A method for enriching circular RNA under denaturing conditions

Abstract

The present disclosure relates to a method for enriching circular polyribonucleotides (circRNAs) from a population of polyribonucleotides, for example, comprising circRNAs and linear polyribonucleotides (linRNAs), wherein the enrichment is performed under denaturing conditions. Also disclosed is a composition comprising a population of polyribonucleotides comprising circRNAs and linRNAs in a solution under denaturing conditions.Furthermore, the scope of the present disclosure includes a composition comprising an enriched population of circRNAs, such as a composition produced by exposing the composition to one or more denaturing conditions.

Description

[Background technology]

[0001] Polyribonucleotides are useful in a variety of therapeutic and modifying applications. Thus, new compositions and methods for isolating and purifying polyribonucleotides would be useful. Summary of the Invention [Means for solving the problem]

[0002] The present disclosure generally relates to a method for enrichment of circular polyribonucleotides (circRNAs), for example, from a population of polyribonucleotides including circRNAs and linear polyribonucleotides (linRNAs), where the enrichment is performed under denaturing conditions. Also disclosed is a composition comprising a population of polyribonucleotides including circRNAs and linRNAs in a solution under denaturing conditions. The scope of the present disclosure further includes compositions comprising an enriched population of circRNAs, such as compositions produced by exposing the composition to one or more denaturing conditions. The present disclosure is based, in part, on the inventors' discovery that separation of circRNAs under denaturing conditions (e.g., heat denaturation, pH, or chemical treatment) is a robust method for purifying and enriching circRNAs from a population of mixed polyribonucleotides including circRNAs, linRNAs, or other impurities or by-products. Furthermore, the disclosed method facilitates the scale-up of the circRNA purification process, thereby enabling the production and purification of large amounts of circRNAs.

[0003] In one aspect, the present disclosure provides a method for producing an enriched population of circRNAs, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNAs and linRNAs; and (b) separating circRNAs from linRNAs under denaturing conditions not involving the use of gel electrophoresis, thereby producing an enriched population of circRNAs. Also contemplated is a method for separating a population of circRNAs from a population of linRNAs under the above conditions, which optionally further comprises quantifying the population of circRNAs and / or quantifying the population of linRNAs.

[0004] In one aspect, the present disclosure provides a method for producing an enriched population of circRNAs, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNAs and linRNAs; and (b) exposing the population of polyribonucleotides to denaturing conditions, thereby enriching the population of circRNAs. Also contemplated is a method for separating the population of circRNAs from the population of linRNAs under the above conditions, which optionally further comprises quantifying the population of circRNAs and / or quantifying the population of linRNAs.

[0005] In some embodiments, (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg (e.g., at least 5 μg, at least 10, at least 25 μg, at least 50 μg, at least 100 μg, at least 250 μg, at least 500 μg, at least 750 μg, at least 1 mg, 5 mg, 10 mg, 100 mg, or 1 g; or between 5 μg and 100 μg, between 5 μg and 500 μg, between 5 μg and 1 mg, between 5 μg and 100 mg, between 500 μg and 100 mg, between 500 μg and 1 g, between 500 μg and 10 g, between 100 mg and 1 g, or between 1 g and 10 g); or (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL (e.g., at least 500 ng / μL, 1 mg / mL, 5 mg / mL, 10 mg / mL, or 50 mg / mL; or 200 ng / μL to 100 mg / mL, 200 ng / μL to 50 mg / mL, or 1 mg / mL to 50 mg / mL).In some embodiments, circRNAs are from about 100 nucleotides to about 20,000 nucleotides (e.g., from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 750 nucleotides, from about 100 nucleotides to about 1,000 nucleotides, from about 100 nucleotides to about 2,500 nucleotides, from about 100 nucleotides to about 5,000 nucleotides, from about 100 nucleotides to about 10,000 nucleotides, from about 500 nucleotides to about 750 nucleotides, from about 750 nucleotides about 1,000 nucleotides, about 750 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides to about 5,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000 nucleotides to about 15,000 nucleotides, about 10,000 nucleotides to about 15,000 nucleotides, About 15,000 nucleotides to about 20,000 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1,000 nucleotides, about 1,250 nucleotides, about 1,500 nucleotides, about 1,750 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,500 nucleotides, about 15,000 nucleotides, about 17,500 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 16,000 nucleotides, about 17,500 nucleotides, about 20,000 nucleotides, about 21,000 nucleotides, about 22,000 nucleotides, about 23,000 nucleotides, about 24,000 nucleotides, about 25,000 nucleotides, about 30,000 nucleotides, about 35,000 nucleotides, about 36,000 nucleotides, about 37,000 nucleotides, about 38,000 nucleotides, about 39,000 nucleotides, about In some embodiments, the circRNA has a length of about 0 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,000 nucleotides, about 13,000 nucleotides, about 14,000 nucleotides, about 15,000 nucleotides, about 16,000 nucleotides, about 17,000 nucleotides, about 18,000 nucleotides, about 19,000 nucleotides, or about 20,000 nucleotides. In some embodiments, the circRNA has a length of less than 1,000 nucleotides.

[0006] In one aspect, the disclosure provides a method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg (e.g., at least 5 μg, at least 10 μg, at least 25 μg, at least 50 μg, at least 100 μg, at least 250 μg, at least 500 μg, at least 750 μg, at least 1 mg, 5 mg, 10 mg, 100 mg, or 1 g; or between 5 μg and 100 μg, between 5 μg and 500 μg, between 5 μg and 1 mg, between 5 μg and 100 mg, between 500 μg and 100 mg, between 500 μg and 1 g, between 500 μg and 10 g, between 100 mg and 1 g, or between 100 mg and 1 g). (ii) the total volume of the sample containing the population of polyribonucleotides is at least 500 μL (e.g., at least 1 mL, 5 mL, 10 mL, 100 mL, or 1 L; or 500 μL to 100 mL, 500 μL to 1 L, 500 μL to 10 L, or 1 L to 10 L); or (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL (e.g., at least 500 ng / μL, 1 mg / mL, 5 mg / mL, 10 mg / mL, or 50 mg / mL; or 200 ng / μL to 100 mg / mL, 200 ng / μL to 50 mg / mL, or 1 mg / mL to 50 mg / mL); and (b) separating circRNA from linRNA under denaturing conditions, thereby producing an enriched population of circRNA. Also contemplated is a method of separating a population of circRNAs from a population of linRNAs under the above conditions, which optionally further comprises a step of quantifying the population of circRNAs and / or a step of quantifying the population of linRNAs.

[0007] In some embodiments, the total weight of polyribonucleotides in the population of polyribonucleotides is 1 μg to 1000 mg (e.g., 5 μg to 10 mg). In some embodiments, the total volume of the sample containing the population of polyribonucleotides is 500 μL to 1000 mL. In some embodiments, the concentration of the population of polyribonucleotides in the sample is 200 ng / μL to 50 mg / mL.

[0008] In some embodiments, circRNAs are from about 100 nucleotides to about 20,000 nucleotides (e.g., from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 750 nucleotides, from about 100 nucleotides to about 1,000 nucleotides, from about 100 nucleotides to about 2,500 nucleotides, from about 100 nucleotides to about 5,000 nucleotides, from about 100 nucleotides to about 10,000 nucleotides, from about 500 nucleotides to about 750 nucleotides, from about 750 nucleotides about 1,000 nucleotides, about 750 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides to about 5,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000 nucleotides to about 15,000 nucleotides, about 10,000 nucleotides to about 15,000 nucleotides, About 15,000 nucleotides to about 20,000 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1,000 nucleotides, about 1,250 nucleotides, about 1,500 nucleotides, about 1,750 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,500 nucleotides, about 15,000 nucleotides, about 17,500 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 16,000 nucleotides, about 17,500 nucleotides, about 20,000 nucleotides, about 21,000 nucleotides, about 22,000 nucleotides, about 23,000 nucleotides, about 24,000 nucleotides, about 25,000 nucleotides, about 30,000 nucleotides, about 35,000 nucleotides, about 36,000 nucleotides, about 37,000 nucleotides, about 38,000 nucleotides, about 39,000 nucleotides, about In some embodiments, the circRNA has a length of about 0 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,000 nucleotides, about 13,000 nucleotides, about 14,000 nucleotides, about 15,000 nucleotides, about 16,000 nucleotides, about 17,000 nucleotides, about 18,000 nucleotides, about 19,000 nucleotides, or about 20,000 nucleotides. In some embodiments, the circRNA has a length of less than 1,000 nucleotides.

[0009] In some embodiments, the separation step (b) is carried out under denaturing conditions that do not involve the use of gel electrophoresis.

[0010] In some embodiments, the enriched population of circRNAs is substantially free of one or more impurities or by-products, in some embodiments, the one or more impurities or by-products include polyacrylamide, boric acid, magnesium, or ethylenediaminetetraacetic acid (EDTA).

[0011] In some embodiments, the denaturing conditions include a temperature of at least 50° C. (e.g., at least 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.). In some embodiments, the denaturing conditions include a temperature of between 50° C. and 85° C. In some embodiments, the denaturing conditions are a temperature of at least 50°C (e.g., at least 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C), followed by a temperature of 8°C or less (e.g., 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -9°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C or less) within a time of 30 seconds or less.

[0012] In some embodiments, the denaturing conditions include a pH less than 5 (e.g., less than 4, 3, 2, or 1) or a pH greater than 9 (e.g., greater than 10, 11, 12, or 13). In some embodiments, the denaturing conditions include a pH less than 5 (e.g., less than 4, 3, 2, or 1). In some embodiments, the denaturing conditions include a pH greater than 9 (e.g., greater than 10, 11, 12, or 13).

[0013] In some embodiments, the denaturing conditions include chemical treatment, which in some embodiments includes treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

[0014] In some embodiments, the acid comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[0015] In some embodiments, the base comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[0016] In some embodiments, the organic solvent is at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75% , 80%, 90%, or more; or 0.01%-10%) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[0017] In some embodiments, the chaotropic agent comprises 100 mM to 8 M urea (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M), guanidinium chloride, lithium perchlorate, or polyethylene glycol (PEG). In some embodiments, the chaotropic agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate.

[0018] In some embodiments, the crowding agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, between 1 M and 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) PEG or urea.

[0019] In some embodiments, the chelating agent comprises 1 mM to 10 mM (e.g., 1 to 2 mM, 2 to 3 mM, 3 to 4 mM, 4 to 5 mM, 5 to 6 mM, 6 to 7 mM, 7 to 8 mM, 8 to 9 mM, or 9 to 10 mM) of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a derivative thereof, EDTA or a derivative thereof, nitrilotriacetic acid (NTA), iminodisuccinic acid (IDS), polyaspartic acid, S,S-ethylenediamine-N,N'-disuccinic acid (EDDS), or methylglycine diacetic acid (MGDA).

[0020] In some embodiments, the detergent comprises 0.005% to 0.05% (v / v) (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) of Nonidet P-40 (NP40), CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween®-20, or Tween®-80.

[0021] In one aspect, the present disclosure provides a method for producing an enriched population of circRNAs, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNAs and linRNAs; and (b) separating circRNAs from linRNAs at a temperature of at least 50°C (e.g., at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C), thereby producing an enriched population of circRNAs. Also contemplated are methods for separating the population of circRNAs from the population of linRNAs under the above conditions, which optionally further comprises quantifying the population of circRNAs and / or quantifying the population of linRNAs.

[0022] In one aspect, the disclosure provides a method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein (i) a total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg (e.g., at least 5 μg, at least 10, at least 25 μg, at least 50 μg, at least 100 μg, at least 250 μg, at least 500 μg, at least 750 μg, at least 1 mg, 5 mg, 10 mg, 100 mg, or 1 g; or between 5 μg and 100 μg, between 5 μg and 500 μg, between 5 μg and 1 mg, between 5 μg and 100 mg, between 500 μg and 100 mg, between 500 μg and 1 g, between 500 μg and 10 g, between 100 mg and 1 g, or between 1 g and 10 g); or (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL (e.g., at least 500 ng / μL, 1 mg / mL, 5 mg / mL, 10 mg / mL, or 50 mg / mL). (b) separating circRNA from linRNA at a temperature of at least 50°C (e.g., at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C), thereby producing an enriched population of circRNA. Also contemplated are methods of separating a population of circRNA from a population of linRNA under the above conditions, which optionally further comprises quantifying the population of circRNA and / or quantifying the population of linRNA.

[0023] In one aspect, the present disclosure provides a method for producing an enriched population of circRNAs, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNAs and linRNAs; and (b) exposing the population of polyribonucleotides to a temperature of at least 50°C (e.g., at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C), thereby enriching the population of circRNAs. In some embodiments, the temperature is 50°C to 85°C. Also contemplated are methods for separating the population of circRNAs from the population of linRNAs under the above conditions, which optionally further comprises quantifying the population of circRNAs and / or quantifying the population of linRNAs.

[0024] In one aspect, the present disclosure provides a method for producing an enriched population of circRNAs, comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNAs and linRNAs; and (b) separating circRNAs from linRNAs at a pH below 5 (e.g., below 4, 3, 2, or 1) or above 9 (e.g., above 10, 11, 12, or 13), thereby producing an enriched population of circRNAs, wherein said separation does not involve the use of gel electrophoresis. Also contemplated is a method for separating the population of circRNAs from the population of linRNAs under the above conditions, which optionally further comprises quantifying the population of circRNAs and / or quantifying the population of linRNAs.

[0025] In one aspect, the disclosure provides a method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein (i) a total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg (e.g., at least 5 μg, at least 10, at least 25 μg, at least 50 μg, at least 100 μg, at least 250 μg, at least 500 μg, at least 750 μg, at least 1 mg, 5 mg, 10 mg, 100 mg, or 1 g; or between 5 μg and 100 μg, between 5 μg and 500 μg, between 5 μg and 1 mg, between 5 μg and 100 mg, between 500 μg and 100 mg, between 500 μg and 1 g, between 500 μg and 10 g, between 100 mg and 1 g, or between 1 g and 10 g); (iii) the total volume of the sample containing the population is at least 500 μL (e.g., at least 1 mL, 5 mL, 10 mL, 100 mL, or 1 L; or 500 μL to 100 mL, 500 μL to 1 L, 500 μL to 10 L, or 1 L to 10 L); or (b) separating circRNA from linRNA at a pH less than 5 (e.g., less than 4, 3, 2, or 1) or greater than 9 (e.g., greater than 10, 11, 12, or 13), thereby producing an enriched population of circRNA. Also contemplated is a method of separating a population of circRNAs from a population of linRNAs under the above conditions, which optionally further comprises a step of quantifying the population of circRNAs and / or a step of quantifying the population of linRNAs.

[0026] In one aspect, the present disclosure provides a method for producing an enriched population of circRNAs, comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNAs and linRNAs; and (b) exposing the population of polyribonucleotides to a pH below 5 (e.g., below 4, 3, 2, or 1) or above 9 (e.g., above 10, 11, 12, or 13), thereby enriching the population of circRNAs. Also contemplated is a method for separating a population of circRNAs from a population of linRNAs under the above conditions, which optionally further comprises quantifying the population of circRNAs and / or quantifying the population of linRNAs.

[0027] In some embodiments, the pH is less than 5 (e.g., less than 4, 3, 2, or 1). In some embodiments, the pH is greater than 9 (e.g., greater than 10, 11, 12, or 13).

[0028] In one aspect, the present disclosure provides a method for producing an enriched population of circRNA, comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; and (b) separating circRNA from linRNA under conditions comprising an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution, thereby producing an enriched population of circRNA, wherein the separation does not comprise the use of gel electrophoresis. Also contemplated is a method for separating a population of circRNA from a population of linRNA under the above conditions, which optionally further comprises quantifying the population of circRNA and / or quantifying the population of linRNA.

[0029] In one aspect, the disclosure provides a method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein (i) a total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg (e.g., at least 5 μg, at least 10, at least 25 μg, at least 50 μg, at least 100 μg, at least 250 μg, at least 500 μg, at least 750 μg, at least 1 mg, 5 mg, 10 mg, 100 mg, or 1 g; or between 5 μg and 100 μg, between 5 μg and 500 μg, between 5 μg and 1 mg, between 5 μg and 100 mg, between 500 μg and 100 mg, between 500 μg and 1 g, between 500 μg and 10 g, between 100 mg and 1 g, or between 1 g and 10 g); (ii) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA, (iii) the total volume of the sample containing is at least 500 μL (e.g., at least 1 mL, 5 mL, 10 mL, 100 mL, or 1 L; or 500 μL to 100 mL, 500 μL to 1 L, 500 μL to 10 L, or 1 L to 10 L); or (iv) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL (e.g., at least 500 ng / μL, 1 mg / mL, 5 mg / mL, 10 mg / mL, or 50 mg / mL; or 200 ng / μL to 100 mg / mL, 200 ng / μL to 50 mg / mL, or 1 mg / mL to 50 mg / mL); and (b) separating circRNA from linRNA under conditions comprising an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution, thereby producing an enriched population of circRNA. Also contemplated is a method of separating a population of circRNAs from a population of linRNAs under the above conditions, which optionally further comprises a step of quantifying the population of circRNAs and / or a step of quantifying the population of linRNAs.

[0030] In one aspect, the present disclosure provides a method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides including circRNA and linRNA; and (b) exposing the population of polyribonucleotides to an acid, base, organic solvent, chaotropic agent, crowding agent, chelating agent, detergent, or salt solution, thereby enriching the population of circRNA. Also contemplated is a method for separating a population of circRNA from a population of linRNA under the above conditions, which optionally further comprises quantifying the population of circRNA and / or quantifying the population of linRNA.

[0031] In some embodiments, the acid is at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109, 109, 102, 103%, 104, 105, 106, 1 %, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) of acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[0032] In some embodiments, the base comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) of sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[0033] In some embodiments, the organic solvent is at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 0.01%-10%) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[0034] In some embodiments, the chaotropic agent comprises 100 mM to 8 M urea (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) urea, guanidinium chloride, lithium perchlorate, or PEG. In some embodiments, the chaotropic agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate.

[0035] In some embodiments, the crowding agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) PEG or urea.

[0036] In some embodiments, the chelating agent comprises 1 mM to 10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) of EGTA or a derivative thereof, EDTA or a derivative thereof, NTA, IDS, EDDS, or MGDA.

[0037] In some embodiments, the detergent comprises 0.005% to 0.05% (v / v) (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) of NP40, CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween®-20, or Tween®-80.

[0038] In some embodiments, step (b) comprises performing column chromatography on the population of polyribonucleotides, wherein performing column chromatography comprises an equilibration step, a sample loading step, a column washing step, and an elution step.

[0039] In some embodiments, separation is performed during the sample loading step. In some embodiments, separation occurs during a column washing step. In some embodiments, separation occurs during an elution step.

[0040] In some embodiments, the column chromatography comprises fast protein liquid chromatography (FPLC), high pressure liquid chromatography (HPLC), hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEC), mixed mode chromatography, or affinity chromatography.

[0041] In some embodiments, AEC involves the use of an anion exchange resin, including styrene-divinylbenzene, silica, sepharose, cellulose, dextran, epoxypolyamine, methacrylate, agarose, or acrylic. In some embodiments, the anion exchange resin includes an ion exchanger, such as quaternary ammonium, aminoethyl, diethylaminoethyl, or diethylaminopropyl. In some embodiments, the anion exchange resin includes beads, the beads having a bead size of 45-165 μm and a pore size of 100-1000 nm in diameter. In some embodiments, AEC involves the use of linear gradient elution or step isocratic elution. In some embodiments, AEC involves the use of a flow rate of 1 mL / min to 150 mL / min.

[0042] In some embodiments, the FPLC is reversed-phase-FPLC (RP-FPLC).

[0043] In some embodiments, step (b) is performed by pooling multiple fractions of purified circRNA.

[0044] In some embodiments, the circRNA and the linRNA have the same ribonucleotide sequence. In some embodiments, the circRNA and the linRNA have the same mass. In some embodiments, the circRNA and the linRNA lack a poly(A) tail.

[0045] In some embodiments, the method comprises exonuclease digestion of linRNA. In some embodiments, the method does not comprise exonuclease digestion of linRNA. In some embodiments, the method does not comprise selective modifications to circRNA or linRNA that improve enrichment of circRNA.

[0046] In some embodiments, the percentage (w / w) of circRNA in the enriched population of circRNA is twice the percentage (w / w) of circRNA in the polyribonucleotide population. In some embodiments, the percentage (w / w) of circRNA in the enriched population of circRNA is at least 65% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the percentage (w / w) of linRNA in the enriched population of circRNA is less than 35% (e.g., less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%).

[0047] In some embodiments, circRNAs are from about 100 nucleotides to about 20,000 nucleotides (e.g., from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 750 nucleotides, from about 100 nucleotides to about 1,000 nucleotides, from about 100 nucleotides to about 2,500 nucleotides, from about 100 nucleotides to about 5,000 nucleotides, from about 100 nucleotides to about 10,000 nucleotides, from about 500 nucleotides to about 750 nucleotides, from about 750 nucleotides about 1,000 nucleotides, about 750 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides to about 5,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000 nucleotides to about 15,000 nucleotides, about 10,000 nucleotides to about 15,000 nucleotides, About 15,000 nucleotides to about 20,000 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1,000 nucleotides, about 1,250 nucleotides, about 1,500 nucleotides, about 1,750 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,500 nucleotides, about 15,000 nucleotides, about 17,500 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 16,000 nucleotides, about 17,500 nucleotides, about 20,000 nucleotides, about 21,000 nucleotides, about 22,000 nucleotides, about 23,000 nucleotides, about 24,000 nucleotides, about 25,000 nucleotides, about 30,000 nucleotides, about 35,000 nucleotides, about 36,000 nucleotides, about 37,000 nucleotides, about 38,000 nucleotides, about 39,000 nucleotides, about In some embodiments, the circRNA has a length of about 0 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,000 nucleotides, about 13,000 nucleotides, about 14,000 nucleotides, about 15,000 nucleotides, about 16,000 nucleotides, about 17,000 nucleotides, about 18,000 nucleotides, about 19,000 nucleotides, or about 20,000 nucleotides. In some embodiments, the circRNA has a length of less than 1,000 nucleotides.

