Method for purifying circular RNA

Abstract

A method for purifying a circular RNA. The purification method comprises: S1. providing a preparation containing the circular RNA; and S2. subjecting the preparation to digestion treatment with Rnase R, cellulose chromatography treatment, and alkaline phosphatase treatment to obtain a purified circular RNA, wherein the cellulose chromatography treatment comprises providing a mixture II of a sample II containing the circular RNA and a buffer solution II, and bringing the mixture II into contact with a cellulose material, followed by separation to obtain a purified product II containing the circular RNA. The buffer solution II comprises an organic alcohol, a salt, and optionally a complexing agent and a buffer substance, and the concentrations of these components allow binding of a double-stranded RNA to the cellulose material while preventing binding of the circular RNA to the cellulose material. A better circular RNA purification effect can be achieved, and a purified circular RNA product with high yield, high purity and strong biological activity is obtained.

Description

A method for purifying circular RNA TECHNICAL FIELD

[0001] The present application belongs to the field of RNA purification, and particularly relates to a method for purifying circular RNA. BACKGROUND

[0002] Circular RNA (circRNA) is a kind of natural (biological) or artificially synthesized closed RNA without 5' or 3' end. The circular RNA molecule is in a closed ring structure, which can prevent exonuclease from degrading RNA, so that it has better drug stability and biological stability than the current standard linear mRNA, and can make the protein level last for several days. Recently, in vitro synthesis of circular RNA has been applied to various therapies such as vaccines, chimeric antigen receptor T cells (CAR-T). Studies have shown that, compared with linear mRNA, engineered circular RNA has unique advantages as a non-viral vector with high expression and low genetic risk.

[0003] However, due to the generation of complex RNA by-products during the in vitro synthesis of circular RNA, including double-stranded RNA, truncated RNA such as transcription termination products, intron RNA fragments generated during circularization, un-circularized linear RNA precursors and broken circular RNA, and these by-products have immunostimulatory properties, which can cause immune responses of cells, so that the purification treatment of circular RNA is essential. However, these by-product components are complex, and it is difficult to purify them using the current linear mRNA method.

[0004] Currently, there are several methods for purifying circular RNA: (1) urea-polyacrylamide gel electrophoresis (Urea-PAGE): PAGE gel has molecular sieve effect and electrophoresis effect, which can distinguish circular RNA from other RNA impurities under the action of electric field, and the purified circular RNA sample can be obtained by gel recovery. However, electrophoresis is suitable for laboratory-scale recovery and purification and analysis and detection, and is not suitable for large-scale production and purification. In addition, heat will be generated during the electrophoresis process, which may affect the stability of RNA; (2) RNase R digestion method: RNase R is an exonuclease, and using RNase R to remove linear RNA and enrich circular RNA is a feasible choice, but RNase R is difficult to remove RNA impurities with high G content or complex secondary structure. In addition, circular RNA exposed to RNase R for a long time will also be degraded; (3) high performance liquid chromatography (HPLC): in general, the high performance liquid chromatography method has the advantages of high sensitivity and process scalability, but the cost is high and the recovery rate is low. Among them, the molecular sieve chromatography (SEC-HPLC) has the advantage of easily removing RNA fragments with large differences (such as intron fragments), but for circular RNA, linear RNA precursors and broken circular RNA with similar molecular sizes, the separation effect of SEC-HPLC is relatively poor, and different base numbers of circular RNA may need to use fillers with specific pore sizes, which is not conducive to the development of universal purification process. Ion pair reversed phase chromatography (IP-RP-HPLC) requires the use of organic solvents, and high temperature conditions are required during separation, which may affect the stability of RNA.

[0005] In the prior art, the combination of RNase R digestion and high performance liquid chromatography is often used for the purification of circular RNA. Although this method can obtain circular RNA with high purity, the yield is low, and the process is complex, which is not conducive to process scaling. In addition, other high-yield circular RNA purification methods have the problems of low purity or high immunogenicity, which leads to poor application effect.

[0006] SUMMARY

[0007] The first aspect of the present application is to overcome the shortcomings of the prior art circular RNA purification method that is difficult to simultaneously ensure high yield and high purity and low immunogenicity, and to provide a circular RNA purification method.

[0008] The second aspect of the present application is to further provide circular RNA prepared by the above purification method.

