairs in length, for example, 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 in length. In some embodiments, the double-stranded nucleic acid is 100 base pairs in length or less, for example, 80, 60, 50, 45, 40, 35, or 30 base pairs in length or less. 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 in length, and optionally, 100, 80, 60, 50, 45, 40, 35, or 30 base pairs in length or less.

[0236] In some embodiments, the double-stranded nucleic acid is 10 to 50 base pairs in length, for example, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 base pairs in length.

[0237] 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.

[0238] 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 target gene, non-coding RNA, primer, or sequence of a component for a 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 target gene, non-coding RNA, primer, or sequence of a component for a molecular assembly) by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or less.

[0239] 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, non-coding RNA, primer, or sequence of a component for a molecular assembly). In some embodiments, the double-stranded nucleic acid comprises a sequence that differs from a sequence complementary to a nucleic acid sequence of interest (e.g., a target gene, non-coding RNA, primer, or sequence of a component for a molecular assembly) by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or less.

[0240] In some embodiments, the ultrafiltration step includes diafiltration. Generally, diafiltration involves separating components in a solution (e.g., double-stranded nucleic acids, single-stranded nucleic acids, salts, solvents, or other molecules described herein) based on their molecular size (e.g., molecular weight), for example, using a permeable filter. In some embodiments, diafiltration includes flowing the solution to be filtered through, for example, a permeable filter described herein. In some embodiments, diafiltration includes repeatedly filtering the solution through, for example, a permeable filter described herein for a certain period of time. In some embodiments, diafiltration is performed for at least 10, 20, 30, 40, or 50 minutes, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the diafiltration volume (DTV) is performed for a 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, or 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, 10 - 40, 15 - 40, 20 - 40, 30 - 40, 35 - 40, 5 - 15, 5 - 20, 5 - 25, 5 - 30, 5 - 35, 10 - 30, 15 - 25, 5 - 10, 10 - 15, 15 - 20, 20 - 25, 25 - 30, 30 - 35, or 35 - 40 psi. In some embodiments, the ultrafiltration is applied at a pressure of about 15 - 30 psi (e.g., about 15 - 25, 15 - 20, 25 - 30, 20 - 30, 15 - 20, 20 - 25, or 25 - 30 psi, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 psi).In some embodiments, ultrafiltration is performed at a temperature of about 10 to 40 °C, such as about 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 35, 20 to 30, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40 °C, such as about 10, 15, 20, 25, 30, 35, 37, or 40 °C.

[0241] In some embodiments, the manufacturing method further includes, for example, analyzing the holding solution by non-denaturing HPLC as described in Examples 1 to 5 (for example, to determine the relative amounts of double-stranded nucleic acids and / or single-stranded nucleic acids in the holding solution). In some embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the holding solution (determined, for example, by non-denaturing HPLC) increases over time and / or with the total diafiltration volume (DTV). In some embodiments, the ratio of double-stranded nucleic acids to single-stranded nucleic acids in the holding solution (determined, for example, by non-denaturing 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.

[0242] 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 solvents.

[0243] In some embodiments, the method reduces the level of double-stranded nucleic acid by only 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 acid in the retention solution to the concentration of double-stranded nucleic acid 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 acid in the retention solution to the concentration of double-stranded nucleic acid in the mixture is the ratio of the concentration of double-stranded nucleic acid in the retention solution to the concentration of double-stranded nucleic acid in the mixture before the purification step (e.g., before the ultrafiltration step).

[0244] In some embodiments, the method reduces the level of single-stranded nucleic acid 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 acid in the retention solution to the concentration of single-stranded nucleic acid 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 acid in the retention solution to the concentration of single-stranded nucleic acid in the mixture is the ratio of the concentration of single-stranded nucleic acid in the retention solution to the concentration of single-stranded nucleic acid in the mixture before the purification step (e.g., before the ultrafiltration step).

[0245] In some embodiments, the method produces a purified double-stranded nucleic acid that meets a purity criterion. In some embodiments, the purity criterion comprises a holding solution 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., 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 criterion comprises a holding solution 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 criterion comprises a holding solution 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 meets the purity criterion at 25, 24, 23, 22, 21, or 20 DTVs or less.

[0246] A. Purified double-stranded nucleic acid The present disclosure is directed, in part, to methods of generating (e.g., producing) double-stranded nucleic acids. Generally, the methods of the present disclosure include one or more steps of selectively retaining a double-stranded nucleic acid from a mixture, e.g., a mixture comprising other mixture components.

[0247] In some embodiments, a method of generating (e.g., manufacturing) a purified double-stranded nucleic acid includes one or more steps of selectively retaining the double-stranded nucleic acid from a mixture (e.g., other mixture components such as a desired product (e.g., a single-stranded nucleic acid), a salt (e.g., a halide salt or a mineral salt), a buffer, a generation reagent or a by-product (e.g., a cleaved nucleic acid sequence or an undesired nucleic acid sequence), a single-stranded nucleic acid of shorter length (e.g., shorter than the first and / or second single-stranded nucleic acid of interest), an organic solvent (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine, but not limited thereto), a thiolation reagent (e.g., xanthane, hydride, or PADS), a capping reagent (e.g., acetic anhydride or NMI), a coupling reagent (e.g., phosphoramidite or ETT), a detritylation reagent (e.g., dichloroacetic acid), a deprotection reagent (e.g., diethylamine, methylamine, or ammonia), and / or a conjugation reagent (e.g., a carboxylic acid, a carboxylic acid ester, a carbonate, and a carboxylic acid activation reagent).