[0048] In one aspect, the present disclosure provides a composition comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein the population of polyribonucleotides is in solution under denaturing conditions; (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg (e.g., at least 5 μg, at least 10, at least 25 μg, at least 50 μg, at least 100 μg, at least 250 μg, at least 500 μg, at least 750 μg, at least 1 mg, 5 mg, 10 mg, 100 mg, or 1 g; or from 5 μg to 100 μg, 5 μg to 500 μg, 5 μg to 1 mg, 5 μg to 10 mg, 500 μg to 100 mg, 500 μg to 1 g, 500 μg (ii) the total volume of the sample containing the population of polyribonucleotides is at least 500 μL (e.g., at least 1 mL, 5 mL, 10 mL, 100 mL, or 1 L; or 500 μL to 100 mL, 500 μL to 1 L, 500 μL to 10 L, or 1 L to 10 L); or (iii) the concentration of the population of polyribonucleotides in the sample is at least 500 ng / μL (e.g., at least 500 ng / μL, 1 mg / mL, 5 mg / mL, 10 mg / mL, or 50 mg / mL; or 200 ng / μL to 100 mg / mL, 200 ng / μL to 50 mg / mL, or 1 mg / mL to 50 mg / mL).

[0049] In one aspect, the present disclosure provides a composition comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein said population of polyribonucleotides is in solution under denaturing conditions, and wherein said solution is substantially free of one or more impurities or by-products.

[0050] In one aspect, the disclosure provides a composition comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein (a) the composition is obtained from a sample comprising a population of nucleic acids; (b) the composition has been exposed to one or more denaturing conditions; and (c) the composition is substantially free of one or more impurities or by-products.

[0051] In one aspect, the disclosure provides a composition comprising an enriched population of circRNA, wherein (a) the composition is obtained from a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; (b) the composition has been exposed to one or more denaturing conditions; and (c) the composition is substantially free of one or more impurities or by-products.

[0052] In one aspect, in some embodiments, circRNAs are from about 100 nucleotides to about 20,000 nucleotides (e.g., from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 750 nucleotides, from about 100 nucleotides to about 1,000 nucleotides, from about 100 nucleotides to about 2,500 nucleotides, from about 100 nucleotides to about 5,000 nucleotides, from about 100 nucleotides to about 10,000 nucleotides, from about 500 nucleotides to about 750 nucleotides, from about 750 nucleotides, nucleotide to about 1,000 nucleotides, about 750 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides to about 5,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000 nucleotides to about 15,000 nucleotides, about 10,000 nucleotides to about 15,000 nucleotides nucleotides, about 15,000 nucleotides to about 20,000 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1,000 nucleotides, about 1,250 nucleotides, about 1,500 nucleotides, about 1,750 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,000 nucleotides, about 13,000 nucleotides, about 14,000 nucleotides, about 15,000 nucleotides, about 16,000 nucleotides, about 17,000 nucleotides, about 18,000 nucleotides, about 19,000 nucleotides, about 21,000 nucleotides, about 22,000 nucleotides, about 23,000 nucleotides, about 24,000 nucleotides, about 25,000 nucleotides, about 26,000 nucleotides, about 27,000 nucleotides, about 28,000 nucleotides, about 29,000 nucleotides, about 30,000 nucleotides, about 31,000 nucleotides, about 32,000 nucleotides, about 33,0 In some embodiments, the circRNA has a length of about 1,000 nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,000 nucleotides, about 13,000 nucleotides, about 14,000 nucleotides, about 15,000 nucleotides, about 16,000 nucleotides, about 17,000 nucleotides, about 18,000 nucleotides, about 19,000 nucleotides, or about 20,000 nucleotides. In some embodiments, the circRNA has a length of 1,000 nucleotides or less.

[0053] In some embodiments, the one or more impurities or by-products include polyacrylamide, boric acid, magnesium, or EDTA.

[0054] In some embodiments, the denaturing conditions include a temperature of at least 50° C. (e.g., at least 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., or 100° C.). In some embodiments, the denaturing conditions include a temperature of between 50° C. and 85° C. In some embodiments, the denaturing conditions are a temperature of at least 50°C (e.g., at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C), followed by a temperature of 8°C or less (e.g., 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -9°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C or less).

[0055] In some embodiments, denaturing conditions include a pH less than 5 (eg, less than 4, 3, 2, or 1) or a pH greater than 9 (eg, greater than 10, 11, 12, or 13).

[0056] In some embodiments, the denaturing conditions include chemical treatment, which in some embodiments includes treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

[0057] In some embodiments, the acid comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[0058] In some embodiments, the base comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) of sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[0059] In some embodiments, the organic solvent is at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 7 ... 5%, 80%, 90% or more; or 0.01% to 10%) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[0060] In some embodiments, the chaotropic agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) urea, guanidinium chloride, lithium perchlorate, or polyethylene glycol (PEG). In some embodiments, the chaotropic agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate.

[0061] In some embodiments, the crowding agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) PEG or urea.

[0062] In some embodiments, the chelating agent comprises 1 mM to 10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) of EGTA or a derivative thereof, EDTA or a derivative thereof, NTA, IDS, EDDS, or MGDA.

[0063] In some embodiments, the detergent comprises 0.005% to 0.05% (v / v) (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) of NP40, CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween®-20, or Tween®-80.

[0064] In some embodiments, the percentage (w / w) of circRNA in the enriched population of circRNA is twice the percentage (w / w) of circRNA in the polyribonucleotide population. In some embodiments, the percentage (w / w) of circRNA in the enriched population of circRNA is at least 65% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the percentage (w / w) of linRNA in the enriched population of circRNA is less than 35% (e.g., less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%).

[0065] In some embodiments, the circRNA and the linRNA have the same ribonucleotide sequence. In some embodiments, the circRNA and the linRNA have the same mass. In some embodiments, the circRNA and the linRNA lack a poly(A) tail.

[0066] In one aspect, the present disclosure provides a method for determining the purity of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; (b) separating circRNA from linRNA by chromatography under denaturing conditions; (c) collecting a chromatogram of the sample comprising a circRNA peak and a linRNA peak; and (d) calculating the area under each peak to determine the purity of circRNA in the sample.

[0067] In some embodiments, the denaturing conditions do not include the use of gel electrophoresis.

[0068] In some embodiments, the denaturing conditions include a temperature of at least 50°C (e.g., at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C). In some embodiments, the denaturing conditions include a temperature of 50°C to 85°C. In some embodiments, the denaturing conditions include a temperature of at least 50°C, followed by a temperature of 8°C or less (e.g., 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -9°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C or less) within a time of 30 seconds or less.

[0069] In some embodiments, the denaturing conditions include a pH less than 5 (e.g., less than 4, 3, 2, or 1) or a pH greater than 9 (e.g., greater than 10, 11, 12, or 13). In some embodiments, the denaturing conditions include a pH less than 5 (e.g., less than 4, 3, 2, or 1). In some embodiments, the denaturing conditions include a pH greater than 9 (e.g., greater than 10, 11, 12, or 13).

[0070] In some embodiments, the denaturing conditions include chemical treatment, which in some embodiments includes treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

[0071] In some embodiments, the acid comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[0072] In some embodiments, the base comprises 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) of sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[0073] In some embodiments, the organic solvent is at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75% , 80%, 90%, or more; or 0.01%-10%) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[0074] In some embodiments, the chaotropic agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) urea, guanidinium chloride, lithium perchlorate, or PEG. In some embodiments, the chaotropic agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate.

[0075] In some embodiments, the crowding agent comprises 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) PEG or urea.

[0076] In some embodiments, the chelating agent comprises 1 mM to 10 mM (e.g., 1 to 2 mM, 2 to 3 mM, 3 to 4 mM, 4 to 5 mM, 5 to 6 mM, 6 to 7 mM, 7 to 8 mM, 8 to 9 mM, or 9 to 10 mM) of EGTA or a derivative thereof, ethylenediaminetetraacetic acid (EDTA) or a derivative thereof, nitrilotriacetic acid (NTA), imino-disuccinic acid (IDS), polyaspartic acid, S,S-ethylenediamine-N,N'-disuccinic acid (EDDS), or methylglycine diacetic acid (MGDA).

[0077] In some embodiments, the detergent comprises 0.005% to 0.05% (v / v) (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) of Nonidet P-40 (NP40), CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween®-20, or Tween®-80.

[0078] In some embodiments, the chromatography comprises liquid chromatography, hi some embodiments, the liquid chromatography is selected from the group consisting of FPLC, HPLC, HIC, AEC, MMC, or affinity chromatography.

[0079] In some embodiments, the relative standard deviation (RSD) of purity is less than 5% (e.g., less than 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%).

[0080] definition To facilitate understanding of this disclosure, certain terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the areas relevant to this disclosure.

[0081] As used herein, "a" and "an" mean "at least one" or "one or more," unless otherwise specified. Additionally, the singular forms "a," "an," and "the" include plural referents unless the context clearly requires otherwise.

[0082] As used herein, "about" refers to an amount of ±10% of the recited value.

[0083] As used herein, the term "carrier" refers to a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., cyclic polyribonucleotide) to a cell, via a partially or fully encapsulated agent, or a combination thereof, by covalent modification of the cyclic polyribonucleotide. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles encapsulated or covalently attached to cyclic polyribonucleotides), liposomes, fusosomes, exosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently attached to polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0084] As used herein, the term "chromatography" refers to various chromatographic methods that can be used to purify and enrich a population of circular polyribonucleotides (circRNAs) from a mixed population of polyribonucleotides, including circRNAs and linear polyribonucleotides (linRNAs). Chromatography includes column and non-column chromatography, e.g., electrophoretic methods such as capillary gel electrophoresis. Chromatography includes low-pressure or normal-pressure liquid chromatographic separation methods. This can include FPLC (reversed-phase (RP)-FPLC and normal-phase (NP)-FPLC), affinity chromatography, hydrophobic interaction chromatography, anion exchange chromatography, or mixed-mode chromatography.

[0085] As used herein, the terms "circRNA", "circular polyribonucleotide", and "circular RNA" are used interchangeably and refer to polyribonucleotide molecules having a structure with no free ends (i.e., free 3' and / or 5' ends), e.g., polyribonucleotide molecules that form a circular or endless structure through covalent or non-covalent bonds. A circular polyribonucleotide may be, for example, a covalently closed polyribonucleotide.

[0086] As used herein, the term "denaturing conditions" refers to any condition or set of conditions, such as physical or chemical conditions, that disrupt the molecular structure of a polynucleotide in solution. Polynucleotides under denaturing conditions can include circRNA, linRNA, linear polydeoxyribonucleotides (linDNA), or circular polydeoxyribonucleotides (circDNA). Denaturing conditions refer to conditions under which hydrogen bonds and other non-covalent forces (e.g., van der Waals forces or hydrophobic interactions) between complementary base pairs are disrupted, thereby reducing or eliminating ordered structures within a polynucleotide, such as secondary or tertiary polymer structures (e.g., double helices, stem-loops, stacking, among others), compared to structures observed under physiological conditions. Denaturing conditions can reduce, eliminate, or reorganize intra- or intermolecular interactions between nucleic acid residues of one or more polynucleotides. Denaturing conditions can also refer to conditions under which covalent bonds between consecutive nucleic acid monomers within a polymer (e.g., phosphodiester bonds between consecutive nucleosides, etc.) are disrupted. Without wishing to be bound by theory, circRNAs may be selectively enriched in samples compared to linRNAs because their ring structure limits the range of possible conformations, making them less susceptible to denaturation, whereas linRNAs may be more flexible and therefore more susceptible to disruption of secondary and tertiary structures by denaturing conditions. Denaturing conditions can be created in situ by manipulating one of several conditions to which circRNAs, linRNAs, linDNAs, circDNAs, or polypeptides are exposed. Non-limiting examples of denaturing conditions include, for example, heat denaturation, shock cooling, acidic or alkaline pH (e.g., pH less than 5 or pH greater than 9), or chemical treatment (e.g., acid, base, organic solvent, chaotropic agent, chelating agent, crowding agent, detergent, or salt solution).

[0087] As used herein, the term "eluate" refers to a fraction containing an analyte (e.g., an enriched population of circRNAs) that is eluted from a medium (e.g., a hydrophobic stationary phase) during a purification step (e.g., a chromatography step such as a RP-FPLC step). The eluate can be released from the medium by applying the eluate to the medium, thereby releasing the analyte. More specifically, the eluate can refer to a fraction containing circRNAs that is released from the medium after application of an eluent (e.g., an elution buffer such as a buffer containing an organic solvent) to the medium.

[0088] As used herein, the terms "linRNA" and "linear polyribonucleotide" are used interchangeably and refer to a polyribonucleotide having a 5' and a 3' end. In some embodiments, a linRNA has a free 5' or 3' end. In some embodiments, a linRNA has a non-covalently linked 5' or 3' end.

[0089] As used herein, the term "modified oligonucleotide" means an oligonucleotide comprising a nucleotide having at least one modification to the sugar, nucleobase, or internucleotide linkage.

[0090] As used herein, the term "modified ribonucleotide" means a ribonucleotide comprising a nucleoside having at least one modification to the sugar, nucleobase, or internucleoside linkage.

[0091] As used herein, the term "naked delivery" refers to a formulation for delivery to cells without the aid of a carrier or covalent modification of moieties that aid in delivery to cells. Naked delivery formulations do not include transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of cyclic polyribonucleotide is a formulation that includes cyclic polyribonucleotide without covalent modification and does not include a carrier.

[0092] As used herein, the term "polynucleotide" refers to a molecule that includes one or more nucleic acid subunits or nucleotides, and can be used interchangeably with "nucleic acid" or "oligonucleotide." A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A polyribonucleotide, ribonucleic acid, or RNA can refer to a polymer that includes multiple ribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0093] As used herein, the phrase "mixed population of polyribonucleotides" refers to a heterogeneous population of polyribonucleotides. Such a heterogeneous population of polyribonucleotides includes circRNA, linRNA, and optionally one or more impurities or by-products (e.g., one or more impurities or by-products described herein).

[0094] As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) linked together, most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs thereof. Polypeptides can be single molecules or multi-molecular complexes such as dimers, trimers, or tetramers. Polypeptides can also include single chain or multi-chain polypeptides such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0095] As used interchangeably herein, the terms "polyA" and "polyA sequence" refer to an untranslated, flanking region of a nucleic acid molecule that is at least 5 nucleotides in length and consists of adenosine residues. In some embodiments, the polyA sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some embodiments, the polyA sequence is located 3' to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide), and the polyA sequence is 3' to a termination element (e.g., a stop codon) such that the polyA is not translated. In some embodiments, the polyA sequence is located 3' to a termination sequence and 3' untranslated region.

[0096] As used herein, the terms "purify," "purifying," and "purification" refer to one or more steps or processes that remove impurities or by-products (e.g., linRNA) from a sample containing a heterogeneous mixture of circRNA and linRNA, among other substances, to produce a composition comprising an enriched population of circRNA with reduced levels of impurities or by-products (e.g., linRNA) compared to the original mixture, or a composition in which the linRNA or substance is reduced by 40% or more (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% or more) by mass compared to the starting mixture.

[0097] As used herein, the terms "pure" and "purity" refer to the degree to which an analyte (e.g., circRNA) is isolated and free from other components. In the context of nucleic acids (e.g., polyribonucleotides), the purity of an isolated nucleic acid (e.g., circRNA) can be expressed in terms of a population of nucleic acids that is free of any contaminants (e.g., linRNA and other substances). For example, the purity of a population of circRNA indicates how much circRNA is present in the population per total mass of isolated material, and can be determined, for example, using a pure circRNA as a reference. The purity levels found in the present disclosure can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, more than 95%, or more than 99% (w / w). A "pure" population of circRNAs of the present disclosure may be greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or up to 70% pure by mass. A "substantially pure" population of circRNAs is substantially free of contaminants or impurities or by-products (e.g., linRNA), and may be, for example, 70%, 75%, 80%, 85%, 90%, 95%, or >99% pure by mass. In some embodiments, the level of contaminants or impurities or by-products is about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (w / w) or less. Purity can be determined by detecting the level of a particular analyte (e.g., circRNA) using gel electrophoresis, spectrophotometry (e.g., NanoDrop by ThermoFisher Scientific), or other techniques suitable for measuring the purity of a population of nucleic acids, and calculating the percentage (w / w) of the analyte relative to the total nucleic acid content (e.g., as determined by assays known in the art).

[0098] As used herein, the phrase "substantially free of one or more impurities or by-products" refers to a characteristic of a sample, e.g., a sample comprising an enriched population of circRNAs, that is free of one or more impurities or by-products (e.g., one or more impurities or by-products disclosed herein) or that contains a minimal amount of one or more impurities or by-products. The minimal amount of one or more impurities or by-products may be 20% (w / w) or less (e.g., 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less, or less). In another example, a sample or enriched circRNA population is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 15% (w / w) (e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less, or less). In another example, a sample or enriched circRNA population is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 10% (w / w) (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less, or less). In another example, a sample or enriched circRNA population is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 5% (w / w) (e.g., 4%, 3%, 2%, 1% (w / w) or less or less). In yet another example, a sample or enriched circRNA population is substantially free of one or more impurities or by-products if the one or more impurities or by-products are present in an amount of less than 1% (0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% (w / w) or less or less).

[0099] As used herein, a "termination element" is a portion, such as a nucleic acid sequence, that terminates translation of an expressed sequence in a circular or linear polyribonucleotide.

[0100] As used herein, a "translation initiation sequence" is a nucleic acid sequence that initiates the translation of an expression sequence in a circular or linear polyribonucleotide.

[0101] As used herein, the term "yield" refers to the relative amount (w / w) of an analyte (e.g., a population of circRNAs) obtained after a purification step or process compared to the amount of the analyte in the starting material (e.g., a mixed population of polyribonucleotides, e.g., circRNAs and linRNAs). Yield can be expressed as a percentage. In the context of the present disclosure, the amount of the analyte (e.g., circRNAs) in the starting material and the analyte obtained after a purification step can be measured using an assay (e.g., gel electrophoresis or spectrophotometry). The method of the present disclosure can be used to provide a yield of an enriched population of circRNAs of about 20% (w / w) or more, for example, compared to the amount present in a mixed population of polyribonucleotides or an enriched population of circRNAs. For example, the method can be used to provide a yield of purified circRNAs of about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more.

[0102] Other features and advantages of the invention will become apparent from the following detailed description, the drawings, and the claims. [Brief description of the drawings]

[0103] [Figure 1] Chromatogram results of purification of urea-free circRNA using a 5 mL DEAE Sepharose weak AEX column. [Diagram 2] Chromatogram results of purification of circRNA using a 5 mL DEAE Sepharose Weak AEX column with the chaotropic salt urea as a denaturing condition. [Diagram 3] This shows the chromatogram results of circRNA purification using urea, a chaotropic salt, as a denaturing condition on an 8 mL DEAE-weak AEX monolith column. [Figure 4] Chromatogram results of purification of 15 mg of circRNA using urea, a chaotropic salt, as a denaturing condition on an 8 mL QA strong AEX monolith column. [Diagram 5] Chromatogram results of purifying 7 mg of circRNA on an 8 mL PrimaS monolith column using chaotropic salt urea and pH as denaturing conditions. [Figure 6] Chromatogram results after purification of circRNA using a 1 mL HIC monolith column under high salt and urea denaturing conditions. [Figure 7] Gel electrophoresis results after purification of circRNA using a 1 mL HIC monolith column under high salt and urea denaturing conditions. [Figure 8] Exemplary chromatogram results of separation of circRNA and linRNA after separation using temperature, chaotropic agent, and organic solvent denaturing conditions are shown. [Figure 9] HPLC analysis results after separation of circRNA and linRNA using temperature, chaotropic agent, and organic solvent denaturing conditions are shown. [Figure 10] HPLC analysis results after separation of circRNA and linRNA using temperature, chaotropic agent, and organic solvent denaturing conditions are shown. [Figure 11] Exemplary chromatogram results after purification of circRNA using temperature and chaotropic agent conditions are shown. [Figure 12] Exemplary gel electrophoresis results after purification of circRNA using temperature and chaotropic agent conditions are shown. [Figure 13] Exemplary chromatogram results following purification of circRNA using high temperature and organic solvent denaturing conditions are shown. [Figure 14] An exemplary chromatogram of a circRNA after two water injections is shown. [Figure 15] An exemplary chromatogram of a blank injection is shown. [Figure 16]Exemplary chromatograms of circRNA and linRNA standards with distinct peaks are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] Disclosed herein are methods for purifying and enriching circular polyribonucleotides (circRNAs) from a heterogeneous population of polyribonucleotides including circRNAs and linear polyribonucleotides (linRNAs), where the purification and enrichment are performed under denaturing conditions. Also disclosed are compositions comprising a population of polyribonucleotides including circRNAs and linRNAs in a solution under denaturing conditions, e.g., a solution substantially free of one or more impurities or by-products. Also included within the scope of the disclosure are compositions comprising an enriched population of circRNAs, e.g., compositions produced by exposure to one or more denaturing conditions. The disclosure is based, in part, on the inventors' discovery that separation of circRNAs under denaturing conditions (e.g., heat denaturation, pH, or chemical treatment) is a robust method for purifying and enriching circRNAs from a population of mixed polyribonucleotides including circRNAs, linRNAs, or other impurities or by-products, thereby improving the purification and yield of the recovered circRNAs compared to other methods. Furthermore, the disclosed methods facilitate the scale-up of the circRNA purification process, thereby enabling the production and purification of large amounts of circRNAs.