[0009] In particular, the method for purifying the circular RNA comprises the following steps: S1. providing a preparation containing the circular RNA; S2. subjecting the preparation to Rnase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase treatment to obtain the purified circular RNA; the cellulose chromatography treatment comprises providing a mixture II containing sample II containing the circular RNA and buffer solution II, and separating the mixture II after contacting with cellulose material to obtain purified product II containing the circular RNA; the buffer solution II contains an organic alcohol and a salt, and optionally a complexing agent and a buffer substance, and the concentrations thereof allow the double-stranded RNA to bind to the cellulose material and do not allow the circular RNA to bind to the cellulose material; the cellulose material is selected from at least one of lignocellulose, microcrystalline cellulose, alpha-cellulose, carboxymethyl cellulose, carboxyethyl cellulose and cellulose acetate.

[0010] In some specific embodiments, the Rnase R digestion treatment comprises providing a mixture I containing sample I containing the circular RNA and Rnase R and buffer solution I, and separating the mixture I after incubation to obtain purified product I containing the circular RNA.

[0011] In some specific embodiments, the alkaline phosphatase treatment comprises providing a mixture III containing sample III containing the circular RNA and buffer solution III, and separating the mixture III after incubation to obtain purified product III containing the circular RNA.

[0012] In some specific embodiments, in the Rnase R digestion treatment, the incubation time is 5-60 min.

[0013] In some specific embodiments, in the Rnase R digestion treatment, the concentration of sample I containing the circular RNA in the mixture I is 0.02-2.5 mg / mL.

[0014] In some specific embodiments, in the Rnase R digestion treatment, the concentration of Rnase R in the mixture I is 0.1-10 IU / mL.

[0015] In some specific embodiments, in the Rnase R digestion treatment, the buffer solution I contains a salt, magnesium chloride and Tris, the concentration of the salt is 0.1-1 M, the concentration of magnesium chloride is 0.1-2 mM, and the concentration of Tris is 10-100 mM.

[0016] In some specific embodiments, the salt in the buffer solution I is selected from at least one of sodium chloride, lithium chloride and potassium chloride.

[0017] In some embodiments, the concentration of the organic alcohol in the buffer solution II is 5-40% (v / v) and is selected from at least one of n-propanol, isopropanol and ethanol.

[0018] In some embodiments, the concentration of the salt in the buffer solution II is 100-300 mM and is selected from at least one of sodium chloride, lithium chloride and potassium chloride.

[0019] In some embodiments, the complexing agent in the buffer solution II is ethylenediaminetetraacetic acid and the concentration is 0.1-1 mM.

[0020] In some embodiments, the buffering substance in the buffer solution II is 4-hydroxyethylpiperazineethanesulfonic acid and / or tris(hydroxymethyl)aminomethane and the concentration is 5-20 mM.

[0021] In some embodiments, the cellulose chromatography process comprises (1) contacting the mixture II of the sample II containing circular RNA and the buffer solution II with the cellulose material in a chromatography column or a micro-separation column, and (2) applying gravity, centrifugal force, pressure or vacuum to the chromatography column or the micro-separation column to separate the liquid phase from the solid phase, and collecting the liquid containing circular RNA, to obtain the purified product II containing circular RNA.

[0022] In some embodiments, the contacting of the mixture II of the sample II containing circular RNA and the buffer solution II with the cellulose material in step (1) is performed under any one of flowing, shaking and stirring.

[0023] In some embodiments, the mass ratio of the sample II containing circular RNA to the cellulose material in the chromatography column is (0.00005-0.001): 1.

[0024] In some embodiments, the mass ratio of the sample II containing circular RNA to the cellulose material in the micro-separation column is (0.0005-0.01): 1.

[0025] In some embodiments, the incubation time in the alkaline phosphatase treatment is 15-120 min.

[0026] In some embodiments, the concentration of the sample III containing circular RNA in the mixture III is 0.02-2.5 mg / mL, and the concentration of the alkaline phosphatase in the mixture III is 50-500 IU / mL.

[0027] In some embodiments, the buffer solution III comprises potassium acetate, magnesium acetate and tris-hydroxymethyl aminomethane, the concentration of potassium acetate is 10-200 mM, the concentration of magnesium acetate is 1-10 mM, and the concentration of tris-hydroxymethyl aminomethane is 10-100 mM.