[0248] In some embodiments, the mixture comprises a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid, and 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 the first single-stranded nucleic acid in single-stranded form and the second single-stranded nucleic acid in single-stranded form. In some embodiments, a method of generating (e.g., producing) a purified double-stranded nucleic acid comprises subjecting a mixture (e.g., the mixture described herein) to an ultrafiltration step that selectively retains the double-stranded nucleic acid comprising the first and second single-stranded nucleic acids rather than single-stranded nucleic acids (e.g., the first and second single-stranded nucleic acids in single-stranded form). In some embodiments, a method of generating (e.g., producing) a purified double-stranded nucleic acid comprises, after subjecting the mixture to an ultrafiltration step, recovering the retentate. In some embodiments, the retentate comprises the double-stranded nucleic acid comprising the first and second single-stranded nucleic acids and does not comprise single-stranded nucleic acids (e.g., the first and second single-stranded nucleic acids in single-stranded form). In some embodiments, a method of generating (e.g., producing) a purified double-stranded nucleic acid comprising the first and second single-stranded nucleic acids comprises separating the double-stranded nucleic acid from the first single-stranded nucleic acid in single-stranded form and / or the second single-stranded nucleic acid in single-stranded form.

[0249] In some embodiments, the double-stranded nucleic acid is at least 10 base pairs in length, 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 in length. In some embodiments, the double-stranded nucleic acid is 100 base pairs in length or less, such as 80, 60, 50, 45, 40, 35, or 30 base pairs in length or less. 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 part 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, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length and optionally 100, 80, 60, 50, 45, 40, 35, or 30 base pairs in length or less.

[0250] In some embodiments, the double-stranded nucleic acid is 10 to 50 base pairs in length, for example, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 base pairs in length.

[0251] 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.

[0252] In some embodiments, the double-stranded nucleic acid is 100 to 200 base pairs in length, for example, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, or 190 to 200 base pairs in length.

[0253] 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 target gene, non-coding RNA, primer, or sequence of a component for a 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 target gene, non-coding RNA, primer, or sequence of a component for a molecular assembly) by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or less.

[0254] 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, non-coding RNA, primer, or sequence of a component for a molecular assembly). In some embodiments, the double-stranded nucleic acid comprises a sequence that differs from a sequence complementary to a nucleic acid sequence of interest (e.g., a target gene, non-coding RNA, primer, or sequence of a component for a molecular assembly) by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or less.

[0255] 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 (e.g., the double-stranded nucleic acid comprises a copy of the single-stranded nucleic acid).

[0256] In some embodiments, the double-stranded nucleic acid comprises a blunt end (e.g., one blunt end). In some embodiments, the double-stranded nucleic acid comprises two blunt ends. In some embodiments, the double-stranded nucleic acid comprises a single-stranded portion (e.g., at one end, e.g., a terminal overhang). In some embodiments, the double-stranded nucleic acid has a single-stranded portion (e.g., at both ends, e.g., terminal overhangs).

[0257] In some embodiments, the double-stranded nucleic acid comprises a conjugate group. In some embodiments, the double-stranded nucleic acid is or comprises a conjugate group such as PEG (polyethylene glycol), an amino linker, GalNAc, or a glycol spacer.

[0258] In some embodiments, the double-stranded nucleic acid comprises a mismatch (e.g., at one of the ends of the double-stranded nucleic acid, within 1 to 2 nucleotides from one end of the double-stranded nucleic acid, or between the ends of the double-stranded nucleic acid).

[0259] In some embodiments, the melting temperature of the double-stranded nucleic acid in the mixture is between about 20 and 85 °C, for example, between about 25 and 80, 30 and 75, 35 and 70, 40 and 65, 45 and 60, 20 and 25, 25 and 30, 30 and 35, 35 and 40, 40 and 45, 45 and 50, 50 and 55, 55 and 60, 60 and 65, 65 and 70, 70 and 75, 75 and 80, or 80 and 85 °C. In some embodiments, the melting temperature of the double-stranded nucleic acid in the mixture is related to the concentration of the salt in the mixture. In some embodiments, the relationship between the melting temperature of the double-stranded nucleic acid in the mixture and the concentration of the salt (e.g., a cation, e.g., a sodium ion) in the mixture is determined according to the method of Owczarzy et al. (2004, Biochemistry 43: 3537-3554; incorporated herein by reference in its entirety).

[0260] In some embodiments, the ratio of the concentration of double-stranded nucleic acid in the holding solution to the concentration 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. In some embodiments, the amount of double-stranded nucleic acid in the holding solution 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 holding solution is between about 500 and 1200 OD / mL (e.g., between about 500 - 550, 550 - 600, 600 - 650, 650 - 700, 700 - 750, 750 - 800, 800 - 850, 850 - 900, 900 - 950, 950 - 1000, 1000 - 1050, 1050 - 1100, 1100 - 1150, or 1150 - 1200 OD / mL). In some examples, the amount of double-stranded nucleic acid in the holding solution 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 holding solution is between about 20 and 48 mg / mL (e.g., between about 20 - 25, 25 - 30, 30 - 35, 35 - 40, 40 - 45, or 45 - 48 mg / mL).

[0261] In some embodiments, the ratio of the amount of double-stranded nucleic acid in the retention solution 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, or 1.00. In some embodiments, the ratio of the amount of single-stranded nucleic acid in the retention solution to the amount of single-stranded nucleic acid 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.