[0105] Purification of cyclic polyribonucleotides The present disclosure features a method for purifying and enriching circRNA from a sample containing a heterogeneous population of polyribonucleotides including circRNA and linRNA, where the purification is performed under denaturing conditions as disclosed herein. In the context of the present disclosure, purification refers to the isolation and enrichment of a target polyribonucleotide population (e.g., circRNA) from a sample containing a mixed population of polyribonucleotides (e.g., linRNA and circRNA) and undesired impurities or by-products (e.g., impurities or by-products as described herein). Thus, after purification, circRNA is present at an increased percent (w / w) of total polyribonucleotides or at a higher concentration compared to in the sample from which the purified population of circRNA was obtained. The undesired impurities or by-products in the sample can be linRNA, polyacrylamide, boric acid, magnesium, or ethylenediaminetetraacetic acid (EDTA), or any combination thereof. The disclosed method purifies and enriches circRNA from a mixed population of polyribonucleotides such that the purity of circRNA is preferably as close to 100% as possible in the enriched population of circRNA. Using this method, enriched populations of circRNAs can be prepared with a purity of about 5% to about 99% or more (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, more than 95%, such as 97% or 99%, or more than 99%). In some embodiments, the purity of the enriched population of circRNAs is measured as a percentage (e.g., percentage amount) of the amount of circRNA in the enriched population relative to the percentage amount of linRNA or one or more impurities or by-products in the enriched population. For example, an enriched population of circRNAs with a purity of 95% contains 95% circRNA and 5% linRNA or one or more impurities or by-products.

[0106] In addition, the method can be used to provide a yield of enriched population of circRNAs of about 20% (w / w) or more relative to the amount present in a mixed population of polyribonucleotides or in an enriched population of circRNAs. For example, the method can be used to provide a yield of enriched population of circRNAs that contains about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more of circRNAs relative to the total amount of polyribonucleotides in the enriched population. Alternatively, the enriched population of circRNAs can contain about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more of circRNAs relative to a mixed population of polyribonucleotides that includes circRNAs and linRNAs. Furthermore, a circRNA enriched population may contain about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more of circRNA relative to the total amount of polynucleotides (e.g., RNA or DNA) in the enriched population or the total amount of polynucleotides in a mixed population of polyribonucleotides.

[0107] The purification method disclosed herein can be used for preparative purification of circRNA, but the disclosed method is also advantageous for analytical purification of circRNA. Preparative purification relates to purification of relatively large amounts of RNA. For example, preparative purification can be used to purify RNA amounts of at least 0.5 mg (e.g., at least 0.6, 0.8, 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, 1000 mg, or more). The advantage of this method is that it can purify larger amounts of circRNA than is possible using other methods suitable for small-scale purification (e.g., agarose gel electrophoresis).

[0108] Denaturing conditions Without wishing to be bound by theory, the present disclosure is based in part on the inventors' surprising discovery that purification of circRNAs can be performed under denaturing conditions. In the context of the present disclosure, denaturing conditions refer to conditions under which hydrogen bonds and other non-covalent forces (e.g., van der Waals forces or hydrophobic interactions) between complementary base pairs are broken, thereby reducing or eliminating ordered structures within a polynucleotide, such as, for example, secondary or tertiary polymer structures (e.g., double helices, stem-loops, stacking, etc., among others), compared to structures observed under physiological conditions. Denaturing conditions also refer to conditions under which covalent bonds between consecutive nucleic acid monomers within a polymer are broken, such as, for example, phosphodiester bonds between consecutive nucleosides. Furthermore, enrichment or separation of circRNAs from linRNAs, among other impurities or by-products, can be improved by denaturing conditions. Such methods are particularly suitable for high-throughput scale-up or scale-out of RNA purification methods known in the art. Thus, the present disclosure provides various denaturing conditions that can be used to process samples containing mixed populations of polyribonucleotides using various purification methods, such as those disclosed herein.

[0109] Thermal denaturation The methods disclosed herein encompass the use of high, low, or variable temperature conditions to enrich circRNAs from a mixed population of polyribonucleotides, including, for example, circRNAs, linRNAs, and various other impurities or by-products (e.g., salts, magnesium, urea, boric acid, etc.). For example, heat denaturation can be performed under high temperature conditions, such as, for example, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, at least 100°C, or higher. In one example, heat denaturation is performed at a temperature of at least 50°C. In another example, heat denaturation is performed at a temperature of at least 50°C and not more than 85°C. Heat denaturation under these temperature conditions can be performed for a time sufficient to denature polynucleotides (e.g., circRNAs and linRNAs, among others), for example, by breaking intramolecular hydrogen bonds within the polynucleotides. For example, heat denaturation can be carried out at the aforementioned temperatures for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, or more. Exposure to heat denaturing conditions can be continuous or discontinuous (e.g., a 10 minute exposure to high temperature conditions in two 5 minute blocks separated by 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or more).

[0110] Thermal denaturation can also be performed under variable temperature conditions. For example, shock cooling can be performed by first exposing a sample containing a heterogeneous mixture of polyribonucleotides, including circRNA and linRNA, to a high temperature condition (e.g., the high temperature conditions described above) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more minutes, and then immediately exposing the sample to a low temperature condition. Cold conditions can include temperatures below 8° C. (e.g., 7° C., 6° C., 5° C., 4° C., 3° ​​C., 2° C., 1° C., 0° C., −1° C., −2° C., −3° C., −4° C., −5° C., −6° C., −7° C., −9° C., −10° C., −15° C., −20° C., −25° C., −30° C., −35° C., −40° C., −45° C., −50° C., −55° C., −60° C., −65° C., −70° C., −75° C., −80° C., or below). Generally, shock cooling is performed such that the time lag between the hot and cold conditions is short. Thus, the time between exposure of the sample to the high temperature condition and exposure to the low temperature condition is generally 1 minute or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 9 seconds or less, 8 seconds or less, 7 seconds or less, 6 seconds or less, 5 seconds or less, 4 seconds or less, 3 seconds or less, 2 seconds or less, 1 second or less. Exposure of the sample to the low temperature condition may be for at least 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or more.

[0111] The aforementioned heat denaturation protocol can be performed on the sample prior to the chromatographic separation methods disclosed herein. Alternatively, heat denaturation can be performed on the sample after chromatographic separation using the disclosed methods. Furthermore, heat denaturation can be performed in parallel with chromatographic purification of the sample (i.e., during the purification process). For example, the sample or mobile phase can be pre-incubated at a high temperature sufficient to denature the intramolecular hydrogen bonds in the polyribonucleotides of the sample before loading the sample onto the chromatographic column. In another example, heat denaturation during the chromatographic separation process can be performed by placing a jacket or sleeve around the column used for purification to achieve the desired temperature conditions sufficient for denaturation of linRNA but not circRNA. Thus, heat denaturation of linRNA in a sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, heat denaturation can be performed on a sample even in the absence of chromatographic purification of the sample.

[0112] pH Denaturation According to the disclosed method, circRNAs can be selectively purified and enriched from a sample containing a mixed population of polyribonucleotides including circRNAs and linRNAs by exposing the sample to denaturing pH conditions. Exemplary pH conditions include acidic conditions (e.g., pH less than 7) or alkaline conditions (pH greater than 7), which cause ionization of ionizable groups in the nucleotides of polyribonucleotides, resulting in loss of secondary structure and selective denaturation of linRNAs but not circRNAs. For example, suitable pH denaturing conditions include a pH less than 5 (e.g., pH 4.5, 4.0, 3.5, 3.0 or less) or a pH greater than 9 (e.g., pH 9.5, 10, 10.5, 11 or more).

[0113] In one example, the pH denaturation is performed at pH 4.9. In another example, the pH denaturation is performed at pH 4.8. In another example, the pH denaturation is performed at pH 4.7. In another example, the pH denaturation is performed at pH 4.6. In another example, the pH denaturation is performed at pH 4.5. In another example, the pH denaturation is performed at pH 4.4. In another example, the pH denaturation is performed at pH 4.3. In another example, the pH denaturation is performed at pH 4.2. In another example, the pH denaturation is performed at pH 4.1. In another example, the pH denaturation is performed at pH 4.0. In another example, the pH denaturation is performed at pH 3.9. In another example, the pH denaturation is performed at pH 3.8. In another example, the pH denaturation is performed at pH 3.8. In another example, the pH denaturation is performed at pH 3.7. In another example, the pH denaturation is performed at pH 3.6. In another example, the pH denaturation is performed at pH 3.5. In another example, the pH denaturation is performed at pH 3.4. In another example, the pH denaturation is performed at pH 3.3. In another example, the pH denaturation is performed at pH 3.2. In another example, the pH denaturation is performed at pH 3.1. In yet another example, the pH denaturation is performed at pH 3.0.

[0114] In yet another example, the pH denaturation is performed at pH 9.1. In another example, the pH denaturation is performed at pH 9.2. In another example, the pH denaturation is performed at pH 9.3. In another example, the pH denaturation is performed at pH 9.4. In another example, the pH denaturation is performed at pH 9.5. In another example, the pH denaturation is performed at pH 9.6. In another example, the pH denaturation is performed at pH 9.7. In another example, the pH denaturation is performed at pH 9.8. In another example, the pH denaturation is performed at pH 9.9. In another example, the pH denaturation is performed at pH 10. In another example, the pH denaturation is performed at pH 10.1. In another example, the pH denaturation is performed at pH 10.2. In another example, the pH denaturation is performed at pH 10.3. In another example, the pH denaturation is performed at pH 10.4. In another example, the pH denaturation is performed at pH 10.5. In another example, the pH denaturation is performed at pH 10.6. In another example, the pH denaturation is performed at pH 10.7. In another example, the pH denaturation is performed at pH 10.8. In yet another example, the pH denaturation is performed at pH 10.9. In another example, the pH denaturation is performed at pH 11.

[0115] The aforementioned pH denaturation protocol can be performed on a sample prior to the chromatographic separation method disclosed herein. Alternatively, pH denaturation can be performed on a sample after chromatographic separation using the disclosed method. Furthermore, pH denaturation can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can be selected or adjusted to have a pH sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, pH denaturation of linRNA in a sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, pH denaturation can be performed on a sample even if the sample is not chromatographically purified.

[0116] Chemical denaturation Another method encompassed by the present disclosure for selectively enriching and purifying circRNA from a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products is denaturation by chemical treatment. In the context of the present disclosure, denaturation by chemical treatment includes treatment with one or more denaturing acids, bases, organic solvents, chaotropic agents, chelating agents, crowding agents, detergents, or salt solutions.

[0117] Acid Denaturant Acid denaturation is a suitable method for denaturing the purification methods disclosed herein on the basis that acidic solutions can protonate ionizable groups in RNA molecules. Suitable acids for use in selective enrichment and purification of circRNA from a sample containing a mixed population of linRNA, circRNA, and polyribonucleotides, optionally including one or more impurities or by-products, are at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 7%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) of acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid. In some embodiments, suitable acids for use in selective enrichment and purification of circRNA from a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products are between 1 mM and 500 mM (e.g., 2-475 mM, 3-450 mM, 4-425 mM, 5-400 mM, 10-375 mM, 15-350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[0118] In one example, selective denaturation of linRNA but not circRNA involves selectively denaturing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products by denaturing the sample at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) of acetic acid. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) acetic acid.

[0119] In another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) hydrochloric acid. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) hydrochloric acid.

[0120] In another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) salicylic acid. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) salicylic acid.

[0121] In another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) phosphoric acid (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) phosphate.

[0122] In another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) boric acid. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) boric acid.

[0123] In yet another example, acid denaturation is performed by exposing the sample to a solution comprising at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) formic acid. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) formic acid.

[0124] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) oxalic acid). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) oxalic acid.

[0125] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) citric acid. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) citric acid.

[0126] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) benzoic acid (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) benzoic acid.

[0127] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) monochloroacetic acid). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) monochloroacetic acid.

[0128] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) dichloroacetic acid (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) dichloroacetic acid.

[0129] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) trichloroacetic acid (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) trichloroacetic acid.

[0130] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) ascorbic acid (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) ascorbic acid.

[0131] In yet another example, acid denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) nitric acid (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) nitric acid.

[0132] The exposure of the sample to the acidic denaturing conditions can be for a time sufficient to denature the polyribonucleotides (e.g., linRNA or circRNA, among others). For example, the exposure of the sample to the acidic denaturing conditions can be for 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or more.

[0133] The acidic denaturation protocol described above can be performed on a sample prior to the chromatographic separation method disclosed herein. Alternatively, acidic denaturation can be performed on a sample after chromatographic separation using the disclosed method. Furthermore, acidic denaturation can be performed in parallel with (i.e., during) the chromatographic purification of a sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the acids listed above in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, acidic denaturation of linRNA in a sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, acidic denaturation can be performed on a sample even if the sample is not chromatographically purified.

[0134] Alkaline Denaturant Alkaline denaturation is a suitable method for denaturing the purification methods disclosed herein on the basis that alkaline solutions can deprotonate ionizable groups in RNA molecules. Suitable alkaline conditions for use in selective enrichment and purification of circRNA from a sample containing a mixed population of linRNA, circRNA, and polyribonucleotides, optionally including one or more impurities or by-products, include at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 and solutions containing (14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, or guanidine. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[0135] In one example, selective denaturation of linRNA but not circRNA involves selectively denaturing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products by denaturing at least 0.5% (v / v) (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, In some embodiments, the method may be performed by exposing the substrate to a solution containing sodium hydroxide (e.g., 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) sodium hydroxide.

[0136] In another example, alkaline denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) potassium hydroxide (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) potassium hydroxide.

[0137] In another example, alkaline denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) imidazole (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) imidazole.

[0138] In another example, alkaline denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) histidine (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) histidine.

[0139] In another example, alkaline denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) sodium bicarbonate (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) sodium bicarbonate.

[0140] In yet another example, alkaline denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) guanidine (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) guanidine.

[0141] In yet another example, alkaline denaturation is performed by exposing the sample to a solution containing at least 0.5% (v / v) tritylamine (e.g., at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more). In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1 mM to 500 mM (e.g., 2 to 475 mM, 3 to 450 mM, 4 to 425 mM, 5 to 400 mM, 10 to 375 mM, 15 to 350 mM, 20 to 325 mM, 30 to 300 mM, 40 to 275 mM, 50 to 250 mM, 60 to 225 mM, 70 to 200 mM, 80 to 175 mM, 90 to 150 mM, 100 to 125 mM, or 110 to 115 mM) tritylamine.

[0142] The sample can be exposed to alkaline denaturing conditions for a time sufficient to denature polyribonucleotides (e.g., circRNA or linRNA, among others). For example, the sample can be exposed to alkaline denaturing conditions for 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or more.

[0143] The aforementioned alkaline denaturation protocol can be performed on a sample prior to the chromatographic separation method disclosed herein. Alternatively, alkaline denaturation can be performed on a sample after chromatographic separation using the disclosed methods. Furthermore, alkaline denaturation can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned bases in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, alkaline denaturation of linRNA in a sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, alkaline denaturation can be performed on a sample even if the sample is not chromatographically purified.

[0144] Organic solvents Organic solvents can be used as denaturants suitable for use with the disclosed methods. Organic solvents suitable for use in selective enrichment and purification of circRNA from a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products, have a concentration of at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[0145] In one example, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products to a solution containing at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) dimethyl sulfoxide.

[0146] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) triethylammonium acetate.

[0147] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) methanol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0148] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) ethanol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0149] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) 2-propanol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0150] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) isopropanol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0151] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) butanol (e.g., 1-butanol, 2-butanol, t-butanol, or isobutanol).

[0152] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) 1-butanol.

[0153] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) 2-butanol.

[0154] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) t-butanol.

[0155] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) isobutanol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0156] In another example, denaturation is performed by exposing the sample to a solution containing at least 0.1% (v / v) phenol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0157] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) chloroform (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0158] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) hexane (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0159] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) acetonitrile).

[0160] In another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) formamide (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0161] In yet another example, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) propylene glycol (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0162] The sample can be exposed to the denaturing solvent for a time sufficient to denature the polyribonucleotides (e.g., circRNA or linRNA, among others). For example, the sample can be exposed to alkaline denaturing conditions for 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or more.

[0163] The aforementioned denaturation protocol can be performed on the sample prior to the chromatographic separation method disclosed herein. Alternatively, denaturation can be performed on the sample after chromatographic separation using the disclosed method. Furthermore, alkaline denaturation can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned organic solvents in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, denaturation of linRNA in the sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, denaturation can be performed on the sample even if the sample is not chromatographically purified.

[0164] Chaotropic Agents Chaotropic agents (e.g., chaotropic salts) can be used as denaturants suitable for use with the disclosed methods. Chaotropic agents (e.g., chaotropic salts) suitable for use in selective enrichment and purification of circRNA from a sample containing a mixed population of linRNA, circRNA, and polyribonucleotides, optionally including one or more impurities or by-products, are at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 88%, 89%, 89%, 90%, 91%, 92%, 9 %, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) of urea, guanidinium chloride, lithium perchlorate, or PEG. In some embodiments, chaotropic agents suitable for use in selective enrichment and purification of circRNA from a sample comprising a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products include 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) urea, guanidinium chloride, lithium perchlorate, or PEG.

[0165] In one example, selective denaturation of linRNA but not circRNA involves selectively denaturing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, by denaturing the sample at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 2%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more guanidinium chloride. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) urea.

[0166] In another embodiment, denaturation is performed by exposing the sample to a solution comprising at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) lithium perchlorate. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) lithium perchlorate.

[0167] In another embodiment, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products to a solution containing less than 1% (e.g., 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less) of n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) deoxycholate.

[0168] In one example, denaturation is performed by exposing the sample to a solution containing at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) n-dodecyl β-d-maltoside. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) β-d-maltoside.

[0169] In another embodiment, denaturation is performed by exposing the sample to a solution containing at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) n-octylglucoside. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) of n-octylglucoside.

[0170] In another embodiment, denaturation is performed by exposing the sample to a solution comprising at least 0.1% (v / v) CHAPS (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more). In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) of CHAPS.

[0171] In another embodiment, denaturation is performed by exposing the sample to a solution containing at least 0.1% (v / v) deoxycholate (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) deoxycholate.

[0172] The exposure of the sample to the denaturing chaotropic agent can be for a time sufficient to denature the polyribonucleotides (e.g., circRNA or linRNA, among others). For example, the exposure of the sample to alkaline denaturing conditions can be for more than 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or more.

[0173] The aforementioned denaturation protocols can be performed on the sample prior to the chromatographic separation methods disclosed herein. Alternatively, denaturation with chaotropic agents can be performed on the sample after chromatographic separation using the disclosed methods. Furthermore, denaturation with chaotropic agents can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned chaotropic agents in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, denaturation of linRNA in the sample can be performed at any stage of the chromatographic separation process, including the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, denaturation can be performed on the sample even if the sample is not chromatographically purified.

[0174] Crowding Agent Crowding agents can be used as denaturants suitable for use with the disclosed methods on the basis that they can crowd the solution so that hydrogen bonding between other molecules does not occur. Crowding agents suitable for use in selective enrichment and purification of circRNA from a sample containing a mixed population of linRNA, circRNA, and polyribonucleotides, optionally including one or more impurities or by-products, include PEG and urea.

[0175] In one example, denaturation is performed by exposing the sample to a solution containing at least 0.1% (v / v) PEG (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 8 M (e.g., 150 mM to 7.5 M, 200 mM to 7 M, 250 mM to 6.5 M, 300 mM to 6 M, 350 mM to 5.5 M, 400 mM to 5 M, 450 mM to 4.5 M, 500 mM to 4 M, 600 mM to 3.5 M, 700 mM to 3 M, 800 mM to 2.5 M, 900 mM to 2 M, 1 M to 1.5 M, 1.1 M to 1.4 M, or 1.2 to 1.3 M) of PEG.

[0176] PEG refers to a group of the general formula (CH2CH2OH)n, where n is an integer, such as PEG2 to PEG 100 In some embodiments, the PEG has a molecular weight of 200 Da to 6000 Da (e.g., 400 Da to 2500 Da, 800 Da to 2200 Da, 1000 Da to 2000 Da, 200 Da, 400 Da, 600 Da, 800 Da, 1000 Da, 1200 Da, 1500 Da, 2000 Da, 2200 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, 5000 Da, 5500 Da, or 6000 Da).

[0177] The aforementioned denaturation protocol can be performed on the sample prior to the chromatographic separation method disclosed herein. Alternatively, denaturation can be performed on the sample after chromatographic separation using the disclosed method. Furthermore, alkaline denaturation can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned crowding agent at a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, denaturation of linRNA in the sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, denaturation can be performed on the sample even if the sample is not chromatographically purified.