[0028] In some embodiments, in step S1, the preparation containing circular RNA is at least one of circular RNA produced in vivo and extracted, circular RNA biosynthesized in vitro, and circular RNA biosynthesized in vitro and pretreated.

[0029] In some embodiments, in the Rnase R digestion treatment, the sample containing circular RNA is at least one of the preparation containing circular RNA provided in step S1, the purified product II containing circular RNA obtained by cellulose chromatography treatment, and the purified product III containing circular RNA obtained by alkaline phosphatase treatment.

[0030] In some embodiments, in the cellulose chromatography treatment, the sample containing circular RNA is at least one of the preparation containing circular RNA provided in step S1, the purified product I containing circular RNA obtained by Rnase R digestion treatment, and the purified product III containing circular RNA obtained by alkaline phosphatase treatment.

[0031] In some embodiments, in the alkaline phosphatase treatment, the sample containing circular RNA is at least one of the preparation containing circular RNA provided in step S1, the purified product I containing circular RNA obtained by Rnase R digestion treatment, and the purified product III containing circular RNA obtained by cellulose chromatography treatment.

[0032] The key of the present application is that the purification of circular RNA is carried out by the combination of RNase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase treatment, and by regulating the conditions of the buffer solution in the cellulose chromatography treatment, the concentration of the buffer solution is allowed to bind double-stranded RNA (dsRNA) to the cellulose material and not to bind circular RNA to the cellulose material, so that the efficient removal of double-stranded RNA is achieved without adversely affecting the circular RNA, the RNase R digestion treatment is mainly to remove most of the linear RNA, and the alkaline phosphatase treatment removes the phosphate in the impurity components, so that the high yield and high purity of the circular RNA purification product can be obtained by the synergistic cooperation of the RNase R digestion treatment, the cellulose chromatography treatment and the alkaline phosphatase treatment, which is more advantageous in operability and time efficiency, improves the efficiency of circular RNA purification, and the circular RNA purification product obtained by the method has the effect of 1+1+1>3 in terms of good biological efficacy (low immunogenicity and good cell expression effect). In addition, the purification method of the present application is simple and reliable, and the cost is relatively low, and can be applied to microseparation column or fast protein liquid chromatography, so that the industrial scale-up application of circular RNA purification can be realized.

[0033] In a preferred embodiment, the components and contents of the preparation containing circular RNA can be analyzed before the purification treatment, and then the appropriate sequence of RNase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase treatment is selected according to the content of impurity components, so that each purification link can maximize the function and not overlap with each other. More preferably, the sequence of RNase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase treatment is used in turn for the purification of circular RNA, at this time, the yield, purity and biological efficacy of the circular RNA purification product are the best. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a capillary electrophoresis result graph of the preparation containing circular Circ-NL before RNase R digestion treatment in Example 1.

[0035] Figure 2 is a capillary electrophoresis result graph of the purification product I-1 containing circular Circ-NL after RNase R digestion treatment in Example 1.

[0036] Figure 3 is a flowchart of cellulose chromatography treatment in Example 1.

[0037] Figure 4 is a chromatogram of fast protein liquid chromatography (FPLC) treatment in Example 1.

[0038] Figure 5 is a LSFA test result graph before and after cellulose chromatography treatment in Example 1.

[0039] Figure 6 is a capillary electrophoresis result graph of the purification product III-1 after alkaline phosphatase digestion in Example 1.

[0040] Figure 7 is a flow chart of the cellulose chromatography treatment in Example 2.

[0041] Figure 8 is a capillary electrophoresis result chart of the purified product III-2 after alkaline phosphatase digestion in Example 2.

[0042] Figure 9 is a capillary electrophoresis result chart of the purified product III-3 after alkaline phosphatase digestion in Example 3.

[0043] Figure 10 is a capillary electrophoresis result chart of the purified product III-4 after alkaline phosphatase digestion in Example 4.

[0044] Figure 11 is a chromatogram of the molecular sieve chromatography treatment in Comparative Example 1.

[0045] Figure 12 is a protein expression comparison chart of the circular RNA purified products obtained by different purification methods in the examples and comparative examples, wherein, RR: Rnase R digestion treatment, WMC: cellulose chromatography treatment, Q: alkaline phosphatase digestion treatment, RR-Q: Rnase R digestion treatment followed by alkaline phosphatase digestion treatment, RR-WMC: Rnase R digestion treatment followed by cellulose chromatography treatment, WMC-Q: cellulose chromatography treatment followed by alkaline phosphatase digestion treatment, RR-WMC-Q (Example 1): Rnase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase digestion, RR-SEC-Q (Comparative Example 1): Rnase R digestion treatment, molecular sieve chromatography treatment and alkaline phosphatase digestion.