[0262] In some embodiments, the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retention solution (e.g., as determined by non-denaturing HPLC) increases over time and / or with the total diafiltration volume (DTV). In certain embodiments, the ratio of double-stranded nucleic acid to single-stranded nucleic acid in the retention solution (e.g., as determined by non-denaturing 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.

[0263] In some embodiments, the ratio of the concentration of double-stranded nucleic acid in the holding solution to the concentration of single-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. In some embodiments, the ratio of the amount of single-stranded nucleic acid in the holding solution 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.

[0264] In some embodiments, the step of providing double-stranded nucleic acid includes the step of annealing a first single-stranded nucleic acid and a second single-stranded nucleic acid. Individual purified aqueous oligonucleotide matrices are introduced into a container equipped with mixing, heating / cooling, and nitrogen atmosphere functions. Each amount of single-stranded nucleic acid is added in equivalents so that the desired molar ratio (e.g., equivalent or excess) is achieved. The ratio is verified by non-denaturing analytical chromatography (e.g., HPLC, SEC). The aqueous mixture of single-stranded nucleic acids is subjected to a thermal cycle (e.g., annealing) to promote hybridization to form double-stranded nucleic acid. Annealing is first performed by cooling the solution to below room temperature (about 5°C to 10°C (e.g., about 5, about 6, about 7, about 8, about 9, or about 10°C)), then heating to a high temperature at a controlled heating rate (gradient) to reach a temperature close to the Tm of the hybridized double-strand, holding at this high temperature for a short time, and then cooling again to below room temperature (about 5°C to 10°C (e.g., about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C)) at a controlled rate (gradient) to obtain a concentrated aqueous solution of double-stranded nucleic acid.

[0265] B. Purified single-stranded nucleic acid The present disclosure is directed, in part, to methods of purifying single-stranded nucleic acids. Generally, the methods of the present disclosure include one or more steps of selectively retaining double-stranded nucleic acids from a mixture, such as, for example, a mixture comprising single-stranded nucleic acids, double-stranded nucleic acids, and / or other mixture components. In some embodiments, the method of purifying single-stranded nucleic acids includes one or more steps of removing single-stranded nucleic acids from a mixture, but does not include the synthesis of single-stranded nucleic acids.

[0266] In some embodiments, a method of producing a purified single-stranded nucleic acid includes one or more steps of selectively retaining double-stranded nucleic acids from a mixture (e.g., a mixture comprising other mixture components (e.g., those that are not the desired product (e.g., single-stranded nucleic acids), salts (e.g., halide salts or mineral salts), buffers, production reagents or by-products (e.g., cleaved nucleic acid sequences or unwanted nucleic acid sequences), shorter single-stranded nucleic acids (e.g., shorter than the first and / or second single-stranded nucleic acids 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 acid or NMI), coupling reagents (e.g., phosphoramidite or ETT), detritylation reagents (e.g., dichloroacetic acid), deprotection reagents (e.g., diethylamine, ethylenediamine, methylamine, or ammonia)), and / or conjugation reagents (e.g., carboxylic acids, carboxylic acid esters, carbonates, amines, and carboxylic acid activation reagents)). In some embodiments, the single-stranded nucleic acid is in the permeate. In some embodiments, the single-stranded nucleic acid is in the permeate and the permeate is recovered.

[0267] In some embodiments, the first single-stranded nucleic acid is at least 10 nucleotides in length, for example, 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 in length. 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 part of the 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, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less. In some embodiments, the first single-stranded nucleic acid is 100 nucleotides in length or less, for example, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less.

[0268] In some embodiments, the first single-stranded nucleic acid is 10 to 50 nucleotides in length, for example, 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides in length.

[0269] 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.

[0270] In some embodiments, the second single-stranded nucleic acid is at least 10 nucleotides in length, 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 in length. In some embodiments, the second single-stranded nucleic acid is 100 nucleotides in length or less, such as 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less. 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, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 80, 60, 50, 45, 40, 35, or 30 nucleotides in length or less.

[0271] In some embodiments, the second single-stranded nucleic acid is 10 to 50 nucleotides in length, such as 12 to 50, 14 to 50, 16 to 50, 18 to 50, 20 to 50, 22 to 50, 24 to 50, 26 to 50, 28 to 50, 30 to 50, 32 to 50, 34 to 50, 36 to 50, 38 to 50, 40 to 50, 42 to 50, 44 to 50, 46 to 50, 48 to 50, 10 to 45, 12 to 45, 14 to 45, 16 to 45, 18 to 45, 20 to 45, 22 to 45, 24 to 45, 26 to 45, 28 to 45, 30 to 45, 32 to 45, 34 to 45, 36 to 45, 38 to 45, 40 to 45, 42 to 45, 10 to 40, 12 to 40, 14 to 40, 16 to 40, 18 to 40, 20 to 40, 22 to 40, 24 to 40, 26 to 40, 28 to 40, 30 to 40, 32 to 40, 34 to 40, 36 to 40, 38 to 40, 10 to 35, 12 to 35, 14 to 35, 16 to 35, 18 to 35, 20 to 35, 22 to 35, 24 to 35, 26 to 35, 28 to 35, 30 to 35, 32 to 35, 10 to 30, 12 to 30, 14 to 30, 16 to 30, 18 to 30, 20 to 30, 22 to 30, 24 to 30, 26 to 30, 28 to 30, 10 to 25, 12 to 25, 14 to 25, 16 to 25, 18 to 25, 20 to 25, 22 to 25, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 10 to 15, or 12 to 15 nucleotides in length.

[0272] 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.

[0273] 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 (e.g., the double-stranded nucleic acid includes a copy of the single-stranded nucleic acid).