[0178] Chelating Agents Chelators are metal ions (among which metal ions, e.g., Ca) that bind and stabilize RNA secondary and tertiary structures within RNA molecules (e.g., linRNA). 2+ , Mg 2+ , Na + , and K +Chelating agents suitable for use in combination with the disclosed methods include ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a derivative thereof, ethylenediaminetetraacetic acid (EDTA) or a derivative thereof, nitrilotriacetic acid (NTA), imino-disuccinic acid (IDS), polyaspartic acid, S,S-ethylenediamine-N,N'-disuccinic acid (EDDS), or methylglycine diacetic acid (MGDA). In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) EGTA or a derivative thereof, EDTA or a derivative thereof, NTA, IDS, EDDS, or MGDA.

[0179] In one example, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing EGTA or a derivative thereof. In some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) EGTA or a derivative thereof.

[0180] In another example, denaturation is performed by exposing the sample to a solution containing EDTA or a derivative thereof, hi some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) EDTA or a derivative thereof.

[0181] In another example, denaturation is performed by exposing the sample to a solution containing NTA, hi some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) NTA.

[0182] In another example, denaturation is performed by exposing the sample to a solution containing IDS, in some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) IDS.

[0183] In another example, denaturation is performed by exposing the sample to a solution containing polyaspartic acid, in some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) polyaspartic acid.

[0184] In another example, denaturation is performed by exposing the sample to a solution containing EDDS, in some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) EDDS.

[0185] In yet another example, denaturation is performed by exposing the sample to a solution containing MGDA, hi some embodiments, the solution contains 1-10 mM (e.g., 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM, or 9-10 mM) MGDA.

[0186] The sample can be exposed to the denaturing chelating agent for a time sufficient to denature the polyribonucleotides (e.g., circRNA or linRNA, among others). For example, the sample can be exposed to the denaturing chelating agent for 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or more.

[0187] The aforementioned denaturation protocols can be performed on the sample prior to the chromatographic separation methods disclosed herein. Alternatively, denaturation can be performed on the sample after chromatographic separation using the disclosed methods. Furthermore, denaturation with a chelating agent can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned chelating agent in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, denaturation of linRNA in the sample can be performed at any stage of the chromatographic separation process, such as the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, denaturation can be performed on the sample even if the sample is not chromatographically purified.

[0188] Detergent Detergents can be used as denaturants suitable for use with the disclosed methods. Detergents suitable for use in selective enrichment and purification of circRNA from a sample containing a mixed population of polyribonucleotides including linRNA, circRNA, and optionally one or more impurities or by-products include Nonidet P-40 (NP40), polysorbates (e.g., Tween-20, Tween-40, Tween-60, or Tween-80), CHAPS, octyl β-D-glucopyranoside, or n-dodecyl β-maltoside. In some embodiments, the selective denaturation of linRNA, but not circRNA, is achieved by a concentration of 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.0 The method is carried out by exposing a sample containing a mixed population of polyribonucleotides to 15-0.016% of NP40, polysorbate (e.g., Tween®-20, Tween®-40, Tween®-60, or Tween®-80), CHAPS, octyl β-D-glucopyranoside, or n-dodecyl β-maltoside.

[0189] In another example, the denaturation is performed by exposing the sample to a solution containing NP40. In some embodiments, the denaturation is performed by exposing the sample to a solution containing 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) NP40.

[0190] In another example, denaturation is performed by exposing the sample to a solution comprising Tween®-20. In some embodiments, denaturation is performed by exposing the sample to a solution comprising 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) Tween®-20.

[0191] In another example, denaturation is performed by exposing the sample to a solution comprising Tween®-80. In some embodiments, denaturation is performed by exposing the sample to a solution comprising 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) Tween®-80.

[0192] In another example, denaturation is performed by exposing the sample to a solution containing CHAPS. In some embodiments, denaturation is performed by exposing the sample to a solution containing 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) CHAPS.

[0193] In another example, denaturation is performed by exposing the sample to a solution containing octyl β-D-glucopyranoside. In some embodiments, denaturation is performed by exposing the sample to a solution containing 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) octyl β-D-glucopyranoside.

[0194] In another example, the denaturation is performed by exposing the sample to a solution containing n-dodecyl β-maltoside. In some embodiments, the denaturation is performed by exposing the sample to a solution containing 0.005-0.05% (e.g., 0.006-0.045%, 0.007-0.04%, 0.008-0.035%, 0.009-0.03%, 0.01-0.025%, 0.011-0.02%, 0.012-0.019%, 0.013-0.018%, 0.014-0.017%, or 0.015-0.016%) dodecyl β-maltoside.

[0195] The sample can be exposed to the denaturing detergent for a time sufficient to denature polyribonucleotides (e.g., circRNA or linRNA, among others). For example, the sample can be exposed to the denaturing detergent conditions for 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or more.

[0196] The aforementioned denaturation protocols can be performed on the sample prior to the chromatographic separation methods disclosed herein. Alternatively, denaturation can be performed on the sample after chromatographic separation using the disclosed methods. Furthermore, denaturation with detergents can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned detergents in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, denaturation of linRNA in the sample can be performed at any stage of the chromatographic separation process, including the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, denaturation can be performed on the sample even if the sample is not chromatographically purified.

[0197] Salt solution The secondary structure formation of nucleic acid polymers is highly dependent on salt concentration, which determines the free energy of base pairing and hydrogen bond formation within polynucleotides. Therefore, the use of salt solutions is a suitable method for denaturing and purifying polyribonucleotides as described herein. Salt solutions suitable for use in selective enrichment and purification of circRNA from a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, include, for example, solutions containing the following: sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl), calcium chloride (CaCl), CsSO4, NaSO4, lithium chloride (LiCl), lithium bromide (LiBr), among others known in the art. In some embodiments, the salt solution comprises 100 mM to 1 M (e.g., 150-950 mM, 200-900 mM, 250-850 mM, 300-800 mM, 350-750 mM, 400-700 mM, 450-650 mM, 500-600 mM, or 525-575 mM) NaCl, KCl, MgCl, CaCl, CsSO4, NaSO4, LiCl, or LiBr.

[0198] In one example, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing NaCl. In some embodiments, selective denaturation of linRNA, but not circRNA, is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) NaCl.

[0199] In another example, denaturation is performed by exposing the sample to a solution containing KCl. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) KCl.

[0200] In another example, denaturation is performed by exposing the sample to a solution containing MgCl. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) MgCl.

[0201] In another example, denaturation is performed by exposing the sample to a solution containing CaCl. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) CaCl.

[0202] In another example, denaturation is performed by exposing the sample to a solution containing CsSO4. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) CsSO4.

[0203] In another example, denaturation is performed by exposing the sample to a solution containing NaSO4. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) NaSO4.

[0204] In another example, denaturation is performed by exposing the sample to a solution comprising LiCl. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample comprising a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution comprising 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) LiCl.

[0205] In another example, denaturation is performed by exposing the sample to a solution containing LiBr. In some embodiments, selective denaturation of linRNA but not circRNA is performed by exposing a sample containing a mixed population of polyribonucleotides, including linRNA, circRNA, and optionally one or more impurities or by-products, to a solution containing 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) LiBr.

[0206] The sample can be exposed to the denaturing salt solution for a time sufficient to denature the polyribonucleotides (e.g., circRNA or linRNA, among others). For example, the sample can be exposed to the denaturing salt solution conditions for 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or more.

[0207] The aforementioned denaturation protocols can be performed on the sample prior to the chromatographic separation methods disclosed herein. Alternatively, denaturation can be performed on the sample after chromatographic separation using the disclosed methods. Furthermore, denaturation with salt solutions can be performed in parallel with (i.e., during) the chromatographic purification of the sample. For example, the sample buffer (e.g., loading buffer) used in the chromatographic column can contain the aforementioned salts in a concentration sufficient to denature the intramolecular hydrogen bonds of the polyribonucleotides of the sample. Thus, denaturation of linRNA in the sample can be performed at any stage of the chromatographic separation process, including the equilibration step, the sample loading step, the column washing step, and the elution step. Furthermore, salt solution denaturation can be performed on the sample even if the sample is not chromatographically purified.

[0208] Combination or multiple modifiers The present disclosure further provides the use of a combination of two, three, four or more of the aforementioned denaturing conditions to produce an enriched population of circRNA from a mixed population of polyribonucleotides including circRNA, linRNA, and other substances. A sample containing a mixed population of circRNA and linRNA can be subjected to two, three, four or more of the disclosed denaturing conditions simultaneously or sequentially (with or without a time interval). The two or more denaturing conditions can be, for example, from the same category of denaturants, such as two or more chaotropic agents selected from the chaotropic agents described herein, two or more acid denaturants selected from the acid denaturants described herein, two or more alkaline denaturants selected from the alkaline denaturants described herein, two or more organic solvents selected from the organic solvents described herein, two or more chelating agents selected from the chelating agents described herein, two or more detergents selected from the detergents described herein, or two or more salt solutions selected from the salt solutions described herein. The two or more denaturing conditions can also be from different categories of denaturants. For example, the combination of two or more denaturing conditions may be as described in Table 1, where the heat denaturation is selected from any heat denaturation condition described herein, the pH denaturation is selected from any pH denaturation condition described herein, the acid denaturant is selected from any acid denaturation condition described herein, the alkaline denaturant is selected from any alkaline denaturation condition described herein, the organic solvent is selected from any organic solvent condition described herein, the chaotropic agent is selected from any chaotropic agent condition described herein, the chelating agent is selected from any chelating agent condition described herein, the detergent is selected from any detergent condition described herein, and the salt solution is selected from any salt solution condition described herein.

[0209] [Table 1]

[0210] The combinations shown in Table 1 may be combined with third, fourth, fifth or more additional denaturing conditions.

[0211] For example, the above denaturing thermal conditions can be combined with the above denaturing pH conditions to produce an enriched population of circRNA from a mixed population of polyribonucleotides including circRNA and linRNA. Alternatively, the denaturing thermal conditions can be combined with one or more chemical denaturants described herein to produce an enriched population of circRNA. Furthermore, an enriched population of circRNA can be produced from a mixed population of polyribonucleotides by contacting the mixed population of polyribonucleotides with a pH denaturant and a chemical denaturant.

[0212] The present disclosure also provides for the use of one or more denaturing conditions described herein in combination with one or more non-denaturing conditions (e.g., temperature, pH, buffer) known in the art. In some embodiments, thermal denaturation as described herein in combination with denaturing pH conditions may be performed on a sample containing a mixed population of polyribonucleotides, including circRNA and linRNA. For example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be denatured at a temperature of at least 50°C, for example, 50°C to 85°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 99°C, 100°C, 102°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, 111°C, 112°C, 113°C, 114°C, 115°C, 116°C, 117°C, 118°C, 119° 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 8.99).

[0213] In some other embodiments, the thermal denaturation described herein may be performed on a sample containing a mixed population of polyribonucleotides including circRNA and linRNA in combination with non-denaturing pH conditions. For example, the polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) may be subjected to a denaturation temperature, such as a temperature of at least 50°C, for example, 50°C to 85°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 9 ... 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9).

[0214] In another example, the polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be purified using denaturing pH conditions (e.g., a pH greater than 5 and less than 9, such as the following: 5.01, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.8, 9.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7 ... The enzyme may be exposed to a pH of 50° C., 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 8.99 in combination with a non-denaturing temperature, e.g., a temperature below 50° C. (e.g., 49° C., 48° C., 47° C., 46° C., 45° C., 44° C., 43° C., 42° C., 41° C., 40° C., 39° C., 38° C., 37° C., 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., 29° C., 28° C., 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C., 20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., or lower).

[0215] In another example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be incubated with denaturing chaotropic agent conditions, e.g., at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 7 ... The cells can be exposed to urea at a concentration of at least 0.5%, 80%, 85%, 90%, 95%, 99% or more), as well as one or more denaturing organic solvent conditions, such as acetonitrile and dimethylsulfoxide at a concentration of at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more).

[0216] In another example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be incubated with denaturing chaotropic agent conditions, e.g., at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, urea at a concentration of at least 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more), plus one or more denaturing organic solvent conditions, e.g., at least 0.1% (v / v) (e.g., at least 0.1% , 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or more) acetonitrile and dimethyl sulfoxide, and The nucleic acid may be exposed to a denaturing temperature such as a temperature of 0°C, for example, a temperature between 50°C and 85°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C).

[0217] In another example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be incubated with denaturing chaotropic agent conditions, e.g., at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 7 ... 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) of urea, as well as a denaturing temperature such as a temperature of at least 50°C, for example, a temperature between 50°C and 85°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C).In another example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be incubated with denaturing chaotropic agent conditions, e.g., at least 1% (v / v) (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 0%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) of urea, as well as denaturing pH conditions (e.g., a pH greater than 5 and less than 9, such as the following: 5.01, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 8.99).

[0218] In another example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be incubated in a denaturing organic solvent condition, e.g., at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 100%, 15 ... %, 75%, 80%, 90% or more) of acetonitrile and dimethylsulfoxide, as well as a denaturing temperature such as a temperature of at least 50°C, for example, a temperature of 50°C to 85°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C).

[0219] In another example, polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) can be incubated with one or more denaturing organic solvent conditions, such as at least 0.1% (v / v) (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 7 ... In some embodiments, the nucleic acid may be exposed to acetonitrile and dimethylsulfoxide at concentrations of 5%, 80%, 90% or more, as well as denaturing pH conditions (e.g., a pH greater than 5 and less than 9, such as the following: 5.01, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 8.99).

[0220] In another example, the polyribonucleotides described herein (e.g., a mixed population of polyribonucleotides including circRNA and linRNA or an enriched population of circRNA) are denatured under denaturing salt conditions, for example, 100 mM to 1 M (e.g., 150 to 950 mM, 200 to 900 mM, 250 to 850 mM, 300 to 800 mM, 350 to 750 mM, 400 to 700 mM, 450 to 650 mM, 500 to 600 mM, or 525 to 575 mM) of NaCl, KCl, MgCl, CaCl, CsSO, NaSO 4, LiCl, or LiBr, as well as exposure to denaturing pH conditions (e.g., a pH greater than 5 and less than 9, such as the following: a pH of 5.01, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 8.99).

[0221] Purification method Disclosed herein are various methods that can be used to purify circRNAs from samples containing a mixed population of circRNAs and linRNAs under denaturing conditions. In some embodiments, the purification method is a column chromatography purification method. Column chromatography methods generally use a process in which a solution containing dissolved substances of interest (commonly referred to as a mobile phase) is flowed through a chromatography column loaded with a stationary phase composed of porous granules. The loaded solution passes through the column and substances are separated based on their interaction with the stationary phase, which is determined by the physicochemical properties of the analytes. Purification methods include column chromatography, such as, for example, fast protein liquid chromatography (FPLC; e.g., reversed-phase (RP)-FPLC), hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEC), mixed-mode chromatography (MMC), or affinity chromatography. These methods are described in more detail below.

[0222] High-performance liquid chromatography The methods described herein include the use of FPLC methods to purify and enrich circRNAs from samples containing one or more distinct populations of circRNAs and linRNAs, or mixed polyribonucleotides containing other substances.

[0223] FPLC is an established method for separating complex mixtures of substances and is commonly used in biochemistry, analytical chemistry, and clinical chemistry applications. There are two general types of FPLC methods: reversed-phase (RP)-FPLC and normal-phase (NP)-FPLC. RP-FPLC differs from NP-FPLC in that RP-FPLC uses a non-polar (i.e., hydrophobic) stationary phase and a polar or medium polarity mobile phase, whereas NP-FPLC uses a polar (hydrophilic) stationary phase and a non-polar mobile phase. FPLC differs from high performance liquid chromatography (HPLC) primarily in the use of lower operating pressures (e.g., <5 bar) and higher flow rates (1-5 mL / min for benchtop systems; liters / min for industrial scale purification) compared to HPLC. In general, FPLC columns can only be used at a maximum pressure of 3-4 MPa (435-580 psi). RP-FPLC is generally performed in an apparatus that includes at least a pump with an eluent reservoir containing the mobile phase, a sample input system, a separation column containing a hydrophobic stationary phase (e.g., resin, beads, film, among others), and a detector. Additional elements of the RP-FPLC apparatus may include a fraction collector for collecting individual fractions collected during the separation process. The core principle underlying RP-FPLC is the interplay between non-polar analytes, non-polar stationary analytes, and the shortening of retention and elution times for more polar analytes. This is because the reduced exposure of the hydrophobic segments of the analytes to the polar solvent reduces the free energy associated with the minimization of the ordered analyte-polar solvent interface. Thus, the hydrophobicity index of an analyte is roughly proportional to its elution time from the chromatographic column. Reducing the polarity of the mobile phase reduces the binding of non-polar analytes, which leads to the elution of the analytes from the chromatographic column. Other factors that affect the retention of analytes include the presence of inorganic salts in the mobile phase, which increase the retention of analytes due to their effect on the mobile phase (i.e., increasing surface tension). Retention can also be influenced by the pH of the mobile phase by modifying the hydrophobicity of the analytes. Mobile phase buffers are generally used to adjust the pH in order to neutralize any residual charge on the analytes and promote hydrophobic interactions between the analytes and the stationary phase.Similarly, the polarity of a charged analyte can be reduced by using an ion pairing agent, which neutralizes the charge of the analyte through ionic interactions.

[0224] The method disclosed herein allows the separation of a circRNA population from a mixed polyribonucleotide population containing circRNA and linRNA by exploiting the difference in functional hydrophobicity between circRNA and linRNA; i.e., circRNA exhibits higher hydrophobicity compared to linRNA, which leads to stronger adsorption of circRNA to the stationary phase and, as a result, longer elution times. Materials suitable for use as reversed phases in RP-FPLC include, but are not limited to, porous polystyrene polymers, (non-alkylated) (porous) polystyrene divinylbenzene polymers, porous silica gel, porous silica gel modified with non-polar residues, in particular porous silica gel modified with alkyl-containing residues, more preferably butyl, octyl or octadecyl-containing residues, porous silica gel modified with phenyl residues, and porous polymethacrylate, in which case, in particular, porous polystyrene polymers or non-alkylated (porous) polystyrene divinylbenzene can be used. Stationary phases using polystyrene divinylbenzene are known per se. Commercially available polystyrene divinylbenzenes known per se and already used in FPLC processes can be used in the process according to the invention.Non-alkylated porous polystyrene divinylbenzenes particularly highly preferred for the process according to the invention can have, but are not limited to, a particle size of 8.0±1.5 μm, in particular 8.0±0.5 μm, and a pore size of 1000-1500 A, in particular 1000-1200 A or 3500-4500 A.

[0225] Hydrophobic interaction chromatography HIC is a purification method that exploits the interaction of the HIC mobile phase with hydrophobic sites on the target molecule of interest (e.g., circRNA). HIC can be performed in bind-elute mode, where the target molecule binds to the stationary phase until eluted in the elution step, or in flow-through mode, where the molecule of interest flows through the mobile phase while impurities or by-products bind to the stationary phase. In some embodiments, HIC can use a combination of bind-elute and flow-through modes.

[0226] HIC can use one or more hydrophobic ligands. Non-limiting examples of HIC hydrophobic ligands include alkyl ligands, aryl ligands, and combinations thereof. For example, the HIC stationary phase can be selected from the group consisting of butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands. In some embodiments, the HIC column is selected from the group consisting of: CaptoPhenyl, low or high substituted Phenyl Sepharose™ 6 Fast Flow, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl, Macro-Prep™ Methyl, Macro-Prep™ t-Butyl, WP HII-Propyl(C3)™, CIMmultus C4 HLD, CIMmultus® OH, Toyopearl™ Ether, Toyopearl™ Phenyl, Toyopearl™ Butyl, ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen Octyl FF, and Tosoh Hexyl.

[0227] According to the methods disclosed herein, HIC can use a loading buffer or washing buffer containing a salt. Non-limiting examples of salts suitable for HIC include ammonium sulfate, sodium sulfate, sodium chloride, ammonium chloride, sodium bromide, or combinations thereof. In some embodiments, the loading and washing buffers include sulfate, citrate, or combinations thereof. In various embodiments, the loading or washing buffers include Ba, Na ... 2+ , Ca 2+ , Mg 2+ , Li + , Cs + , Na + , K + , Rb + , or NH4 + or PO4 3- , SO4 2- , CH3CO3 - , Cl - , Br - , NO3 - , ClO4 - , I - , or SCN -or combinations thereof. The hydrophobic interaction is stronger with higher salt concentrations. Adsorption of the population of circRNAs of interest to the HIC column is facilitated by high salt concentrations, but the actual concentration can vary widely depending on the nature of the circRNAs of interest, the type of salt, and the particular HIC ligand selected. In some embodiments, the salt concentration ranges from about 50 mM to about 5000 mM, about 100 mM to about 4000 mM, about 1000 mM to about 4000 mM, about 50 mM to about 2000 mM, depending in part on the type of salt and the HIC adsorbent. In one embodiment, the salt concentration is about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1200 mM, about 1400 mM, about 1600 mM, about 1800 mM, or about 2000 mM. In some embodiments, the load buffer and wash buffer have a pH of about 4.0 to 8.5, or about 5.0 to 7.0. In certain embodiments, the load buffer and wash buffer have a pH of about 4.0, about 4.5, about 5.0, about 5.5, about 6, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the load buffer and wash buffer are the same or substantially the same.