[0046] Figure 13 is a comparison chart of the changes in RIG-I and TLR3 / 7 / 8 transcripts after transfection of A549 cells for 6h by the circular RNA purified products before and after treatment with alkaline phosphatase in the examples and comparative examples (using the chemiluminescence value of the unpurified sample for normalization), wherein, RR-WMC: Rnase R digestion treatment followed by cellulose chromatography treatment, RR-WMC-Q (Example 1): Rnase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase digestion, RR-SEC-Q (Comparative Example 1): Rnase R digestion treatment, molecular sieve chromatography treatment and alkaline phosphatase digestion. DETAILED DESCRIPTION

[0047] In the present application, the purification method of the circular RNA comprises the following steps: S1. providing a preparation containing circular RNA; S2. subjecting the preparation to Rnase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase treatment to obtain purified circular RNA.

[0048] In the present application, the order of the Rnase R digestion treatment, the cellulose chromatography treatment and the alkaline phosphatase treatment can be selected according to the actual situation. In a preferred embodiment, the components and contents of the preparation containing circular RNA can be analyzed before the purification treatment, and the order of the Rnase R digestion treatment, the cellulose chromatography treatment and the alkaline phosphatase treatment can be selected according to the content of the impurity components. Specifically, when the content of the linear RNA precursor that is not circularized in the impurity components is detected to be the highest, the Rnase R digestion treatment can be performed first, and the order of the purification treatment is the Rnase R digestion treatment, the cellulose chromatography treatment and the alkaline phosphatase treatment in sequence; when the content of the double-stranded RNA in the impurity components is detected to be the highest, the cellulose chromatography treatment can be performed first, and the order of the purification treatment is the cellulose chromatography treatment, the Rnase R digestion treatment and the alkaline phosphatase treatment in sequence. The order of the Rnase R digestion treatment, the cellulose chromatography treatment and the alkaline phosphatase treatment can be selected according to the content of the impurity components in the preparation containing circular RNA, so that the function of each purification treatment step can be maximized without overlapping, thereby more favorably improving the yield, purity and biological function of the circular RNA purification product.

[0049] In the present application, in step S1, the preparation containing circular RNA can be at least one of circular RNA produced in vivo and extracted, circular RNA biosynthesized in vitro, and circular RNA biosynthesized in vitro and pretreated. Preferably, the purification method provided by the present application significantly improves the purification effect of circular RNA biosynthesized in vitro. The impurities in the preparation include RNA (such as double-stranded RNA, high-molecular-weight RNA impurities, linear RNA precursor that is not circularized, broken circular RNA and intron RNA small fragments generated by circularization), DNA (such as template DNA from IVT), protein (such as RNA polymerase, capping enzyme, DNase, RNase inhibitor), pyrophosphate and free nucleotides in addition to circular RNA. The pretreatment method can be various existing pretreatment methods, and LiCl precipitation method is preferably used for pretreating circular RNA. The specific embodiment of the LiCl precipitation method can be: mixing the preparation containing circular RNA with a LiCl solution, cooling at -80°C for more than 30 min, centrifuging the sample in a high-speed refrigerated centrifuge after cooling, adding ethanol after removing the supernatant, and obtaining a white precipitate after centrifuging, solid-liquid separation, blowing dry in a nuclease-free environment, and obtaining a transparent precipitate. The centrifugation conditions include a rotation speed of preferably 14000-18000 rpm and a centrifugation time of preferably 15-25 min.

[0050] In the present application, the circular RNA can be circular coding RNA and / or circular non-coding RNA. The purification method provided by the present application can be used for purifying a preparation containing circular coding RNA, a preparation containing circular non-coding RNA, or a preparation containing both. Preferably, the purification method is used for purifying a preparation containing circular coding RNA.

[0051] In the present application, the length and sequence of the circular RNA are not specifically limited. Preferably, the length of the circular RNA is preferably at least 200 nucleotides. More preferably, the circular RNA comprises at least one of a phosphodiester bond, a phosphorothioamidate bond, a phosphorothioate bond, and a methylphosphonate bond.