[0274] III. Apparatus In some embodiments, the ultrafiltration step includes applying a mixture (e.g., the mixtures described herein) to a filtration device (e.g., a device including a barrier (e.g., a membrane, e.g., a semipermeable membrane)). In some embodiments, the device includes a first chamber, a second chamber, and / or a filtration element disposed between the first chamber and the second chamber, e.g., the filtration elements described herein, e.g., a barrier (e.g., a membrane (e.g., a semipermeable membrane)). In some embodiments, the first chamber holds the mixture to be filtered (e.g., the mixtures described herein) and is optionally configured to capture the retention fluid. In some embodiments, the second chamber is configured to capture the permeate. In some examples, the mixture includes double-stranded nucleic acids (e.g., including a first single-stranded nucleic acid and a second single-stranded nucleic acid) and single-stranded nucleic acids (e.g., 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). In some embodiments, the filtration element (e.g., a barrier (e.g., a membrane, e.g., a semipermeable membrane)) selectively retains double-stranded nucleic acids (e.g., including the first and second single-stranded nucleic acids) rather than single-stranded nucleic acids (e.g., the first and / or second single-stranded nucleic acids in single-stranded form). In some embodiments, the device does not substantially retain, e.g., does not retain a detectable amount of, one or more other mixture components described herein.

[0275] In some embodiments, the device is pressurized (e.g., at a pressure of about 5 - 40 psi, in some examples in a medium psi range (about 15 - 30 psi)) to concentrate the matrix on the retention fluid side of the membrane (e.g., a semipermeable membrane). Thereafter, in some embodiments, treated water may be introduced under pressure to facilitate filtration (e.g., diafiltration) of other mixed components.

[0276] In some embodiments, the ultrafiltration step produces a retentate and a permeate. In some embodiments, the retentate is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% enriched for a desired molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) relative to, for example, the mixture subjected to ultrafiltration. In some embodiments, the retentate is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% enriched for double-stranded nucleic acid relative to, for example, the mixture subjected to ultrafiltration. In some embodiments, the retentate is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% enriched for single-stranded nucleic acid relative to, for example, the mixture subjected to ultrafiltration.

[0277] In some embodiments, the desired molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) is concentrated to a purity of from 2% to at least 90%, from 5% to at least 90%, from 10% to at least 90%, from 15% to at least 90%, from 20% to at least 90%, from 25% to at least 90%, from 30% to at least 90%, from 35% to at least 90%, from 40% to at least 90%, from 45% to at least 90%, from 50% to at least 90%, from 55% to at least 90%, from 60% to at least 90%, from 65% to at least 90%, from 70% to at least 90%, from 75% to at least 90%, from 80% to at least 90%, from 85% to at least 90%, from 90% to at least 92.5%, from 95% to at least 97.5%, or from 97.5% to at least 99%. In some embodiments, the desired molecule of interest is concentrated to a purity of from 50% to at least 90%, from 55% to at least 90%, from 60% to at least 90%, from 65% to at least 90%, from 70% to at least 90%, from 75% to at least 90%, from 80% to at least 90%, from 85% to at least 90%, from 90% to at least 92.5%, from 92.5% to at least 95%, from 95% to at least 97.5%, or from 97.5% to at least 99%.

[0278] In some embodiments, the purity of the desired molecule of interest (e.g., double-stranded nucleic acid or single-stranded nucleic acid) increases 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.

[0279] In some embodiments, the ultrafiltration step produces a retentate and a permeate. In some embodiments, the retentate is enriched, for example, by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% with respect to the permeate for a desired molecule of interest (e.g., double-stranded or single-stranded nucleic acid). In some embodiments, the retentate is enriched, for example, by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% with respect to the permeate for double-stranded nucleic acid. In some embodiments, the permeate is enriched, for example, by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% with respect to the permeate for single-stranded nucleic acid.

[0280] In some embodiments, the retentate contains one or more of a salt, buffer, or product reagent or byproduct at a different concentration than the permeate, mixture, or permeate and mixture. In some embodiments, the retentate has a lower concentration of one or more of a salt, buffer, or product reagent or byproduct than the permeate, mixture, or permeate and mixture.

[0281] In some embodiments, the second chamber contains the permeate. In some embodiments, the second chamber contains, for example, a permeate containing 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.

[0282] In some embodiments, the single-stranded nucleic acid in the permeate is mainly composed of the sense strand (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the single-stranded nucleic acid in the permeate consists of the sense strand). In some embodiments, the single-stranded nucleic acid in the permeate is mainly composed of the antisense strand (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the single-stranded nucleic acid in the permeate consists of the antisense strand).

[0283] A. Membrane In some embodiments, the membrane of the filtration device or filtration element comprises one or more of polysulfone, polypropylene, cellulose acetate, polylactic acid, nitrocellulose, mixed cellulose ester cellulose nitrate, regenerated cellulose, polyethersulfone, polyamide, cellulose derivative (HYDROSART®), polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track-etched membrane. In some embodiments, the filtration device or filtration element is or comprises a hollow fiber, tubular, spiral wound, cassette, plate, or frame membrane type filter.