[0228] In an exemplary method of performing HIC separation, a sample (e.g., a sample containing a mixed population of polyribonucleotides such as circRNA and linRNA) is contacted with a HIC medium, e.g., using a batch purification technique or using column or membrane chromatography or monolith materials (referred to as HIC medium or resin). For example, for chromatographic separation, a generally cylindrical shaped chromatographic device is used to contain a chromatographic support medium (e.g., HIC medium) prepared with an appropriate buffer. Once the chromatographic material is added to the chromatographic device, the sample containing the population of circRNA of interest is contacted with the chromatographic material in the presence of a loading buffer so that a substantial portion of the circRNA of interest or impurities or by-products can be bound to the HIC medium. The population of circRNA of interest in the sample binds to the HIC medium, while impurities or by-products such as, e.g., linRNA, flow through to form a flow-through fraction containing impurities or by-products. The bound circRNA is eluted in an elution step, thereby forming an enriched population of circRNA. In some embodiments, the population of circRNAs bound to the HIC medium is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% of the total amount of polyribonucleotides in the sample.

[0229] Anion exchange chromatography AEC separates molecules based on the difference between the local charge of the nucleic acid of interest (e.g., circRNA) and the local charge of the chromatographic material. Packed AEC columns or membrane devices can be operated in bind-elute, flow-through, or hybrid modes. After washing the column or membrane device with equilibration buffer or another buffer of different pH or conductivity, product recovery is achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the AEC matrix. Changing the pH, and thereby the charge of the solute, is another way to achieve elution of the solute. The change in conductivity or pH can be gradual (gradient elution) or stepwise (step elution).

[0230] Anionic or cationic substituents can be attached to the matrix to form anionic or cationic supports for chromatography. Non-limiting examples of anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ion exchange media such as DE23™, DE32™, DE52™, CM-23™, CM-32™ and CM-52™ are available from Whatman Ltd., Maidstone, Kent, UK. SEPHADEX™ based and cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX®, as well as DEAE-, Q-, CM-, and S-SEPHAROSE®, as well as SEPHAROSE® Fast Flow, and Capto™ S are all available from GE Healthcare. Additionally, both DEAE and CM derivatized ethylene glycol-methacrylate copolymers, such as TOYOPEARL™ DEAE-650S or M and TOYOPEARL™ CM-650S or M, are available from Toso Haas Co., Philadelphia, Pa., CIMmultus™ QA, CIMmultus™ DEAE, CIMmultus™ EV, CIMmultus™ EDA, and CIMmultus™ SO3 are available from Sartorius, Nuvia S and UNOSphere™ S are available from BioRad, Hercules, Calif., and Eshmuno™ S is available from EMD Millipore, Billerica, Calif.

[0231] Mixed-mode chromatography MMC is chromatography using mixed-mode media, such as, but not limited to, CaptoAdhere available from GE Healthcare, CIMmultus® PrimaS available from Sartorius, and CIMmultus® H-Bond. Such media include MMC ligands. In some embodiments, such ligands refer to ligands that can provide at least two distinct, but cooperative, sites that interact with the substance to be bound. One of these sites confers an attractive type of charge-charge interaction between the ligand and the nucleic acid of interest. The other site typically confers an electron acceptor-donor interaction, or a hydrophobic or hydrophilic interaction. Electron donor-acceptor interactions include hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, and other interactions, among others. Mixed-mode functionality can confer different selectivities compared to traditional anion exchangers. MMC ligands are also known as "multimodal" chromatography ligands.

[0232] According to the present disclosure, the MMC medium may comprise a mixed mode ligand bound directly or via a spacer to an organic or inorganic support, sometimes referred to as the base matrix. The support may be in the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, etc. In certain embodiments, the support is prepared from native polymers, such as cross-linked carbohydrate materials, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginates, etc., among others. To obtain high adsorption capacity, the support can be made porous, and then the ligand is bound to the pore surfaces as well as to the external surface. Such native polymer supports can be prepared according to standard methods, such as the inverse suspension gelation method (S Hjerten: BIOCHIM BIOPHYS ACTA 79(2), 393-98 (1964). Alternatively, the supports can be prepared from synthetic polymers, among others, for example crosslinked synthetic polymers, such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinyl amides, etc. Such synthetic polymers can be manufactured according to standard methods, see for example "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Porous native or synthetic polymer supports are also available from commercial suppliers, such as Amersham Bioscience, Uppsala, Sweden.

[0233] Purity Assessment The evaluation of the purity of the enriched population of circRNAs can be performed by methods well known in the art. In one embodiment, the circRNAs have a purity of at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by mass. Purity can be measured, for example, by mass spectrometry, HPLC, chip-based electrophoresis, microscopy, circular dichroism (CD) spectroscopy, spectrophotometry, fluorometry (e.g., Qubit), polyacrylamide gel electrophoresis imaging, UV-Vis spectrophotometry, RNA electrophoresis, RNAse H analysis, or any combination thereof. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total mass of polyribonucleotides in a preparation described herein is comprised in circRNA molecules.

[0234] In some embodiments, the circRNA preparation comprises less than a threshold amount (e.g., the threshold amount is a reference standard for the circRNA preparation, e.g., a pharmaceutical release specification) of linRNA molecules as measured by mass spectrometry, HPLC, chip-based electrophoresis, microscopy, CD spectroscopy, spectrophotometry, fluorometry (e.g., Qubit), polyacrylamide gel electrophoresis imaging, UV-Vis spectrophotometry, RNA electrophoresis, or RNAse H analysis. In some embodiments, the circRNA preparation comprises undetectable levels of linRNA molecules.

[0235] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has a mononucleotide content of less than 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL.

[0236] In one embodiment, circRNAs have a mononucleotide content of less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, or any percentage therebetween of total nucleotides by mass, where total nucleotide content is the total mass of deoxyribonucleotide and ribonucleotide molecules.

[0237] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has a linRNA content, e.g., a linRNA counterpart or RNA fragment, of less than 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 1 mg / mL, 1.5 mg / mL, or 2 mg / mL.

[0238] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has less than 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% linRNA.

[0239] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has a DNA content, e.g., template DNA, e.g., cellular DNA (e.g., host cell DNA), of less than 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL.

[0240] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or an intermediate in a circRNA production process) has a DNA content of less than 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of total nucleotides by mass, where total nucleotide molecules is the total mass of deoxyribonucleotide molecules and ribonucleotide molecules.

[0241] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has protein (e.g., cellular protein (CP), e.g., enzymes) contamination of less than 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, or 500 ng / mL.

[0242] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has less than 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of protein (e.g., CP, e.g., enzyme) contamination per milligram (mg) of circRNA.

[0243] In one embodiment, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or a circRNA production process intermediate) has a circRNA concentration of at least 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, 1000 μg / mL, 5000 μg / mL, 10,000 μg / mL, or 100,000 μg / mL.

[0244] In some embodiments, the circRNA is purified by chromatography, e.g., liquid chromatography, e.g., FPLC (e.g., normal phase or reverse phase FPLC), HPLC, HIC, AEC, MMC, or affinity chromatography.

[0245] Preparative separation method In some embodiments, any of the conditions described herein can be used as a method for sorting circRNAs from a mixed population of polyribonucleotides including circRNAs and linRNAs or an enriched population of circRNAs.

[0246] In some embodiments, the preparative method produces amounts of circRNA of at least 0.5 mg (e.g., at least 0.6, 0.8, 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, 1000 mg, or more). The methods of the present invention are advantageous in that they are highly scalable.

[0247] Analysis method In some embodiments, any of the conditions described herein can be used as an analytical method to determine the purity of circRNAs from a mixed population of polyribonucleotides including circRNAs and linRNAs or a circRNA-enriched population. The present invention includes methods for iteratively determining the purity of a batch of enriched circRNAs using one or more denaturing conditions described herein.

[0248] In some embodiments, the analytical method determines the purity of the circRNA using chromatography, e.g., liquid chromatography, e.g., FPLC (e.g., normal phase or reverse phase FPLC), HPLC, HIC, AEC, MMC, or affinity chromatography.

[0249] In some embodiments, the analytical method has a relative standard deviation (RSD) of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. In some embodiments, the RSD is calculated based on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 circRNA samples.

[0250] In some embodiments, the analytical method includes the use of a mobile phase, hi some embodiments, the mobile phase includes urea, bis-trispropane (BTP), acetonitrile, NaCl, water, hydrochloric acid (HCl), or DMSO.

[0251] In some embodiments, the denaturing conditions are found in the mobile phase.

[0252] In some embodiments, the chromatography column is heated to provide denaturation. In some embodiments, the circRNA sample is heated for denaturation prior to introduction into the column.

[0253] In some embodiments, the circRNA sample is denatured using a combination of denaturing agents as described above, for example, a combination of urea and acetonitrile.

[0254] In some embodiments, the analytical method includes determining impurity or by-product levels.

[0255] In some embodiments, the analytical method comprises quantifying the purity of circRNA in an amount of at least 0.5 mg (e.g., at least 0.6, 0.8, 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, 1000 mg, or more).

[0256] Impurities and By-products The disclosed methods are suitable for producing preparations that contain a population of circRNAs and have reduced levels of impurities or by-products, for example, compared to the level of impurities or by-products in a sample prior to purification by the disclosed methods, or compared to the level of impurities or by-products in another sample containing 100% (w / w) circRNAs. In some embodiments, the methods of the present invention produce compositions that contain a population of circRNAs of interest and have reduced levels of total impurities or by-products. In some embodiments, preparations with reduced levels of total impurities or by-products are free of or substantially free of impurities or by-products. For example, low impurity or by-product compositions may contain about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, or less of total impurities or by-products. In some embodiments, low impurity or by-product compositions contain about 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1% or less total impurities or by-products.

[0257] In some embodiments, the impurity or by-product is a process-related impurity or by-product. As used herein, the term "process-related impurity or by-product" refers to an impurity or by-product that is present in a composition comprising a population of circRNAs of interest, but is not derived from the circRNA itself. Process-related impurities or by-products include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatography materials, and media components. For example, process-related impurities or by-products may include polyacrylamide, boric acid, magnesium, EDTA. As used herein, a "low impurity composition" refers to a composition that includes a low level of process-related impurities or by-products compared to a composition in which the impurities or by-products are not reduced. For example, a low process-related impurity composition may include about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less of process-related impurities or by-products. In one embodiment, a low process-related impurity composition is free of process-related impurities or by-products or is substantially free of process-related impurities or by-products.

[0258] In some embodiments, the circRNA (e.g., a circRNA pharmaceutical preparation or composition or an intermediate in the circRNA production process) is substantially free of impurities or by-products. In various embodiments, the level of at least one impurity or by-product in a composition comprising the circRNA is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the composition prior to purification or processing to remove the impurity or by-product. In some embodiments, the level of at least one process-related impurity or by-product is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the composition prior to purification or processing to remove the impurity or by-product. In some embodiments, the level of at least one product-related substance is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the composition prior to purification or processing to remove the impurity or by-product.

[0259] Methods known in the art can be used to quantify impurities or by-products present in a sample. For example, spectroscopic methods such as ultraviolet (UV), near infrared spectroscopy (NIR), Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy, or Raman spectroscopy can be used to monitor the levels of impurities or by-products in online, at-line, or in-line modes. In some embodiments, online, at-line, or in-line monitoring methods can be used either in the wash line of the chromatography step or in the collection vessel to enable the achievement of the desired circRNA quality / recovery. In some embodiments, UV signals can be used as an alternative to achieve appropriate product quality / recovery, and the UV signal can be appropriately processed, including but not limited to process techniques such as integration, differentiation, moving average, etc., so that normal process variability can be addressed and the target product quality can be achieved. In some embodiments, such measurements can be combined with in-line dilution methods to allow the ion concentration / conductivity of the load / wash to be controlled by feedback, thereby facilitating product quality control.

[0260] Production of circular polyribonucleotides The present disclosure provides methods for producing circular RNA, such as, for example, recombinant techniques or chemical synthesis. For example, the DNA molecules used to produce the RNA circle may include DNA sequences of native naturally occurring nucleic acid sequences, modified versions thereof, or DNA sequences encoding synthetic polypeptides not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of nucleic acid sequences, synthesis of oligonucleotide mixtures, and ligation of mixtures to "build" mixtures of nucleic acid molecules, and combinations thereof.

[0261] circRNAs can be prepared according to available techniques, including but not limited to chemical synthesis and enzymatic synthesis. In some embodiments, linear primary constructs or linear RNAs can be circularized or ligated to generate circRNAs described herein. The mechanism of circularization or ligation can occur by methods such as, for example, chemical, enzymatic, splint ligation, or ribozyme catalysis. The newly formed 5'-3' bond can be either intramolecular or intermolecular. For example, in the case of splint ligation, a splint ligase such as SplintR® ligase can be used. According to this method, a single-stranded polynucleotide (splint), such as a single-stranded DNA or RNA, can be designed to hybridize with both ends of a linRNA, thereby juxtaposing the two ends upon hybridization with the single-stranded splint. Thus, the splint ligase can catalyze the ligation of the two ends of the juxtaposed linRNA to generate circRNA. In some embodiments, a DNA or RNA ligase can be used to synthesize a circular polynucleotide. As a non-limiting example, the ligase can be a circ ligase or a circular ligase.

[0262] In another example, either the 5' or 3' end of the linRNA can encode a ligase ribozyme sequence, such that during in vitro transcription, the resulting linear circRNA contains an active ribozyme sequence that can ligate the 5' end of the linRNA to the 3' end of the linRNA. The ligase ribozyme can be derived from group I introns, hepatitis delta virus, hairpin ribozymes, or selected by SELEX (systematic evolution of ligands by exponential enrichment).

[0263] In another example, the linRNA can be circularized or linked by using at least one non-nucleic acid moiety. For example, the at least one non-nucleic acid moiety can react with a region or feature near the 5' end or near the 3' end of the linRNA to circularize or link the linRNA. In another example, the at least one non-nucleic acid moiety can be located at or linked to or near the 5' or 3' end of the linRNA. The non-nucleic acid moiety can be either homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety can be a linkage, such as a hydrophobic linkage, an ionic linkage, a biodegradable linkage, or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety can be a linking moiety. As yet another non-limiting example, the non-nucleic acid moiety can be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.

[0264] In another example, the linRNA may be circularized or ligated by self-splicing. In some embodiments, the linRNA may include a loop E sequence for self-ligation. In another embodiment, the linRNA may include a self-circularizing intron, such as a 5' and 3' slice junction, or a self-circularizing catalytic intron, such as a group I, group II, or group III intron. Non-limiting examples of group I intron self-splicing sequences include the T4 bacteriophage gene td, and the self-splicing replacement intron-exon sequences from the intervening sequence (IVS) rRNA of Tetrahymena, the cyanobacterium Anabaena pre-tRNA-Leu gene, or Tetrahymena pre-rRNA.

[0265] In some embodiments, the polyribonucleotide may include a catalytic intron fragment, such as the 3' half of a group I catalytic intron fragment and the 5' half of a group I catalytic intron fragment. The first and second annealing regions may be located within the catalytic intron fragment. Group I catalytic introns are self-splicing ribozymes that catalyze their own cleavage from mRNA, tRNA, and rRNA precursors via a two-metal ion phosphoryl transfer mechanism. Importantly, the RNA itself self-catalyzes intron removal without the need for an exogenous enzyme such as ligase.

[0266] In some embodiments, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from a cyanobacterial Anabaena pre-tRNA-Leu gene or a Tetrahymena pre-rRNA.

[0267] In some embodiments, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are from a cyanobacterium Anabaena pre-tRNA-Leu gene, the 3' exon fragment comprises a first annealing region, and the 5' exon fragment comprises a second annealing region. The first annealing region can comprise, for example, 5 to 50, e.g., 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides, and the second annealing region can comprise, for example, 5 to 50, e.g., 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides.

[0268] In some embodiments, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from Tetrahymena pre-RNA, and the 3' half of the group I catalytic intron fragment comprises a first annealing region and the 5' exon fragment comprises a second annealing region. In some embodiments, the 3' exon fragment comprises a first annealing region and the 5' half of the group I catalytic intron fragment comprises a second annealing region. The first annealing region can comprise, for example, 6 to 50, e.g., 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides and the second annealing region can comprise, for example, 6 to 50, e.g., 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0269] In some embodiments, the 3' half of the group I catalytic intron fragment and the 5' half of the group I catalytic intron fragment are derived from the cyanobacterium Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, or T4 phage td gene.

[0270] In some embodiments, the 3' half of the group I catalytic intron fragment and the 5' group I catalytic intron fragment are from the T4 phage td gene. The 3' exon fragment may comprise a first annealing region and the 5' half of the group I catalytic intron fragment may comprise a second annealing region. The first annealing region may comprise, for example, 2-16, e.g., 10-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides and the second annealing region may comprise, for example, 2-16, e.g., 10-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0271] In some embodiments, the 3' half of the Group I catalytic intron fragment is the 5' end of the linear polynucleotide.

[0272] In some embodiments, the 5' half of the Group I catalytic intron fragment is the 3' end of a linear polyribonucleotide.

[0273] In another example, linRNAs can be cyclized or linked by atoms at or near the 5' and 3' ends of the linRNA, non-nucleic acid moieties that cause attraction between molecular surfaces. One or more linRNAs can be cyclized or linked by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, agnostic bonds, dipolar bonds, conjugated bonds, hyperconjugated bonds, and antibonds.

[0274] In another example, the linRNA may contain ribozyme RNA sequences near the 5' and 3' ends. The ribozyme RNA sequences may be covalently linked to a peptide when the sequence is exposed to the remaining ribozymes. The ribozyme RNA sequences and the peptides covalently linked near the 5' and 3' ends associate with each other, thereby circularizing or linking the linRNA. In another example, the peptides covalently linked to the ribozyme RNA near the 5' and 3' ends are ligated using a variety of methods known in the art, including but not limited to protein ligation, and then circularizing or linking the linear primary construct or linear mRNA. A non-limiting list of examples of ribozymes used in the linear primary constructs or linRNAs of the present invention, or methods for incorporating or covalently linking peptides to peptides, are described in U.S. Patent Application Publication No. 20030082768, the contents of which are incorporated herein by reference in their entirety.

[0275] In yet another example, chemical cyclization methods can be used to generate circRNAs, including but not limited to click chemistry (e.g., alkyne and azide-based methods or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, or any combination thereof.

[0276] In another example, a circular polyribonucleotide is produced using a deoxyribonucleotide template transcribed in a cell-free system (e.g., by in vitro transcription) to produce a linear RNA. The linear polyribonucleotide produces a splicing-competent polyribonucleotide, which can self-splice to produce a circular polyribonucleotide.

[0277] In some embodiments, a circular polyribonucleotide is produced (e.g., in a cell-free system) by providing a linear polyribonucleotide; and allowing the linear polyribonucleotide to self-splice under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.

[0278] In some embodiments, the circular polyribonucleotide is produced by providing deoxyribonucleotides encoding a linear polyribonucleotide; transcribing the deoxyribonucleotides in a cell-free system to produce a linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and allowing the linear polyribonucleotide to self-splice under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide.

[0279] In some embodiments, the circular polyribonucleotide is produced by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce a linear polyribonucleotide, the transcription occurring in solution under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a 5' split intron and a 3' split intron (e.g., a self-splicing construct for producing circRNA). In some embodiments, the linear polyribonucleotide comprises a 5' annealing region and a 3' annealing region.

[0280] In some embodiments, the linear polyribonucleotide is produced from a deoxyribonucleic acid, e.g., a DNA vector, a linearized DNA vector, or a deoxyribonucleic acid described herein, such as a cDNA. In some embodiments, the linear polyribonucleotide is transcribed from the deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).

[0281] In another example, circular polyribonucleotides can be produced in a cell, for example, a prokaryotic or eukaryotic cell. In some embodiments, an exogenous polyribonucleotide is provided to the cell (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding the transcription of a linear polyribonucleotide described herein). The linear polyribonucleotide can be transcribed within the cell from an exogenous DNA molecule provided to the cell. The linear polyribonucleotide can be transcribed within the cell from an exogenous recombinant DNA molecule transiently provided to the cell. In some embodiments, the exogenous DNA molecule is not integrated into the genome of the cell. In some embodiments, the linear polyribonucleotide is transcribed within the cell from a recombinant DNA molecule that is integrated into the genome of the cell.

[0282] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell comprising the polyribonucleotide described herein may be a bacterial cell or an archaeal cell. For example, prokaryotic cells comprising the polyribonucleotides described herein can be selected from the group consisting of E. coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis, Bacillus anthracis, Bacillus subtil ... The prokaryotic cell may be Bacillus anthracis, Bacillus cereus, betaproteobacteria species (e.g., Burkholderia), alphaproteobacterial species (e.g., Agrobacterium), Pseudomonas species (e.g., Pseudomonas putida), and enterobacteria. The prokaryotic cell may be grown in a medium. The prokaryotic cell may be introduced into a bioreactor.

[0283] The cell may be a eukaryotic cell. In some embodiments, the eukaryotic cell is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic organism is a unicellular fungal cell, such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces species, Brettanomyces species, Schizosaccharomyces species, Torulaspora species, and Pichia species). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. The unicellular animal cell may be a cell isolated from a multicellular animal and then grown in culture, or a daughter cell thereof. In some embodiments, the unicellular animal cell may be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. The unicellular plant cell may be a cell isolated from a multicellular plant and then grown in culture, or a daughter cell thereof. In some embodiments, the unicellular plant cell may be dedifferentiated. In some embodiments, the unicellular plant cell is derived from a plant callus. In some embodiments, the unicellular organism cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algae cell, such as a unicellular green alga, a diatom, a euglenoid, or a dinoflagellate.Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris species, Protosiphon botryoides, Botryococcus braunii, Cryptococcus species, Chlamydomonas reinhardtii and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cell.