[0284] 1. Molecular weight cut-off (MWCO) In some embodiments, a barrier (e.g., a membrane, e.g., a semipermeable membrane) includes a molecular weight cut-off (MWCO). In some embodiments, a barrier having an MWCO selectively retains double-stranded nucleic acids (e.g., preferentially over single-stranded nucleic acids). In some embodiments, a barrier having an MWCO selectively permits the passage of single-stranded nucleic acids (e.g., preferentially over 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 not 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 the double-stranded nucleic acid and not 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, 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 the single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the 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 the 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 the single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the 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 the double-stranded nucleic acid.In some embodiments, the MWCO is at least about 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, or 1 to 2 kilodaltons larger than the molecular weight of the single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both). In some embodiments, the MWCO is about 1 to 30, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 1 to 25, 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 15 to 25, 20 to 25, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 1 to 15, 2 to 15, 3 to 15, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, or 1 to 2 kilodaltons smaller 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 the single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the 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 the single-stranded nucleic acid (e.g., the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both) and at least about 5 kilodaltons less than the molecular weight of the double-stranded nucleic acid.

[0285] 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 - 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 - 100, 15 - 80, 15 - 60, 15 - 50, 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. 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 certain embodiments, the MWCO is about 8-15 kDa. In certain embodiments, the MWCO is about 10 kDa.

[0286] In some embodiments, a barrier having an 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 substantial denaturation of the double-stranded nucleic acids (e.g., without detectable denaturation of the double-stranded nucleic acids), e.g., at an intermembrane 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, about 10 kDa (e.g., about 7, 8, 9, 10, 11, 12, 13, or 14 kDa) selectively retains double-stranded nucleic acids, and the double-stranded nucleic acids in the mixture to be denatured are less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, 0.0001%, or 0.00001%.

[0287] 2. Average pore size In some embodiments, a barrier (e.g., a membrane, e.g., a semipermeable membrane) includes 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.

[0288] 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 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 or less.

[0289] In some embodiments, the average pore size is from about 0.05 to 20, 0.1 to 20, 0.5 to 20, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 15 to 20, 0.05 to 10, 0.1 to 10, 0.5 to 10, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 0.05 to 8, 0.1 to 8, 0.5 to 8, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 0.05 to 6, 0.1 to 6, 0.5 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 0.05 to 4, 0.1 to 4, 0.5 to 4, 1 to 4, 2 to 4, 3 to 4, 0.05 to 2, 0.1 to 2, 0.5 to 2, 1 to 2, 0.05 to 1, 0.1 to 1, or 0.5 to 1 μm.

[0290] 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, and X is the minimum pore size that allows at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both to permeate the membrane (e.g., at 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, or 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV)); and Y is the minimum pore size that allows at least 90% of the double-stranded nucleic acid to permeate the membrane (e.g., at 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, or 65, 70, 75, 80, 81, 85, or 90 DTVs).

[0291] B. Parameter While not wishing to be bound by theory, in some embodiments, certain process parameters are thought to be important in combination with each other to determine the effectiveness of a method of generating (e.g., manufacturing) oligonucleotides (e.g., purified double-stranded oligonucleotides and / or purified single-stranded oligonucleotides). In some embodiments, combinations of process parameters have, for example, an optimal configuration in relation to each other and do not have an optimal configuration when considered alone. Exemplary combinations of process parameters are described below.

[0292] C. Mixture Components In some embodiments, the mixture comprises double-stranded nucleic acids and single-stranded nucleic acids. 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 sufficiently complementary to form a double-stranded nucleic acid, and the mixture comprises the double-stranded nucleic acid. In some embodiments, the mixture comprises a first single-stranded nucleic acid, a second single-stranded nucleic acid, a double-stranded nucleic acid comprising the first single-stranded nucleic acid and the second single-stranded nucleic acid, and other mixture components.

[0293] In some embodiments, the mixture comprises other mixture components. In some embodiments, the other mixture components include components that are not the desired product (e.g., molecules present in the holding solution or the permeate, e.g., molecules of the desired target). In some embodiments, the product comprises double-stranded nucleic acid. In some embodiments, the product comprises single-stranded nucleic acid. In some embodiments, the other mixture components include salts (e.g., halide salts or mineral salts), buffers, production reagents or by-products (e.g., cleaved nucleic acid sequences or undesired nucleic acid sequences), single-stranded nucleic acids of shorter length (e.g., shorter than the first and / or second single-stranded nucleic acids of interest), organic solvents (e.g., acetonitrile, ethanol, dimethylformamide, DMSO, toluene, pyridine, or lutidine, but not limited thereto), 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), deprotection reagents (e.g., diethylamine, methylamine, or ammonia), conjugation reagents (e.g., carboxylic acids, carboxylic acid esters, amines, carbonates, and carboxylic acid activation reagents), or any combination thereof, but not limited thereto.

[0294] In some embodiments, the mixture comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml or less, or 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 L or less (and optionally at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 L).

[0295] In some embodiments, the mixture comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml, or at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 L (and optionally, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μl, or at most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml, or at most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 L).

[0296] In some embodiments, the mixture is 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 1 to 20, 5 to 20, 10 to 20, or 1 to 10 μl; 0.1 to 100, 0.5 to 100, 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 0.1 to 50, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20, 0.1 to 10, 0.5 to 10, 1 to 10, 0.1 to 1, 0.5 to 1, or 0.1 to 0.5 ml; or 0.1 to 500, 0.5 to 500, 1 to 500, 5 to 500, 10 to 500, 20 to 500, 30 to 500, 40 to 500, 50 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 0.1 to 250, 0.5 to 250, 1 to 250, 5 to 250, 10 to 250, 20 to 250, 30 to 250, 40 to 250, 50 to 250, 60 to 250, 70 to 250, 80 to 250, 90 to 250, 100 to 250, 150 to 250, 200 to 250, 0.1 to 100, 0.5 to 100, 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 0.1 to 50, 0.5 to 50, 1 to 50, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 0.1 to 20, 0.5 to 20, 1 to 20, 5 to 20, 10 to 20, 0.1 to 10, 0.5 to 10, 1 to 10, 0.1 to 1, 0.5 to 1, or 0.1 to 0.5 L.