[0284] In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryotic organism. For example, the multicellular eukaryotic organism may be selected from the group consisting of a vertebrate, an invertebrate, a multicellular fungus, a multicellular alga, and a multicellular plant. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryotic organism is an invertebrate. In some embodiments, the eukaryotic organism is a multicellular fungus. In some embodiments, the eukaryotic organism is a multicellular plant. In some embodiments, the eukaryotic cell is a human cell or a cell of a non-human mammal, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., bovine, including cow, buffalo, bison, sheep, goat, and muskox; pig; camelid, including camel, llama, and alpaca; deer, antelope; and equine, including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare). In some embodiments, the eukaryotic cell is a cell of an avian species, e.g., a member of the avian order Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleaognathae (e.g., ostrich, emu), Columbiformes (e.g., pigeon, dove), or Psittaciformes (e.g., parrot). In some embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, worm, or mollusc. In some embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm (which may be a dicotyledonous or monocotyledonous plant), a gymnosperm (e.g., a conifer, a cycad, a ginkgo), a fern, a horsetail, a club moss, or a bryophyte, etc. In several embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular alga.

[0285] The eukaryotic cells can be grown in a culture medium. The eukaryotic cells may be contained in a bioreactor.

[0286] Examples of bioreactors include, but are not limited to, stirred tank (e.g., well mixed) and flat plate (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibratory mixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of a bioreactor may be a batch or continuous process. A bioreactor is continuous if the streams of reagents and products are continuously fed and removed from the system. A batch bioreactor may have a continuous recirculation flow, but no continuous feed of reagents or product harvest.

[0287] Some methods of the present disclosure relate to large-scale production of cyclic polyribonucleotides. For large-scale production methods, the methods can be carried out in volumes ranging from 1 liter (L) to 50 L or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more). In some embodiments, the method can be carried out in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L. In some embodiments, the bioreactor is capable of producing at least 1 g of circular RNA. In some embodiments, a bioreactor can produce 1-200 g of circular RNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, or 50-200 g of circRNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200 g per liter), per batch or reaction (e.g., 1-200 g per batch or reaction), or per unit of time (e.g., 1-200 g per hour or day). In some embodiments, two or more bioreactors can be utilized in series to increase production capacity (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 bioreactors can be used in series).

[0288] Methods for producing circRNAs described herein are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); Muller and Appel, from RNA Biol, 2017, 14(8):1018-1027; and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012). Other methods for producing cyclic polyribonucleotides are described, for example, in WO 2022 / 247943, U.S. Pat. No. 11000547, WO 2018 / 191722, WO 2019 / 236673, WO 2020 / 023595, WO 2022 / 204460, WO 2022 / 204464, and WO 2022 / 204466.

[0289] Various methods for synthesizing circRNAs have been described elsewhere (e.g., U.S. Pat. Nos. 6,210,931, 5,773,244, 5,766,903, 5,712,128, 5,426,180, U.S. Patent Publication No. 20100137407, WO 1992001813, WO 2010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015); the contents of each of which are incorporated herein by reference in their entirety).

[0290] Circular Polyribonucleotides The present disclosure features circRNA compositions and methods for making and purifying circRNA. In some embodiments, circRNA is produced from linRNA (e.g., by methods known in the art, including by enzymatic ligation or autocatalytic RNA). In some embodiments, linRNA is transcribed from a deoxyribonucleotide template (e.g., a vector, a linearized vector, or a cDNA).

[0291] circRNAs may include features such as one or more coding sequences, one or more non-coding sequences, or a combination thereof. CircRNAs may include one or more coding sequences, such as, for example, a coding sequence that codes for the expression of a polypeptide. Each coding sequence may be operably linked to an internal ribosome entry site (IRES) or one or more regulatory sequences, or a combination thereof. CircRNAs may include one or more non-coding sequences, such as a non-coding sequence that specifically binds to a target, such as a protein or a nucleic acid. Characteristics of circRNAs are described, for example, in International Patent Publication Nos. 2019 / 118919, 2020 / 023655, 2020 / 180751, 2020 / 180752, 2020 / 181013, 2020 / 198403, 2020 / 257730, 2020 / 257727, and 2020 / 252436, each of which is incorporated by reference with respect to the circRNAs described therein.

[0292] The size of the circRNA to be purified may be any size suitable for the purification methods disclosed herein.

[0293] For example, the circRNA to be purified may have a length of at least 20,000 nucleotides, at least 19,000 nucleotides, at least 18,000 nucleotides, at least 17,000 nucleotides, at least 16,000 nucleotides, at least 15,000 nucleotides, at least 14,000 nucleotides, at least 13,000 nucleotides, at least 12,000 nucleotides, at least 11,000 nucleotides, at least 10,000 nucleotides, at least 9,000 nucleotides, at least 8,000 nucleotides, at least 7,000 nucleotides, at least 6,000 nucleotides, at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 900 nucleotides, at least 800 nucleotides, at least 700 nucleotides, at least 600 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, or at least 100 nucleotides.

[0294] For example, the circRNA to be purified may be less than about 20,000 nucleotides, less than about 19,000 nucleotides, less than about 18,000 nucleotides, less than about 17,000 nucleotides, less than about 16,000 nucleotides, less than about 15,000 nucleotides, less than about 14,000 nucleotides, less than about 15,000 nucleotides, less than about 14,000 nucleotides, less than about 13,000 nucleotides, less than about 12,000 nucleotides, less than about 11,000 nucleotides, less than about 10,000 nucleotides, less than about 9,000 nucleotides, less than about It may have a length of less than 8,000 nucleotides, less than about 7,000 nucleotides, less than about 6,000 nucleotides, less than about 5,000 nucleotides, less than about 4,000 nucleotides, less than about 3,000 nucleotides, less than about 2,000 nucleotides, less than about 1,000 nucleotides, less than about 900 nucleotides, less than about 800 nucleotides, less than about 700 nucleotides, less than about 600 nucleotides, less than about 500 nucleotides, less than about 400 nucleotides, less than about 300 nucleotides, less than about 200 nucleotides, or less than about 100 nucleotides.

[0295] For example, the circRNA to be purified may be from about 100 nucleotides to about 20,000 nucleotides (e.g., from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 750 nucleotides, from about 100 nucleotides to about 1,000 nucleotides, from about 100 nucleotides to about 2,500 nucleotides, from about 100 nucleotides to about 5,000 nucleotides, from about 100 nucleotides to about 10,000 nucleotides, from about 500 nucleotides to about 750 nucleotides, from about 750 nucleotides to about about 1,000 nucleotides, about 750 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 1,250 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides to about 5,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000 nucleotides to about 15,000 nucleotides, about 10,000 nucleotides to about 15,000 nucleotides, About 15,000 nucleotides to about 20,000 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1,000 nucleotides, about 1,250 nucleotides, about 1,500 nucleotides, about 1,750 nucleotides, about 2,000 nucleotides, about 3,000 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides nucleotides, about 6,000 nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about 9,000 nucleotides, about 10,000 nucleotides, about 11,000 nucleotides, about 12,000 nucleotides, about 13,000 nucleotides, about 14,000 nucleotides, about 15,000 nucleotides, about 16,000 nucleotides, about 17,000 nucleotides, about 18,000 nucleotides, about 19,000 nucleotides, or about 20,000 nucleotides in length.

[0296] According to the methods disclosed herein, purification of circRNAs from a mixed population of polyribonucleotides can be performed on any type of circRNA, including single- or double-stranded circRNAs, circRNAs with and without secondary structure, labeled or unlabeled circRNAs (e.g., fluorescently labeled, radioactively labeled, antibody labeled, etc., among others).

[0297] The circRNA selected for purification may contain naturally occurring DNA or RNA nucleosides (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine), or the circRNA may contain non-naturally occurring (i.e., modified) nucleobases, internucleoside linkages, or sugars. In some embodiments, the circRNA selected for purification contains naturally occurring nucleosides. If the circRNA is modified, it may contain modified ribonucleosides (e.g., containing modified nucleobases or modified ribose moieties) or modified internucleoside linkages (e.g., phosphorothioates and phosphoramidates, among others). Generally, such modifications are incorporated to stabilize the RNA molecule and reduce hydrolysis by nucleases. Modifications of circRNAs specifically contemplated by the present disclosure include the nucleobase modifications described in WO2020 / 198403.

[0298] In some embodiments, substantially all of the nucleosides or internucleoside linkages of the disclosed circRNA are modified nucleosides. In some embodiments, all of the nucleosides or internucleoside linkages of the disclosed circRNA are modified nucleosides. A circRNA in which "substantially all nucleosides are modified nucleosides" is mostly, but not entirely, modified and may contain no more than 5, 4, 3, 2, or 1 naturally occurring nucleosides. In some embodiments, a circRNA may contain no more than 5, 4, 3, 2, or 1 alternative nucleosides. In some embodiments, modified circRNAs comprise at least one (e.g., at least 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000 or more) modified nucleosides. The aforementioned modifications, including modifications to the nucleobase, sugar moiety, or internucleoside linkages, can be incorporated into the circRNAs described herein to the extent that they preserve the information encoded in the unmodified RNA sequence (e.g., the amino acids encoded by each codon) and do not interfere with protein translation.

[0299] Polypeptide Expression Sequences In some embodiments, the polyribonucleotides described herein comprise one or more expression sequences, where each expression sequence encodes a polypeptide. In some embodiments, the circular polyribonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more expression sequences.

[0300] Each encoded polypeptide may be linear or branched. The polypeptide may have a length of about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, polypeptides have a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less that may be useful.

[0301] The polypeptides included herein may include naturally occurring or non-naturally occurring polypeptides. In some examples, the polypeptide is or includes a functional fragment or variant of a reference polypeptide (e.g., an enzymatically active fragment or variant of an enzyme). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a polypeptide described herein or a naturally occurring polypeptide, for example, over a specific region or over the entire sequence. In some cases, a polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a protein of interest.

[0302] Some examples of polypeptides include, but are not limited to, a fluorescent tag or marker, an antigen, a therapeutic polypeptide, or a polypeptide for agricultural use.

[0303] The therapeutic polypeptide may be a hormone, a neurotransmitter, a growth factor, an enzyme (e.g., oxidoreductases, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, desaturases), a cytokine, an antigen-binding polypeptide (e.g., an antigen-binding antibody or antibody-like fragment, such as a single chain antibody, nanobody or other polypeptide comprising an Ig heavy or light chain), an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an interferon, an interleukin, or a thrombolytic agent.

[0304] The polypeptide for agricultural use may be a bacteriocin, a lysin, an antibacterial polypeptide, an antifungal polypeptide, a nodule C-rich peptide, a bacteriocyte regulatory peptide, a peptide toxin, a pesticidal polypeptide (e.g., an insecticidal or nematicidal polypeptide), an antigen-binding polypeptide (e.g., an antigen-binding antibody or antibody-like fragment, such as a single chain antibody, nanobody or other Ig heavy or light chain-containing polypeptide), an enzyme (e.g., a nuclease, amylase, cellulase, peptidase, lipase, chitinase), a peptide pheromone, or a transcription factor.

[0305] In some cases, the polyribonucleotide expresses a human protein. In some cases, the polyribonucleotide expresses a non-human protein.

[0306] In some embodiments, the polyribonucleotide expresses an antibody, such as an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the cyclic polyribonucleotide can be any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. In some embodiments, the cyclic polyribonucleotide expresses a portion of an antibody, such as a light chain, a heavy chain, an Fc fragment, a CDR (complementarity determining region), an Fv fragment, or a Fab fragment, or further portions thereof. In some embodiments, the cyclic polyribonucleotide expresses one or more portions of an antibody. For example, the cyclic polyribonucleotide can include two or more expression sequences, each of which expresses a portion of an antibody, the sum of which can constitute an antibody. In some cases, the cyclic polyribonucleotide includes one expression sequence that encodes a heavy chain of the antibody, and another expression sequence that encodes a light chain of the antibody. In some cases, when the cyclic polyribonucleotide is expressed in a cell or in a cell-free environment, the light and heavy chains can undergo appropriate modification, folding, or other post-translational modifications to form a functional antibody.

[0307] In embodiments, the polypeptide comprises multiple polypeptides, e.g., multiple copies of a single polypeptide sequence, or multiple different polypeptide sequences, In embodiments, the multiple polypeptides are connected by linker or spacer amino acids.

[0308] In some embodiments, the expressed sequence includes a polyA sequence (e.g., at the 3' end of the expressed sequence). In some embodiments, the length of the polyA sequence is more than 10 nucleotides in length. In one embodiment, the polyA sequence is more than 15 nucleotides in length (e.g., is at least or more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides). In some embodiments, the polyA sequence is designed according to the description of polyA sequences in paragraphs

[0202] to

[0204] of WO 2019 / 118919 A1, the entirety of which is incorporated herein by reference. In some embodiments, the expression sequence lacks a polyA sequence (e.g., at the 3' end of the expression sequence).

[0309] In some embodiments, the cyclic polyribonucleotide comprises a polyA, lacks a polyA, or has a modified polyA to modulate one or more characteristics of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacks a polyA or has a modified polyA to improve one or more functional properties, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency.

[0310] Internal ribosome entry sites In some embodiments, the polyribonucleotide comprises one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence.

[0311] IRES elements suitable for inclusion within a polyribonucleotide comprise an RNA sequence capable of associating with a eukaryotic ribosome, hi some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0312] In some embodiments, the IRES element is derived from DNA of an organism, including, but not limited to, a virus, a mammal, or a Drosophila. Such viral DNA can be derived from, but is not limited to, EMCV cDNA and poliovirus cDNA, as well as picornavirus complementary DNA (cDNA). In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster.

[0313] In some embodiments, if present, the IRES sequence is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invictavirus 1, Rhopalopsis aphid virus, Reticuloendotheliosis virus, Human poliovirus 1, German winged stink bug enteric virus, Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Tea budworm picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila melanogaster C virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen brood virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus (AEV), Acute bee paralysis virus, Hibiscus chlorotic ringspot virus virus), classical swine fever virus, human FGF2, human SFTPAl, human AMLl / RUNXl, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, dog Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Salivirus, cosavirus, parechovirus, Drosophila hairless, yeast (S. cerevisiae) TFIID, yeast (S.cerevisiae YAP1, human c-src, human FGF-1, picomavirus, Turnip crinkle virus, Aichi virus, Krohi virus, Echovirus 11, aptamer for eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2) IRES sequence. In yet another embodiment, the IRES is a Coxsackievirus B3 (CVB3) IRES sequence. In a further embodiment, the IRES is an encephalomyocarditis virus (EMCV) IRES sequence. In yet another embodiment, the IRES is a Theiler's encephalomyelitis virus IRES sequence.

[0314] In some embodiments, the IRES sequence has a modified sequence compared to the wild-type IRES sequence. In some embodiments, if the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified so that it is a cytosine residue. For example, in some embodiments, the IRES sequence is a CVB3 IRES sequence with the terminal adenosine residue modified to a cytosine residue. In some embodiments, the IRES sequence is an Enterovirus 71 (EV17) IRES with the terminal guanosine residue modified to a cytosine residue. In some embodiments, the polyribonucleotide comprises at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expressed sequence. In some embodiments, the IRES flanks at least one (e.g., 2, 3, 4, 5 or more) expressed sequence on both sides. In some embodiments, the polyribonucleotide comprises one or more IRES sequences on one or both sides of each expressed sequence, resulting in the separation of the resulting peptides and / or polypeptides.

[0315] In some embodiments, a polyribonucleotide described herein comprises an IRES (eg, an IRES operably linked to a coding region). For example, polyribonucleotides are described in the following: Chen et al. Mol. Cell 81(20):4300-4318,2021; Jopling et al. Oncogene 20:2664-2670,2001; Baranick et al. PNAS 105(12):4733-4738,2008; Lang et al. Molecular Biology of the Cell 13(5):1792-1801,2002; Dorokhov et al. PNAS 99(8):5301-5306,2002; Wang et al. Nucleic Acids Research 33(7):2248-2258,2005; Petz et a. Nucleic Acids Research 35(8):2473-2482,2007; Chen et al. Science 268:415-417, 1995; Fan et al. Nature Communication 13(1):3751-3765, 2022, and WO 2021 / 263124, each of which is incorporated by reference in its entirety into this specification.

[0316] Adjustment element In some embodiments, the polyribonucleotides described herein include one or more regulatory elements. In some embodiments, the polyribonucleotides include regulatory elements, such as sequences that regulate the expression of an expression sequence within the polyribonucleotide.

[0317] A regulatory element may comprise a sequence located adjacent to an expression sequence that codes for an expression product. The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of the expressed product compared to the amount or number of products expressed in the absence of the regulatory element. Furthermore, one regulatory element may increase the amount of the expressed product for multiple expression sequences linked side by side. Thus, one regulatory element can promote the expression of one or more expression sequences. Multiple regulatory elements can also be used, for example, to differentially regulate the expression of different expression sequences.

[0318] In some embodiments, the regulatory element is a translation modulator. The translation modulator may regulate the translation of an expressed sequence in the polyribonucleotide. The translation modulator may be a translation enhancer or suppressor. In some embodiments, the polyribonucleotide comprises at least one translation modulator adjacent to at least one expressed sequence. In some embodiments, the polyribonucleotide comprises a translation modulator adjacent to each expressed sequence. In some embodiments, the translation modulator is on one or both sides of each expressed sequence, resulting in the separation of the expression products, e.g., peptides and / or polypeptides.

[0319] In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

[0320] Further examples of regulatory elements are described, for example, in paragraphs

[0154] to

[0161] of International Publication No. 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0321] Translation initiation sequence In some embodiments, the polyribonucleotides described herein comprise at least one translation initiation sequence. In some embodiments, the polyribonucleotides comprise a translation initiation sequence operably linked to an expression sequence.

[0322] In some embodiments, the polyribonucleotide may encode a polypeptide and include a translation initiation sequence, e.g., a start codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide includes a translation initiation sequence, e.g., a Kozak sequence, flanking the expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, a translation initiation sequence, e.g., a Kozak sequence, is present on one or both sides of each expression sequence to provide separation of the expression products. In some embodiments, the polyribonucleotide includes at least one translation initiation sequence flanking the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the circular polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] to

[0165] of WO 2019 / 118919, which is incorporated herein by reference in its entirety.

[0323] A polyribonucleotide may comprise two or more start codons, such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may begin at the first start codon or may begin downstream of the first start codon.

[0324] In some embodiments, the polyribonucleotide may initiate at the first start codon, e.g., a codon that is not AUG. Translation of the polyribonucleotide may initiate at an alternative translation start sequence, e.g., but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG. In some embodiments, translation initiates at an alternative translation start sequence under selective conditions, e.g., stress-inducing conditions. As a non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, e.g., ACG. As another non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, CTG / CUG. As another non-limiting example, translation of the polyribonucleotide may initiate at an alternative translation start sequence, GTG / GUG. As another non-limiting example, a polyribonucleotide may initiate translation at alternative translation initiation sequences, including repeat-associated non-AUG (RAN) sequences, e.g., short stretches of repetitive RNA, e.g., CGG, GGGGCC, CAG, CTG.

[0325] Termination sequence In some embodiments, the polyribonucleotide described herein comprises at least one termination sequence. In some embodiments, the polyribonucleotide comprises a termination sequence operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination sequence.

[0326] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence. In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence, such that the polyribonucleotide is continuously translated. Exclusion of the termination sequence may result in rolling circle translation or continuous expression of the expression product.

[0327] In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination sequence. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may lack a termination sequence, such that the circular polyribonucleotide is translated continuously. The exclusion of the termination sequence may result in rolling circle translation or continuous expression of expression products, such as peptides or polypeptides, due to lack of ribosome stalling or shedding. In such embodiments, the rolling circle translation expresses continuous expression products through each expression sequence. In some other embodiments, the termination sequence of the expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the circular polyribonucleotide include a termination sequence. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the circular polyribonucleotide is performed. In such cases, the expression product may be shedding from the ribosome when the ribosome encounters a termination sequence, such as a stop codon, and terminates translation. In some embodiments, translation is terminated while the ribosome, eg, at least one subunit of the ribosome, remains in contact with the circular polyribonucleotide.

[0328] In some embodiments, the circular polyribonucleotide comprises a termination sequence at the end of one or more expressed sequences. In some embodiments, the one or more expressed sequences comprise two or more subsequent termination sequences. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome is completely separated from the circular polyribonucleotide. In some such embodiments, the generation of the subsequent (e.g., second, third, fourth, fifth, etc.) expressed sequences in the circular polyribonucleotide may require the ribosome to reassociate with the circular polyribonucleotide before the start of translation. Generally, the termination sequence comprises an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, that signals the termination of translation. In some embodiments, one or more termination sequences in the circular polyribonucleotide are frame-shifted termination sequences, such as, but not limited to, off-frame or -1 and +1 shifted reading frames (e.g., cryptic stops), that may terminate translation. Frameshifted termination sequences include the triple nucleotides TAA, TAG, and TGA that occur in the second and third reading frames of the expressed sequence.Frameshifted termination sequences can be important to prevent misreading of mRNA, which is often harmful to cells.In some embodiments, the termination sequence is a stop codon.