[0297] 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., OD at 250 - 260 nm, e.g., 258 - 260 nm) (and optionally, 2500, 2000, 1500, 1400, 1300, 1200, 1100, or 1000 OD / mL or less).

[0298] 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 about 900 OD / mL (e.g., about 900 OD / mL). e

[0299] 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 about 1100 OD / mL (e.g., about 1100 OD / mL).

[0300] In certain 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 about 400 OD / mL (e.g., about 400 OD / mL).

[0301] In some embodiments, the mixture comprises an amount of single-stranded nucleic acid that 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 of the amount of double-stranded nucleic acid. In certain embodiments, the amount of single-stranded nucleic acid is about 5% in excess of the amount of double-stranded nucleic acid.

[0302] In some embodiments, the concentration of salt (e.g., cation, e.g., 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 is substantially salt-free. In some embodiments, the concentration of salt (e.g., cation, e.g., sodium ion) in the mixture is from about 50 mM to about 1.5 M, such as from about 60 nM to about 1.2 M, or from 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 and 85 °C, such as between about 25 and 80, 30 and 75, 35 and 70, 40 and 65, 45 and 60, 20 and 25, 25 and 30, 30 and 35, 35 and 40, 40 and 45, 45 and 50, 50 and 55, 55 and 60, 60 and 65, 65 and 70, 70 and 75, 75 and 80, or 80 and 85 °C.

[0303] D. Total amount and time of diafiltration Without wishing to be bound by theory, 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 each considered process parameters that can be important in determining the effectiveness of the method for purifying double-stranded oligonucleotides. These process parameters together include combinations of process parameters where optimal configurations are related to each other and, for example, are dependent on each other.

[0304] In some embodiments, the ultrafiltration step includes the step of applying (e.g., sequentially) 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. In some embodiments, the ultrafiltration step includes 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 or less. In some embodiments, the ultrafiltration step includes the step of applying (e.g., sequentially) 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, and optionally 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 9 DTVs or less of diafiltration volume (DTV) to the filtration device.In some embodiments, the ultrafiltration step includes 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 DTV to the filtration device (e.g., sequentially).In some embodiments, the ultrafiltration step comprises applying (e.g., sequentially) 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 to the filtration device (e.g., sequentially).

[0305] In some embodiments, the ultrafiltration step comprises applying the DTV to the filtration device (e.g., sequentially) until a stable low conductivity threshold is achieved. In some embodiments, the ultrafiltration is performed until a stable low conductivity threshold (e.g., about 30, 40, 50, 60, 70, or 80 μS / cm or less, e.g., a conductivity threshold of about 50 μS / cm) is achieved, e.g., the DTV is applied until a stable low conductivity threshold is achieved.

[0306] E. Pressure Although not wishing to be bound by theory, the pressure at which the mixture is ultrafiltered, the total diafiltration volume (DTV) and / or time of the apparatus, and the temperature at which the mixture is ultrafiltered are each considered important process parameters to determine the effectiveness of the method for purifying double-stranded oligonucleotides. These process parameters together have an optimal configuration that is related to each other and includes, for example, combinations of process parameters that are dependent on each other.

[0307] In some embodiments, the ultrafiltration step includes 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 (intermembrane pressure) to the mixture of double-stranded nucleic acid products, for example, within a filtration device. In some embodiments, the ultrafiltration step includes applying a force of 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 or less to the mixture of double-stranded nucleic acid products, for example, within a filtration device. In some embodiments, the ultrafiltration step includes 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 (intermembrane pressure), optionally 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 or less to the mixture of double-stranded nucleic acid products, for example, within a filtration device. In some embodiments, the ultrafiltration step includes 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 (intermembrane pressure). In some embodiments, the ultrafiltration step includes applying a force within the pressure operating conditions recommended by the manufacturer of the filtration device.

[0308] In some embodiments, the ultrafiltration step comprises achieving a recycle rate under a membrane 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 recycle rate under a membrane pressure of 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 recycle rate under a membrane 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 and optionally, achieving a recycle rate under a membrane pressure of 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 The ultrafiltration step comprises achieving a recycle rate under a membrane pressure of as follows.

[0309] In some embodiments, the ultrafiltration step comprises achieving a recycle rate under a membrane 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 recycle rate under a membrane pressure of 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 2Including achieving a recirculation rate under the pressure of the following membranes. In some embodiments, the ultrafiltration step is 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 , and optionally, 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 Including the step of achieving a recirculation rate under the pressure of the following membranes. In some embodiments, the ultrafiltration step is at least 5, 6, 7, 8, 9, 10, 11, or 12 L / min / m 2 Including achieving a recirculation rate under the pressure of the following membranes.

[0310] In some embodiments, the ultrafiltration step is 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 Including the step of achieving a recirculation rate under the pressure of...

[0311] F. Temperature Although not wishing to be bound by theory, 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 apparatus are each considered important process parameters for determining the effectiveness of the method for purifying double-stranded oligonucleotides. These process parameters together have an optimal configuration that is related to and, for example, includes combinations of process parameters that are dependent on each other.

[0312] In some embodiments, ultrafiltration is carried out at a temperature of about 10 to 40 °C, for example, about 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 35, 20 to 30, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40 °C, for example, about 10, 15, 20, 25, 30, 35, 37, or 40 °C. In some embodiments, ultrafiltration is carried out at a temperature 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 lower than the oligonucleotide melting point. In some embodiments, ultrafiltration is carried out at a temperature where the oligonucleotide melting point is not reached or exceeded (e.g., less than it) anywhere within the system. In some embodiments, the temperature at which ultrafiltration is carried out does not change by more than about 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, or 5 °C during the filtration process.

Example

[0313] IV. Example The present invention will be further illustrated by the following examples. These examples are provided for illustrative purposes only and are not to be construed as limiting the scope or content of the present invention in any way.

[0314] [Example 1] Exemplary purification method In this example, a double-stranded oligonucleotide composed of two sufficiently complementary strands is synthetically formed by annealing equimolar amounts of the individual strands together. Analysis by non-denaturing HPLC is used to measure the content of the double-stranded and unreacted single-stranded species. Analysis of the double-stranded oligonucleotide sample is performed at 35 °C using an Agilent 1290 UHPLC equipped with a Waters XBridge BEH C4 300A, 2.1×50 mm, 3.5 μM analytical column. To compare the abundance of the excess single-stranded and double-stranded species, mobile phase A (95 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mM triethylamine, 24 mM t-butylamine, 1 μM ethylenediaminetetraacetic acid) and mobile phase B (acetonitrile) are used, and peaks are eluted over 26.25 minutes.

[0315] [Example 2] Exemplary ultrafiltration or crossflow filtration The oligonucleotide matrix is introduced into an ultrafiltration or crossflow ultrafiltration device. A standard ultrafiltration device consisting of a retentate tank, a pump, an ultrafiltration membrane with a specific molecular weight cut-off aspect (e.g., 2, 5, 10 kD), a conductivity meter, and a waste liquid container (Figure 1) is used. The system is pressurized at medium pressure (about 15 - 30 psi) to concentrate the matrix on the retentate side of the permeation membrane. Next, treated water is introduced under pressure to facilitate diafiltration of low molecular weight contaminants.

[0316] Diafiltration is continued until a stable low conductivity threshold, typically (≤50 μS / cm), is achieved and up to a minimum target of system volume exchange.

[0317] [Example 3] Double-stranded nucleic acid with a molecular weight greater than 10 kDa at a concentration of approximately 900 OD / mL An ultrafiltration device was equipped with a cut-off membrane with a molecular weight cut-off of 10 kDa (HYDROSART (registered trademark) Ultrafilter, Sartorius). Double-stranded oligonucleotides with a molecular weight exceeding 10 kDa, containing about 5% excess single-stranded and a total oligonucleotide concentration of about 900 OD / mL, were ultrafiltered as described in Example 1 and Example 2. Diafiltration was carried out as a function of the total diafiltration volume (DTV) and time. Samples of the retentate matrix were analyzed by UV and non-denaturing HPLC as a function of time for the double-stranded and single-stranded composition. As shown in Table 1 and Figures 2A - 2C below, the ratio of low molecular weight single-stranded to the ratio of double-stranded decreased over time.

[0318]

Table 1

[0319] [Example 4] Double-stranded nucleic acids with a molecular weight exceeding 10 kDa at a concentration of about 400 OD / mL An ultrafiltration device was equipped with a cut-off membrane with a molecular weight cut-off of 10 kDa. Double-stranded oligonucleotides with a molecular weight exceeding 10 kDa, containing about 5% excess single-stranded and an oligonucleotide concentration of about 400 OD / mL, were ultrafiltered as described in Example 1 and Example 2. Diafiltration was carried out as a function of the total diafiltration volume (DTV) and time. Samples of the retentate matrix were analyzed by UV and non-denaturing HPLC as a function of time for the double-stranded and single-stranded composition. As shown in Table 2 below, the ratio of low molecular weight single-stranded to the ratio of double-stranded did not decrease over time.

[0320]

Table 2

[0321] [Example 5] Double-stranded nucleic acids with a molecular weight exceeding 10 kDa at a concentration of about 1100 OD / mL An ultrafiltration device was equipped with a cut-off membrane having a molecular weight cut-off of 10 kDa. Double-stranded oligonucleotides having a molecular weight exceeding 10 kDa, containing an excessive amount of about 5% single-stranded and an oligonucleotide concentration of 1,100 to 1,400 OD / mL, were ultrafiltered as described in Example 1 and Example 2. Diafiltration was carried out as a function of the total amount of diafiltration (DTV) and time. Samples of the retentate matrix were analyzed by UV and non-denaturing HPLC as a function of time for the composition of double-stranded and single-stranded. As shown in Table 3 below, the ratio of low molecular weight single-stranded to the ratio of double-stranded decreased with the passage of time. [Table 3]

[0322] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described from the perspective of preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the steps or the order of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that the same or similar results can be achieved by replacing specific chemically and physiologically related agents with the agents described herein. All such similar alternatives and modifications that are apparent to those skilled in the art are considered to be within the scope of the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. 1. A method for separating double-stranded nucleic acids from single-stranded nucleic acids, comprising: providing a mixture of double-stranded and single-stranded nucleic acids; and subjecting said mixture to an ultrafiltration step that selectively retains double-stranded nucleic acids over single-stranded nucleic acids. Including, the ultrafiltration step comprises applying the mixture to a filtration device, the filtration device comprising a membrane having a molecular weight (MW) cutoff 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; Thereby separating double-stranded nucleic acids from single-stranded nucleic acids.