[0329] Further examples of termination sequences are described in paragraphs

[0169] to

[0170] of International Publication No. 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0330] Untranslated Regions In some embodiments, the circular polyribonucleotide comprises an untranslated region (UTR). The UTR of a genomic region that comprises a gene may be transcribed but not translated. In some embodiments, the UTR may be included upstream of the translation initiation sequence of the expression sequence described herein. In some embodiments, the UTR may be included downstream of the expression sequence described herein. In some cases, one UTR for a first expression sequence is the same as, contiguous with, or overlaps with another UTR for a second expression sequence. In some embodiments, the intron is a human intron.

[0331] Exemplary untranslated regions are described in paragraphs

[0197] to

[0201] of International Publication No. 2019 / 118919, the entirety of which is incorporated herein by reference.

[0332] In some embodiments, the cyclic polyribonucleotide comprises a polyA sequence. Exemplary polyA sequences are described in paragraphs

[0202] to

[0205] of WO 2019 / 118919, the entirety of which is incorporated herein by reference. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence.

[0333] In some embodiments, the cyclic polyribonucleotide comprises a UTR that contains one or more stretches of adenosines and uridines. These AU-rich signatures may increase the turnover rate of the expression product.

[0334] The introduction, removal, or modification of AU-rich elements (AREs) in the UTRs can be useful for modulating the stability or immunogenicity (e.g., the level of one or more markers of immune or inflammatory response) of a cyclic polyribonucleotide. When modifying a particular cyclic polyribonucleotide, one or more copies of an ARE may be introduced into the cyclic polyribonucleotide, and the copies of the ARE may modulate the translation and / or production of the expression product. Similarly, AREs can be identified and removed or modified into a cyclic polyribonucleotide to modulate the intracellular stability, which in turn affects the translation and production of the resulting protein.

[0335] It should be understood that any UTR from any gene may be incorporated into each flanking region of the circular polyribonucleotide.

[0336] In some embodiments, the circular polyribonucleotide lacks a 5'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 3'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a cap and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5'UTR, a 3'UTR, and an IRES and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic, a regulatory element (e.g., a translation modulator, e.g., a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids (e.g., siRNA, lncRNA, shRNA) targeting an endogenous gene, and a sequence encoding a therapeutic mRNA or protein.

[0337] In some embodiments, the cyclic polyribonucleotide lacks a 5' UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3' UTR. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination sequence. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks susceptibility to degradation by exonucleases. In some embodiments, the fact that the cyclic polyribonucleotide lacks susceptibility to degradation can mean that the cyclic polyribonucleotide is not degraded by exonucleases or is degraded to a limited extent in the presence of exonucleases, e.g., is equivalent or similar in the absence of exonucleases. In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, the cyclic polyribonucleotide has reduced degradation when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks binding to cap-binding proteins. In some embodiments, the circular polyribonucleotide lacks a 5' cap.

[0338] Stagger Element In some embodiments, the cyclic polyribonucleotide comprises at least one stagger element adjacent to the expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element adjacent to each expressed sequence. In some embodiments, the stagger element is on one or both sides of each expressed sequence, resulting in separation of the expression products, e.g., peptides and / or polypeptides. In some embodiments, the stagger element is a portion of one or more expressed sequences. In some embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences, each of which is separated from the subsequent expressed sequence by a stagger element in the cyclic polyribonucleotide. In some embodiments, the stagger element prevents the generation of a single polypeptide from (a) two translations of a single expressed sequence, or (b) one or more translations of two or more expressed sequences. In some embodiments, the stagger element is a sequence separated from one or more expressed sequences. In some embodiments, the stagger element comprises a portion of an expressed sequence of one or more expressed sequences.

[0339] Non-coding sequences In some embodiments, the polyribonucleotides described herein include one or more non-coding sequences, e.g., sequences that do not code for expression of a polypeptide. In some embodiments, the polyribonucleotides include two, three, four, five, six, seven, eight, nine, ten or more non-coding sequences. In some embodiments, the polyribonucleotides do not code for a polypeptide expression sequence.

[0340] The non-coding sequence may be a natural or synthetic sequence. In some embodiments, the non-coding sequence may modify cell behavior, such as lymphocyte behavior. In some embodiments, the non-coding sequence is antisense to a cellular RNA sequence.

[0341] In some embodiments, the polyribonucleotide comprises a regulatory nucleic acid that is typically an RNA or RNA-like structure of about 5-500 base pairs (bp) (depending on the particular RNA structure (e.g., miRNA of 5-30 bp, lncRNA of 200-500 bp) and may have a nucleobase sequence that is identical (complementary) or nearly identical (substantially complementary) to a coding sequence in an expressed target gene in a cell. In embodiments, the circular polyribonucleotide comprises a regulatory nucleic acid that encodes an RNA precursor, e.g., a miRNA precursor, that is processable into a smaller RNA, which may be about 50 to about 1000 bp, that is processable into a smaller miRNA intermediate or mature miRNA.

[0342] Long non-coding RNA (IncRNA) is defined as a non-protein-coding transcript longer than 100 nucleotides. Many IncRNAs are characterized as tissue-specific. Diverse IncRNAs that are transcribed in the opposite direction to nearby protein-coding genes comprise a significant proportion (e.g., about 20% of all IncRNAs in mammalian genomes) and likely regulate the transcription of nearby genes. In one embodiment, the polyribonucleotide provided herein comprises the sense strand of an IncRNA. In one embodiment, the polyribonucleotide provided herein comprises the antisense strand of an IncRNA.

[0343] Protein Binding Sequences In some embodiments, the cyclic polyribonucleotide comprises one or more protein binding sites that allow proteins, such as ribosomes, to bind to internal sites within the RNA sequence.By designing a protein binding site, such as a ribosome binding site, into the cyclic polyribonucleotide, the cyclic polyribonucleotide can evade or reduce detection by the host's immune system by masking the cyclic polyribonucleotide from components of the host's immune system, regulating degradation, or regulating translation.

[0344] In some embodiments, the cyclic polyribonucleotide comprises at least one immunity protein binding site, e.g., to evade an immune response, e.g., a cytotoxic T lymphocyte (CTL) response. In some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and aids in masking the cyclic polyribonucleotide as exogenous. In some embodiments, the immunity protein binding site is a nucleotide sequence that binds to an immunity protein and aids in masking the cyclic polyribonucleotide as exogenous or foreign.

[0345] The traditional mechanism of ribosome association to linear RNA involves ribosome binding to the capped 5' end of the RNA. The first peptide bond is formed as soon as the ribosome moves from the 5' end to the start codon. According to the present disclosure, internal initiation of translation of a circular polyribonucleotide (i.e., cap-independent) does not require a free or capped end. Rather, the ribosome binds to an uncapped internal site, whereby the ribosome initiates polypeptide elongation at the start codon. In some embodiments, the circular polyribonucleotide comprises one or more RNA sequences that include a ribosome binding site, e.g., a start codon.

[0346] Natural 5'UTRs have characteristics that play a role in translation initiation. They contain signatures such as the Kozak sequence, which is commonly known to be involved in the process by which the ribosome initiates the translation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0347] In some embodiments, the cyclic polyribonucleotide encodes a protein binding sequence that binds to a protein. In some embodiments, the protein binding sequence targets or localizes the cyclic polyribonucleotide to a specific target. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a protein.

[0348] In some embodiments, the protein binding sites include, but are not limited to, ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, EL AVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28B , m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBFOX 2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF 1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1, or any other protein that binds RNA.

[0349] How to use In some embodiments, polyribonucleotides (e.g., circular polyribonucleotides) generated as described herein are used as effectors in therapy or agriculture.

[0350] For example, polyribonucleotides purified by the methods described herein may be administered to a subject (e.g., in a pharmaceutical, veterinary, or agricultural composition). In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In embodiments, the subject is an avian, such as a member of an avian taxonomic group such as Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is an invertebrate, such as an arthropod (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks. In embodiments, the subject is an invertebrate that is an invertebrate agricultural pest or a parasite on an invertebrate or vertebrate host. In embodiments, the subject is a plant, such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifers, cycads, Gnetophytes, ginkgo), fern, horsetail, club moss, or bryophyte. In embodiments, the subject is a eukaryotic alga (unicellular or multicellular). In embodiments, the subject is an agriculturally or horticulturally important plant, such as row crops, fruit-bearing plants and trees, vegetables, trees, and ornamentals, e.g., ornamental flowers, shrubs, trees, ground covers, and turf.

[0351] In some embodiments, the disclosure provides a method of modifying a subject by providing a composition or preparation described herein to the subject. In some embodiments, the composition or preparation is or comprises a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell that contains a nucleic acid described herein.

[0352] In some embodiments, the disclosure provides a method of treating a condition in a subject in need thereof by providing to the subject a composition or preparation described herein. In some embodiments, the composition or preparation is or comprises a nucleic acid molecule (e.g., a DNA molecule or polyribonucleotide described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is or comprises a eukaryotic or prokaryotic cell that contains a nucleic acid described herein.

[0353] In some embodiments, the present disclosure provides methods of providing a polyribonucleotide (e.g., a circular polyribonucleotide) to a subject by providing to a subject a eukaryotic or prokaryotic cell that comprises a polynucleotide described herein.

[0354] preparation In some embodiments of the present disclosure, the polyribonucleotides described herein (e.g., cyclic polyribonucleotides) or preparations thereof prepared by the present methods can be formulated in compositions, e.g., agricultural, veterinary, or pharmaceutical compositions, for delivery to a cell, a plant, an invertebrate, a non-human vertebrate, or a human subject. In some embodiments, the polyribonucleotides are formulated in pharmaceutical compositions. In some embodiments, the compositions include polyribonucleotides and a diluent, carrier, adjuvant, or combinations thereof. In certain embodiments, the compositions include polyribonucleotides described herein and a carrier or a diluent without any carrier. In some embodiments, a composition including polyribonucleotides and a diluent without any carrier is used for naked delivery of polyribonucleotides (e.g., cyclic polyribonucleotides) to a subject.

[0355] The pharmaceutical composition may optionally include one or more additional active substances, e.g., therapeutic and / or prophylactic active substances. The pharmaceutical composition may optionally include an inert substance that serves as a vehicle or medium for the composition described herein (e.g., a cyclic polyribonucleotide, e.g., a composition that includes any one of the inert ingredients approved by the United States Food and Drug Administration (FDA) and listed in the inactive ingredient database). The pharmaceutical composition of the present invention may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersion agents, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersing agents, granulating agents, disintegrating agents, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof.

[0356] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans that make the compositions suitable for administration to various animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or implement such modifications with no more than routine (if possible) experimentation. Subjects to which the pharmaceutical compositions are intended to be administered include, but are not limited to, humans and / or other primates; mammals, including commercially important mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially important birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0357] The preparations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, forming and / or packaging the product.

[0358] In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the preparation is 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL of linear polyribonucleotide molecules. , 200ng / mL, 300ng / mL, 400ng / mL, 500ng / mL, 600ng / mL, 1μg / ml, 10μg / mL, 50μg / mL, 100μg / mL, 200g / mL, 300μg / mL, 400μg / mL, 500μg / mL, 600μg / mL, 700μg / mL, 800μg / mL, 900μg / mL, 1mg / mL, 1.5mg / ml, or 2mg / ml or less.

[0359] In some embodiments, a reference standard for the amount of circular polyribonucleotide molecules present in a preparation is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 100% (w / w), 110% (w / w), 120% (w / w), 130% (w / w), 140% (w / w), 150% (w / w), 160% (w / w), 170% (w / w), 180% (w / w), 190% (w / w), 200% (w / w), 21% (w / w), 22% (w / w), 23% (w / w), 24% (w / w), 25% (w / w), 26% (w / w), 27% (w / w), 28% (w / w), 29% (w / w), 30% (w / w), 31% (w / w), 32% (w / w), 33% (w / w), 34% (w / w), 35% (w / w), 36% (w / w), 37% (w / w), 38% (w / w), 39% (w / w), 40% (w / w), 40% (w / w), 40% (w / w), 40% (w / w), 40% (w / w), 40% (w / w 94%(w / w), 95%(w / w), 96%(w / w), 97%(w / w), 98%(w / w), 99%(w / w), 99.1%(w / w), 99.2%(w / w), 99.3%(w / w), 99.4%(w / w), 99.5%(w / w), 99.6%(w / w), 99.7%(w / w), 99.8%(w / w), 99.9%(w / w) or 100%(w / w) of the molecule.

[0360] In some embodiments, a reference standard for the amount of linear polyribonucleotide molecules present in a preparation is less than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) or less linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation.

[0361] In some embodiments, a reference standard for the amount of nicked polyribonucleotide molecules present in a preparation is no more than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), or 15% (w / w) of nicked polyribonucleotide molecules of total ribonucleotide molecules in the pharmaceutical preparation.

[0362] In some embodiments, the reference standard for the amount of combination of nicks and linear polyribonucleotide molecules present in the preparation is 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) or less of combination of nicks and linear polyribonucleotide molecules of the total ribonucleotide molecules in the pharmaceutical preparation. In some embodiments, the pharmaceutical preparation is an intermediate pharmaceutical preparation of the final circular polyribonucleotide formulation. In some embodiments, the pharmaceutical preparation is a bulk drug or active pharmaceutical ingredient (API). In some embodiments, the pharmaceutical preparation is a formulation for administration to a subject.

[0363] In some embodiments, the preparation of circular polyribonucleotides is further processed (before, during or after reduction of linear RNA) to substantially remove DNA, protein contaminants (e.g., cellular proteins such as host cell proteins or process impurity proteins), endotoxins, mononucleotide molecules, and / or process-related impurities.

[0364] salt In some cases, the compositions or pharmaceutical compositions provided herein include one or more salts. To control osmolality, physiological salts such as sodium salts can be included in the compositions provided herein. Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, and the like. In some cases, the compositions are formulated with one or more pharma- ceutically acceptable salts. The one or more pharma-ceutically acceptable salts can include those of inorganic ions, such as, for example, sodium, potassium, calcium, and magnesium ions. Such salts can include salts of inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. Polyribonucleotides can exist in either linear or cyclic form.

[0365] Buffer / pH The compositions or pharmaceutical compositions provided herein may include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (e.g., with aluminum hydroxide adjuvant), or citrate buffer, etc. The buffers are in some cases within the range of 5-20 mM.

[0366] The compositions or pharmaceutical compositions provided herein can have a pH between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8. The compositions or pharmaceutical compositions can have a pH of about 7. Polyribonucleotides can exist in either linear or circular form.

[0367] Diluent In some embodiments, a composition of the present disclosure comprises a polyribonucleotide, or a preparation thereof prepared by a method described herein, and a diluent.

[0368] The diluent may be a non-carrier excipient. The non-carrier excipient serves as a vehicle or medium for the composition, such as the cyclic polyribonucleotide described herein. The non-carrier excipient serves as a vehicle or medium for the composition, such as the linear polyribonucleotide described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonicity agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, or mixtures thereof. The non-carrier excipient may be any of the inactive ingredients listed in the Inactive Ingredient Database that are approved by the United States Food and Drug Administration (FDA) and do not exhibit cell-penetrating effects. A non-carrier excipient can be, for example, any inactive ingredient suitable for administration to a non-human animal suitable for veterinary use. Modifications of compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood and a veterinary pharmacologist of ordinary skill can design and / or make such modifications with only routine experimentation, if any.

[0369] In certain embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) can be delivered as naked delivery formulations, such as those that include a diluent. Naked delivery formulations deliver polyribonucleotides to cells without a carrier and without modification or partial or complete encapsulation of polyribonucleotides, capped polyribonucleotides, or complexes thereof.

[0370] A naked delivery formulation is a formulation that does not contain a carrier, and the polyribonucleotide (e.g., cyclic polyribonucleotide) does not have a covalent modification attached to a moiety that aids in delivery to a cell, or is without partial or complete encapsulation of the polyribonucleotide. In some embodiments, the polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell is a polyribonucleotide that is not covalently attached to a protein, small molecule, particle, polymer, or biopolymer. The polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell does not contain a modified phosphate group. For example, the polyribonucleotide that does not have a covalent modification attached to a moiety that aids in delivery to a cell does not contain a phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoroamidate, phosphorodiamidate, alkyl or aryl phosphonate, or phosphotriester.

[0371] In some embodiments, the naked delivery formulation does not include any or all of a transfection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier. In certain embodiments, the naked delivery formulation is selected from the group consisting of phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydrous modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3 -(2-Hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

[0372] In some embodiments, the naked delivery formulation comprises a non-carrier excipient. In some embodiments, the non-carrier excipient comprises an inactive ingredient that does not exhibit a cell-penetrating effect. In some embodiments, the non-carrier excipient comprises a buffer, such as PBS. In some embodiments, the non-carrier excipient is a solvent, a non-aqueous solvent, a diluent, a suspending aid, a surfactant, an isotonicity agent, a thickener, an emulsifier, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersant, a granulating agent, a disintegrant, a binder, a buffer, a lubricant, or an oil.

[0373] In some embodiments, the naked delivery formulation comprises a diluent. The diluent can be a liquid diluent or a solid diluent. In some embodiments, the diluent is an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, or 2-propanol. Examples of buffers include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of isotonicity agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

[0374] Lipid Nanoparticles The compositions, methods, and delivery systems provided by the present disclosure may use any suitable carrier or delivery modality, including, in certain embodiments, lipid nanoparticles (LNPs) as described herein. The lipid nanoparticles, in certain embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or amphoteric lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers as described in Table 5 of International Publication No. WO2019217941, which is incorporated herein by reference in its entirety); and one or more sterols (e.g., cholesterol).

[0375] Lipids (e.g., lipid nanoparticles) that can be used in nanoparticle formation include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference - for example, a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941. The lipid nanoparticle can include additional elements, such as a polymer, for example, a polymer described in Table 5 of WO2019217941, which is incorporated by reference.

[0376] In certain embodiments, the conjugated lipid, if present, is selected from the group consisting of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium, Additional exemplary PEG-lipid conjugates may include one or more of the following: PEG-lipid salts, and those described in Table 2 of WO2019051289 (incorporated by reference), or combinations of the above. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, U.S. Patent Publication Nos. 2003 / 0077829 and 2005 / 0175682. , International Publication Nos. 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, 2017 / 0119904, 2018 / 0028664, and WO 2017 / 099823, the contents of all of which are incorporated herein by reference in their entireties.

[0377] In some embodiments, sterols that may be incorporated into the lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or U.S. Patent Publication No. 2010 / 0130588, which are incorporated by reference. Further exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated by reference herein.

[0378] In certain embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of the particles, and a sterol.

[0379] Exemplary ionizable lipids that may be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of International Publication No. WO2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulas: X of US2016 / 0311759; I of US20150376115 or US2016 / 0376224; I, II, or III of US20160151284; I, IA, II, or IIA of US20170210967; No. Ic; U.S. Patent Application Publication No. 2013 / 0178541, No. A; U.S. Patent Application Publication No. 2013 / 0303587 or U.S. Patent Application Publication No. 2013 / 0123338, No. I; U.S. Patent Application Publication No. 2015 / 0141678, No. I; U.S. Patent Application Publication No. 2015 / 0239926, No. II, III, IV, or V; U.S. Patent Application Publication No. 2017 / 0119904, No. I; WO 2017 / 117528, No. I or II; U.S. Patent Application Publication No. 2012 / 0149894 A; U.S. Patent Application Publication No. 2015 / 0057373 A; WO 2013 / 116126 A; U.S. Patent Application Publication No. 2013 / 0090372 A; U.S. Patent Application Publication No. 2013 / 0274523 A; U.S. Patent Application Publication No. 2013 / 0274504 A; U.S. Patent Application Publication No. 2013 / 0053572 A; WO 2013 / 0 16058, A; WO 2012 / 162210, A; U.S. Patent Application Publication No. 2008 / 042973, I; U.S. Patent Application Publication No. 2012 / 01287670, I, II, III, or IV; U.S. Patent Application Publication No. 2014 / 0200257, I or II; U.S. Patent Application Publication No. 2015 / 0203446, I, II, or III; U.S. Patent Application Publication No. 2015 / 0005363, I or III;U.S. Patent Application Publication No. 2014 / 0308304, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV; U.S. Patent Application Publication No. 2013 / 0338210; WO 2009 / 132131, I, II, III, or IV; U.S. Patent Application Publication No. 2012 / 01011478, A; U.S. Patent Application Publication No. 2012 / 0027796, I or XXXV; U.S. Patent Application Publication No. 2012 / 0058144, XIV or XVII; U.S. Patent Application Publication No. 2012 / 0058144, XIV or XVII; No. 2013 / 0323269; U.S. Patent Application Publication No. I of U.S. Patent Application Publication No. 2011 / 0117125; U.S. Patent Application Publication No. I, II, or III of U.S. Patent Application Publication No. 2011 / 0256175; U.S. Patent Application Publication No. I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII of U.S. Patent Application Publication No. 2012 / 0202871; U.S. Patent Application Publication No. I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of U.S. Patent Application Publication No. 2011 / 0076335; U.S. Patent Application Publication No. 2006 / 008378 I or II of the specification; I of U.S. Patent Application Publication No. 2013 / 0123338; ​​I or XAYZ of U.S. Patent Application Publication No. 2015 / 0064242; XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013 / 0022649; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; I, II, or III of U.S. Patent Application Publication No. 2013 / 0116307; I or II of U.S. Patent Application Publication No. 2010 / 0062967; U.S. Patent Application Publication No. 2013 / 0189351 I-X of US Patent Application Publication No. 2014 / 0039032; V of US Patent Application Publication No. 2018 / 0028664; I of US Patent Application Publication No. 2016 / 0317458; I of US Patent Application Publication No. 2013 / 0195920; 5, 6, or 10 of US Patent Application Publication No. 10,221,127; III-3 of WO 2018 / 081480; I-5 or I-8 of WO 2020 / 081938; 18 or 25 of US Patent Application Publication No. 9,867,888;US2019 / 0136231A;WO2020 / 219876II;US2012 / 0027803A1;US2019 / 0240349OF-02;US10,086,013A23;cKK-E12 / A6 of Miao et al (2020);C12-200 of WO2010 / 053572;7C1 of Dahlman et al (2017);Whitehead et al al., 304-O13 or 503-O13; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO 2020 / 106946; I of WO 2020 / 106946; and (1), (2), (3) or (4) of WO 2021 / 113777. Exemplary lipids further include any one of the lipids in Tables 1 to 16 of WO 2021 / 113777.