2. a first chamber configured to hold the mixture to be filtered and, optionally, to capture a retentate, a second chamber configured to capture a permeate, and a filtration element disposed between the first and second chambers; An apparatus comprising: the mixture comprises double-stranded nucleic acids and single-stranded nucleic acids; A device wherein the filtration element selectively retains double-stranded nucleic acids over single-stranded nucleic acids, and optionally the filtration element comprises a membrane having a molecular weight (MW) cutoff 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.

3. 10. The method of claim 1, wherein the ultrafiltration step or filtration 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 reagents / by-products, capping reagents, coupling reagents, conjugation reagents, detritylation reagents / by-products, deprotection reagents / by-products, carbohydrates, peptides, lipids, polyethylene glycol, or fluorescent labels.

4. 10. The method of claim 1, wherein the ultrafiltration step produces a retentate and a permeate, and the retentate comprises a different concentration of one or more of salt, buffer, deprotection base, or product reagent or by-product than the permeate, the mixture, or both.

5. providing a mixture, providing a first single-stranded nucleic acid and a second single-stranded nucleic acid comprising sequences sufficiently complementary to each other to hybridize under conditions suitable for hybridization; a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a 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 both combining said first single-stranded nucleic acid and said second single-stranded nucleic acid under conditions suitable for hybridization to produce a mixture comprising The method of claim 1 , comprising:

6. 10. The method of claim 1, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both, is or comprises DNA, RNA, UNA, PNA, or LNA.

7. The first single-stranded nucleic acid, the second single-stranded nucleic acid, or both, may be selected from the group consisting of MOE, 2'fluoro, 2'OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- 2. The method of claim 1, comprising one or more modified and / or non-canonical nucleotides selected from propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, nucleotides containing modified sugars, or combinations thereof.

8. The apparatus of claim 2 , wherein the filtration element comprises a crossflow filter.

9. 10. The method of claim 1, wherein the ultrafiltration step comprises applying a force of 5 to 30 psi (transmembrane pressure).

10. 10. The method of claim 1, wherein the ultrafiltration step comprises applying 20 to 90 DTV to the filtration device.

11. 10. The method of claim 1, wherein the ultrafiltration is performed until a stable low conductivity threshold is achieved.

12. The method of claim 1 , wherein the double-stranded nucleic acid comprises a conjugate group.

13. 10. The method of claim 1, wherein the ultrafiltration step comprises diafiltration.

14. 14. The method of claim 13, wherein the diafiltration is carried out for a 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, or 65, 70, 75, 80, 81, 85, or 90 diafiltration volumes (DTV).

15. 10. The method of claim 1, wherein the ultrafiltration is applied at a pressure of about 5 to 40 psi.

16. 10. The method of claim 1, wherein the ultrafiltration is carried out at a temperature of about 10 to 40°C.

17. 10. The method of claim 1, wherein the mixture comprises a nucleic acid concentration of at least 100 OD / mL.

18. 10. The method of claim 1, wherein the ratio of double-stranded to single-stranded nucleic acids in the retentate increases over time and / or with total diafiltration volume (DTV).

19. 10. The method of claim 1, 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 mixture.

20. 1. A method for optimizing a technique for producing purified double-stranded nucleic acids, comprising: (i) a double-stranded nucleic acid comprising a first single-stranded nucleic acid and a second single-stranded nucleic acid at a first concentration; and single-stranded nucleic acids, e.g., a first single-stranded nucleic acid in single-stranded form, a second concentration of a second single-stranded nucleic acid in single-stranded form, or both providing a mixture comprising: the mixture having a predetermined concentration of salt; (ii) subjecting the mixture to an ultrafiltration process, the ultrafiltration process comprising applying the mixture to a filtration device having a predetermined molecular weight cutoff for a predetermined length of time; (iii) recovering the retentate; (iv) varying one or more of the first concentration, the second concentration, the predetermined molecular weight cutoff, the predetermined length of time, or the salt concentration; (v) repeating steps (i)-(iii) one or more times, each using varying one or more of said first concentration, second concentration, predetermined molecular weight cutoff, predetermined length of time, and / or salt concentration; (vi) determining the ratio of double-stranded to single-stranded nucleic acids in the recovered retentate in each repetition of steps (i) to (iii); and (vii) identifying the first concentration, second concentration, predetermined molecular weight cutoff, predetermined length of time, and / or salt concentration that results in the highest ratio of double-stranded to single-stranded nucleic acids in the recovered retentate. Including, A method thereby optimizing the technique for producing purified double-stranded nucleic acids.

21. The apparatus of claim 2, wherein the ultrafiltration process or filtration element also does not retain one or more other mixture components, the other mixture components being selected from the group consisting of shorter length single-stranded nucleic acids, organic solvents, thiolation reagents / by-products, capping reagents, coupling reagents, conjugation reagents, detritylation reagents / by-products, deprotection reagents / by-products, carbohydrates, peptides, lipids, polyethylene glycol, or fluorescent labels.

22. 3. The device of claim 2, wherein the first single-stranded nucleic acid, the second single-stranded nucleic acid, or both, is or comprises DNA, RNA, UNA, PNA, or LNA.

23. The first single-stranded nucleic acid, the second single-stranded nucleic acid, or both of them are selected from the group consisting of MOE, 2'fluoro, 2'OMe, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- 3. The device of claim 2, comprising one or more modified and / or non-canonical nucleotides selected from propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, nucleotides containing modified sugars, or combinations thereof.

24. The device of claim 2, wherein the double-stranded nucleic acid comprises a conjugate group.

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