[0380] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl 16-O-dimethyl-1, ... PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl ... dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylethanolamine phatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Further exemplary lipids include, in certain embodiments, but are not limited to, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, which is incorporated herein by reference. Such lipids include, in certain embodiments, plant lipids that have been shown to improve hepatic transfection with mRNA (e.g., DGTS).

[0381] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, the entire contents of which are incorporated herein by reference.

[0382] Numbered embodiments [1] A method for producing an enriched population of circular polyribonucleotides (circRNAs), the method comprising: (a) providing a sample containing a population of polyribonucleotides that includes circRNAs and linear polyribonucleotides (linRNAs); and (b) separating the circRNAs from the linRNAs under denaturing conditions that do not include the use of gel electrophoresis, thereby producing an enriched population of circRNAs. [2] A method for producing an enriched population of circRNA, the method comprising: (a) providing a sample containing a population of polyribonucleotides including circRNA and linRNA; and (b) exposing the population of polyribonucleotides to denaturing conditions, thereby enriching the population of circRNA. [3] The method of embodiment [1] or [2], wherein: (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) the total volume of the sample containing the population of polyribonucleotides is at least 500 μL; or (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL. [4] A method for producing an enriched population of circRNA, the method comprising: (a) preparing a sample containing a population of polyribonucleotides including circRNA and linRNA, wherein (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) the total volume of the sample containing the population of polyribonucleotides is at least 500 μL; or (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL; and (b) separating circRNA from linRNA under denaturing conditions, thereby producing an enriched population of circRNA. [5] The method according to any one of embodiments [1] to [4], wherein the total weight of polyribonucleotides in the population of polyribonucleotides is 1 μg to 1000 mg. [6] The method according to any one of embodiments [1] to [5], wherein the total amount of polyribonucleotides in the population of polyribonucleotides is 500 μL to 1000 mL. [7] The method according to any one of embodiments [1] to [6], wherein the concentration of polyribonucleotides in the population of polyribonucleotides is 200 ng / μL to 50 mg / mL. [8] The method of any one of embodiments [4] to [7], wherein the separation step (b) is carried out under denaturing conditions that do not involve the use of gel electrophoresis. [9] The method of any one of embodiments [1] to [8], wherein the enriched population of circRNA is substantially free of one or more impurities or by-products.

[10] The method of embodiment [9], wherein the one or more impurities or by-products include polyacrylamide, boric acid, magnesium, or ethylenediaminetetraacetic acid (EDTA).

[11] The method according to any one of embodiments [1] to

[10] , wherein the denaturing conditions include a temperature of at least 50°C.

[12] The method of embodiment

[11] , wherein the denaturing conditions include a temperature of 50°C to 85°C.

[13] The method according to any one of embodiments [1] to

[12] , wherein the denaturing conditions comprise a temperature of at least 50°C, followed by a temperature of 8°C or less for a period of 30 seconds or less.

[14] The method according to any one of embodiments [1] to

[13] , wherein the denaturing conditions include a pH of less than 5 or a pH of more than 9.

[15] The method of embodiment

[14] , wherein the denaturing conditions include a pH of less than 5.

[16] The method of embodiment

[14] , wherein the denaturing conditions are greater than 9.

[17] The method according to any one of embodiments [1] to

[16] , wherein the denaturing conditions include a chemical treatment.

[18] The method of embodiment

[17] , wherein the chemical treatment comprises treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

[19] The method of embodiment

[18] , wherein the acid comprises 1 mM to 500 mM acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[20] The method of embodiment

[18] or

[19] , wherein the base comprises 1 mM to 500 mM sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[21] The method according to any one of embodiments

[18] to

[20] , wherein the organic solvent comprises at least 0.1% (v / v) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[22] The method according to any one of embodiments

[18] to

[21] , wherein the chaotropic agent comprises 100 mM to 8 M urea, guanidinium chloride, lithium perchlorate, or polyethylene glycol (PEG).

[23] The method according to any one of embodiments

[18] to

[22] , wherein the chaotropic agent comprises 100 mM to 8 M of n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate.

[24] The method according to any one of embodiments

[18] to

[23] , wherein the crowding agent comprises 100 mM to 8 M PEG or urea.

[25] The method according to any one of embodiments

[18] to

[24] , wherein the chelating agent comprises 1 mM to 10 mM of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a derivative thereof, ethylenediaminetetraacetic acid (EDTA) or a derivative thereof, nitrilotriacetic acid (NTA), iminodisuccinic acid (IDS), polyaspartic acid, S,S-ethylenediamine-N,N'-disuccinic acid (EDDS), or methylglycine diacetic acid (MGDA).

[26] The method according to any one of embodiments

[18] to

[25] , wherein the detergent comprises 0.005% to 0.05% (v / v) of Nonidet P-40 (NP40), CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween-20, or Tween-80.

[27] A method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; and (b) separating circRNA from linRNA at a temperature of at least 50°C, thereby producing an enriched population of circRNA, wherein the separation step does not include the use of gel electrophoresis.

[28] A method for producing an enriched population of circRNA, the method comprising: (a) preparing a sample comprising a population of polyribonucleotides including circRNA and linRNA, wherein (i) a total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) a total volume of the sample comprising the population of polyribonucleotides is at least 500 μL; or (iii) a concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL; and (b) separating circRNA from linRNA at a temperature of at least 50°C, thereby producing an enriched population of circRNA.

[29] A method for producing an enriched population of circRNAs, the method comprising: (a) providing a sample containing a population of polyribonucleotides including circRNAs and linRNAs; and (b) exposing the population of polyribonucleotides to a temperature of at least 50°C, thereby enriching the population of circRNAs.

[30] The method according to any one of

[27] to

[29] , wherein the temperature is 50°C to 85°C.

[31] A method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; and (b) separating circRNA from linRNA at a pH below 5 or above 9, thereby producing an enriched population of circRNA, wherein the separation does not include the use of gel electrophoresis.

[32] A method for producing an enriched population of circRNA, the method comprising: (a) providing a sample containing a population of polyribonucleotides including circRNA and linRNA, wherein (i) a total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) a total volume of the sample containing the population of polyribonucleotides is at least 500 μL; or (iii) a concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL; and (b) separating circRNA from linRNA at a pH less than 5 or greater than 9, thereby producing an enriched population of circRNA.

[33] A method for producing an enriched population of circRNAs, the method comprising: (a) providing a sample comprising a population of polyribonucleotides including circRNAs and linRNAs; and (b) exposing the population of polyribonucleotides to a pH below 5 or above 9, thereby enriching the population of circRNAs.

[34] The method according to any one of embodiments

[31] to

[33] , wherein the pH is less than 5.

[35] The method according to any one of embodiments

[31] to

[33] , wherein the pH is greater than 9.

[36] A method for producing an enriched population of circRNA, the method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; and (b) separating circRNA from linRNA under conditions comprising an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution, thereby producing an enriched population of circRNA, wherein the separation does not include the use of gel electrophoresis.

[37] A method for producing an enriched population of circRNA, the method comprising: (a) preparing a sample comprising a population of polyribonucleotides including circRNA and linRNA, wherein (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) the total volume of the sample comprising the population of polyribonucleotides is at least 500 μL; or (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL; and (b) separating circRNA from linRNA under conditions comprising an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution, thereby producing an enriched population of circRNA.

[38] A method for producing an enriched population of circRNA, the method comprising: (a) providing a sample containing a population of polyribonucleotides including circRNA and linRNA; and (b) exposing the population of polyribonucleotides to an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution, thereby enriching the population of circRNA.

[39] The method according to any one of embodiments

[36] to

[38] , wherein the acid comprises at least 0.5% (v / v) acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid.

[40] The method according to any one of embodiments

[36] to

[38] , wherein the base comprises 1 mM to 500 mM sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine.

[41] The method of any one of embodiments

[36] to

[38] , wherein the organic solvent comprises at least 0.1% (v / v) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol.

[42] The method according to any one of embodiments

[36] to

[38] , wherein the chaotropic agent comprises 100 mM to 8 M urea, guanidinium chloride, lithium perchlorate, or polyethylene glycol (PEG).

[43] The method according to any one of

[36] to

[38] , wherein the chaotropic agent comprises 100 mM to 8 M n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate.

[44] The method according to any one of

[36] to

[38] , wherein the crowding agent comprises 100 mM to 8 M polyethylene glycol (PEG) or urea.

[45] The method according to any one of embodiments

[36] to

[38] , wherein the chelating agent comprises 1 mM to 10 mM EGTA or a derivative thereof, EDTA or a derivative thereof, NTA, IDS, EDDS, or MGDA.

[46] The method according to any one of embodiments

[36] to

[38] , wherein the detergent comprises 0.005% to 0.05% (v / v) of NP40, CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween®-20, or Tween®-80.

[47] The method of any one of embodiments [1] to

[46] , wherein step (b) includes performing column chromatography on the population of polyribonucleotides, the step of performing column chromatography including an equilibration step, a sample loading step, a column washing step, and an elution step.

[48] ​​The method of embodiment

[47] , wherein the separation step is performed during the sample loading step.

[49] The method of embodiment

[46] or

[47] , wherein the separation step is performed during a column washing step.

[50] The method according to any one of embodiments

[47] to

[49] , wherein the separation step is carried out during the elution step.

[51] The method of any one of embodiments

[47] to

[50] , wherein the column chromatography comprises fast protein liquid chromatography (FPLC), high pressure liquid chromatography (HPLC), hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEC), mixed mode chromatography, or affinity chromatography.

[52] The method of embodiment

[51] , wherein the AEC comprises the use of an anion exchange resin selected from the group consisting of styrene-divinylbenzene, silica, sepharose, cellulose, dextran, epoxypolyamine, methacrylate, agarose, and acrylic.

[53] The method of embodiment

[52] , wherein the anion exchange resin comprises an ion exchanger selected from the group consisting of quaternary ammonium, aminoethyl, diethylaminoethyl, or diethylaminopropyl.

[54] The method according to embodiment

[52] or [53, wherein the anion exchange resin comprises beads, the beads having a bead diameter of 45 to 165 μm and a pore size of 100 to 1000 nm.

[55] The method according to any one of embodiments

[51] to

[54] , wherein the AEC comprises the use of linear gradient elution or step isocratic elution.

[56] The method of any one of embodiments

[51] to

[55] , wherein the AEC comprises using a flow rate of 1 mL / min to 150 mL / min.

[57] The method of embodiment

[51] , wherein the FPLC is reversed-phase FPLC (RP-FPLC).

[58] The method of any one of embodiments [1] to

[57] , wherein step (b) is carried out by pooling multiple fractions of purified circRNA.

[59] The method of any one of embodiments [1] to

[58] , wherein the circRNA and the linRNA have the same ribonucleotide sequence.

[60] The method of any one of embodiments [1] to

[59] , wherein the circRNA and the linRNA have the same mass.

[61] The method of any one of embodiments [1] to

[60] , wherein the circRNA and linRNA lack a poly(A) tail.

[62] The method according to any one of embodiments [1] to

[61] , wherein the method comprises exonuclease digestion of linRNA.

[63] The method according to any one of embodiments [1] to

[61] , wherein the method does not include exonuclease digestion of linRNA.

[64] The method according to any one of embodiments [1] to

[63] , wherein the method does not include selective modifications to circRNA or linRNA that improve the enrichment of circRNA.

[65] The method of any one of embodiments [1]-

[64] , wherein the percentage (w / w) of circRNA in the enriched population of circRNA is twice the percentage (w / w) of circRNA in the polyribonucleotide population.

[66] The method of any one of embodiments [1] to

[65] , wherein the percentage (w / w) of circRNA in the enriched population of circRNA is at least 65%.

[67] The method of any one of embodiments [1] to

[66] , wherein the percentage (w / w) of linRNA in the enriched population of circRNA is less than 35%.

[68] The method of any one of embodiments [1] to

[67] , wherein the circRNA has a length of about 1,000 nucleotides or less.

[69] A composition comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein the population of polyribonucleotides is in a solution under denaturing conditions; (i) the total weight of polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) the total volume of a sample comprising the population of polyribonucleotides is at least 500 μL; or (iii) the concentration of the population of polyribonucleotides in the sample is at least 500 ng / μL.

[70] A composition comprising a population of polyribonucleotides comprising circRNA and linRNA, wherein the population of polyribonucleotides is in a solution under denaturing conditions, and the solution is substantially free of one or more impurities or by-products.

[71] A composition comprising a population of polyribonucleotides comprising circRNA and linRNA, (a) a composition is obtained from a sample that includes a population of nucleic acids; (b) the composition has been exposed to one or more denaturing conditions; (c) The composition is substantially free of one or more impurities or by-products.

[72] A composition comprising a population of polyribonucleotides comprising circRNA and linRNA, (a) a composition is obtained from a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; (b) the composition has been exposed to one or more denaturing conditions; (c) The composition is substantially free of one or more impurities or by-products.

[73] The composition of any one of embodiments

[69] -

[72] , wherein the one or more impurities or by-products include polyacrylamide, boric acid, magnesium, or EDTA.

[74] The composition of any one of embodiments

[69] to

[73] , wherein the denaturing conditions include a temperature of at least 50°C.

[75] The composition according to any one of embodiments

[69] to

[74] , wherein the denaturing conditions comprise a temperature of 50°C to 85°C.

[76] The composition of any one of embodiments

[69] to

[75] , wherein the denaturing conditions comprise a temperature of at least 50°C, followed by a temperature of 8°C or less.

[77] The composition of any one of embodiments

[69] to

[73] , wherein the denaturing conditions include a pH of less than 5 or a pH of more than 9.

[78] The composition of any one of embodiments

[69] to

[73] , wherein the denaturing conditions include chemical treatment.

[79] The composition of embodiment

[78] , wherein the chemical treatment comprises treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

[80] The composition of embodiment

[79] , wherein the acid comprises 1 mM to 500 mM acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic ac...

Claims

1. 1. A method for producing an enriched population of circular polyribonucleotides (circRNA), said method comprising: (a) providing a sample containing a population of polyribonucleotides including circRNA and linear polyribonucleotides (linRNA); (b) separating the circRNA from the linRNA under denaturing conditions that do not involve the use of gel electrophoresis, thereby producing an enriched population of circRNA; A method comprising:

2. 1. A method for producing an enriched population of circRNA, said method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; (b) exposing said population of polyribonucleotides to denaturing conditions that do not involve the use of gel electrophoresis, thereby enriching said population of circRNA; A method comprising:

3. (i) the total weight of the polyribonucleotides in the population of polyribonucleotides is at least 1 μg; (ii) the total volume of the sample containing the population of polyribonucleotides is at least 500 μL; (iii) the concentration of the population of polyribonucleotides in the sample is at least 200 ng / μL; or (iv) The method of claim 1 or claim 2, wherein the enriched population of circRNA is substantially free of one or more impurities or by-products, wherein the one or more impurities or by-products comprise polyacrylamide, boric acid, magnesium, or ethylenediaminetetraacetic acid (EDTA).

4. 3. The method of claim 1 or claim 2, wherein the denaturing conditions comprise a temperature of at least 50°C; or wherein the denaturing conditions comprise a temperature of at least 50°C followed by a temperature of 8°C or less within a period of 30 seconds or less.

5. 3. The method of claim 1 or claim 2, wherein the denaturing conditions comprise a pH below 5 or a pH above 9.

6. 3. The method of claim 1 or claim 2, wherein the denaturing conditions comprise chemical treatment, including treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

7. a) the acid comprises 1 mM to 500 mM acetic acid, hydrochloric acid, salicylic acid, phosphoric acid, boric acid, formic acid, oxalic acid, citric acid, benzoic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, or nitric acid; b) the base comprises 1 mM to 500 mM sodium hydroxide, potassium hydroxide, imidazole, histidine, sodium bicarbonate, guanidine, or triethylamine; c) the organic solvent comprises at least 0.1% (v / v) of dimethyl sulfoxide, triethylammonium acetate, methanol, ethanol, 2-propanol, isopropanol, 1-butanol, 2-butanol, t-butanol, isobutanol, phenol, chloroform, hexane, acetonitrile, formamide, acetone, denatonium, or propylene glycol; d) the chaotropic agent comprises 100 mM to 8 M urea, guanidinium chloride, lithium perchlorate, or polyethylene glycol (PEG); and / or the chaotropic agent comprises 100 mM to 8 M n-dodecyl β-d-maltoside, n-octylglucoside, CHAPS, or deoxycholate; e) the crowding agent comprises 100 mM to 8 M PEG or urea; f) the chelating agent comprises 1 mM to 10 mM of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) or a derivative thereof, ethylenediaminetetraacetic acid (EDTA) or a derivative thereof, nitrilotriacetic acid (NTA), iminodisuccinic acid (IDS), polyaspartic acid, S,S-ethylenediamine-N,N′-disuccinic acid (EDDS), or methylglycine diacetic acid (MGDA); or g) The method of claim 6, wherein the detergent comprises 0.005% to 0.05% (v / v) of Nonidet® P-40 (NP40), CHAPS, octyl β-D-glucopyranoside, n-dodecyl β-d-maltoside, Tween®-20, or Tween®-80.

8. 2. The method of claim 1, wherein step (b) comprises performing column chromatography on the population of polyribonucleotides, wherein performing column chromatography comprises an equilibration step, a sample loading step, a column washing step, and an elution step, wherein the separating occurs during the equilibration step, the sample loading step, the column washing step, and / or during the elution step, and wherein the column chromatography comprises fast protein liquid chromatography (FPLC), high pressure liquid chromatography (HPLC), hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEC), mixed mode chromatography, or affinity chromatography.

9. The method of claim 1 or claim 2, wherein the circRNA and linRNA have the same ribonucleotide sequence; and / or the circRNA and linRNA have the same mass; and / or the circRNA and linRNA lack a poly(A) tail.

10. The method of claim 1 or claim 2, wherein the method comprises exonuclease digestion of the linRNA; or the method does not comprise exonuclease digestion of the linRNA; and / or the method does not comprise selective modifications to the circRNA or the linRNA that improve enrichment of the circRNA.

11. The method of claim 1 or claim 2, wherein the percentage (w / w) of circRNA in the enriched population of circRNA is twice the percentage (w / w) of circRNA in the polyribonucleotide population; and / or the percentage (w / w) of circRNA in the enriched population of circRNA is at least 65%; and / or the percentage (w / w) of linRNA in the enriched population of circRNA is less than 35%.

12. A composition comprising a population of polyribonucleotides comprising circRNA and linRNA, said population of polyribonucleotides being in solution under denaturing conditions; (i) the total weight of polyribonucleotides in said population of polyribonucleotides is at least 1 μg; (ii) the total volume of the sample containing the population of polyribonucleotides is at least 500 μL; or (iii) The composition, wherein the concentration of the population of polyribonucleotides in the sample is at least 500 ng / μL.

13. 1. A composition comprising an enriched population of circRNA, (a) the composition is obtained from a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; (b) the composition is exposed to one or more denaturing conditions; (c) the composition is substantially free of one or more impurities or by-products.

14. 14. The method of claim 12 or claim 13, wherein the denaturing conditions comprise a temperature of at least 50°C; or wherein the denaturing conditions comprise a temperature of at least 50°C followed by a temperature of 8°C or less.

15. 14. The method of claim 12 or claim 13, wherein the denaturing conditions comprise a pH below 5 or a pH above 9.

16. 14. The method of claim 12 or claim 13, wherein the denaturing conditions comprise chemical treatment, including treatment with an acid, a base, an organic solvent, a chaotropic agent, a crowding agent, a chelating agent, a detergent, or a salt solution.

17. The method of claim 12 or claim 13, wherein the percentage (w / w) of circRNA in the enriched population of circRNA is twice the percentage (w / w) of circRNA in the polyribonucleotide population; and / or the percentage (w / w) of circRNA in the enriched population of circRNA is at least 65%; and / or the percentage (w / w) of linRNA in the enriched population of circRNA is less than 35%.

18. The method of claim 12 or 13, wherein the circRNA and the linRNA have the same ribonucleotide sequence; and / or the circRNA and the linRNA have the same mass; and / or the circRNA and the linRNA lack a poly(A) tail.

19. 1. A method for determining the purity of circRNA, said method comprising: (a) providing a sample comprising a population of polyribonucleotides comprising circRNA and linRNA; (b) separating the circRNA from the linRNA by chromatography under denaturing conditions, wherein the denaturing conditions do not include the use of gel electrophoresis; and (c) collecting a chromatogram of the sample comprising the circRNA peak and the linRNA peak; (d) calculating the area under each peak to determine the purity of the circRNA in the sample; A method comprising:

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