[0052] In the present application, the cellulose chromatography process comprises providing a mixture II containing a sample II containing circular RNA and a buffer solution II, and separating the mixture II from the cellulose material to obtain a purified product II containing circular RNA. The buffer solution II contains an organic alcohol and a salt, and optionally a complexing agent and a buffer substance, at a concentration that allows double-stranded RNA to bind to the cellulose material and does not allow circular RNA to bind to the cellulose material. In the buffer solution II, the concentration of the organic alcohol is preferably 5-40% (v / v), more preferably 8-25% (v / v), such as 8% (v / v), 10% (v / v), 12% (v / v), 15% (v / v), 18% (v / v), 20% (v / v), 22% (v / v), 25% (v / v), or any value therebetween. The concentration of the salt in the buffer solution II is preferably 100-300 mM, more preferably 120-250 mM, such as 120 mM, 150 mM, 180 mM, 200 mM, 220 mM, 250 mM, or any value therebetween. The concentration of the complexing agent in the buffer solution II is preferably 0.1-1 mM, such as 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, or any value therebetween. The concentration of the buffer substance in the buffer solution II is preferably 5-20 mM, such as 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, or any value therebetween. The organic alcohol in the buffer solution II is preferably at least one of n-propanol, isopropanol, and ethanol. The salt in the buffer solution II is preferably at least one of sodium chloride, lithium chloride, and potassium chloride. The complexing agent of the buffer solution II is preferably ethylenediaminetetraacetic acid (EDTA). The buffer substance of the buffer solution II is preferably 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and / or tris(hydroxymethyl)aminomethane. Under the conditions of the buffer solution II at this time, it is more advantageous to achieve efficient adsorption removal of double-stranded RNA by cellulose material and to maintain the stability of circular RNA and high yield purification efficiency.

[0053] In the cellulose chromatography process, the cellulose material is provided as a washed cellulose material. The washing of the cellulose material can be performed by (1) mixing the cellulose material with a washing solution under shaking and / or stirring for 5-10 min; (2) collecting the washed cellulose material after solid-liquid separation; and optionally (3) repeating steps (1) and (2) one, two or more times. The washing solution contains the components and concentrations as described in Buffer Solution II.

[0054] In a preferred embodiment, the cellulose chromatography process can comprise the following steps: loading the cellulose material in a chromatography column, contacting the cellulose material in the chromatography column with a mixture II of Sample II containing circular RNA and Buffer Solution II in a flowing state, the contacting time is preferably 10-60 min, such as 10 min, 20 min, 30 min, 40 min, 50 min, 60 min or any value therebetween, applying pressure or vacuum to the chromatography column to separate the liquid phase from the solid phase, collecting the eluate containing circular RNA, and obtaining the purified product II containing circular RNA after separation. The separation method can be any of the existing methods for enriching RNA, such as LiCl precipitation, isopropanol precipitation, sodium acetate precipitation, nucleic acid purification magnetic beads, nucleic acid purification column, gel recovery, etc. Preferably, the separation method can be LiCl precipitation method to concentrate the eluate to enrich the purified product, i.e. to obtain the purified product II containing circular RNA. The specific embodiments of the LiCl precipitation method can be as described above. In the chromatography column, the mass ratio of Sample II containing circular RNA to cellulose material is preferably (0.00005-0.001):1, such as 0.00005:1, 0.00008:1, 0.0001:1, 0.0002:1, 0.0005:1, 0.0008:1, 0.001:1 or any value therebetween.

[0055] In a preferred embodiment, the cellulose chromatography process can comprise the following steps: (1) loading a mixture II containing circular RNA and buffer solution II into a micro-separation column with cellulose material, and shaking vigorously for 5-10 min under shaking conditions; (2) collecting the supernatant containing circular RNA after solid-liquid separation; and optionally (3) repeating steps (1) and (2) one, two or more times; (4) concentrating the supernatant using a LiCl precipitation method to enrich the purified product, and optionally adding ultrapure water (DNase / RNase free) to resuspend the enriched purified product in an aqueous solution, i.e. to obtain a purified product II containing circular RNA. In each cycle of steps (1) and (2), new cellulose material is used in step (1). In the micro-separation column, the mass ratio of the mixture II containing circular RNA to the cellulose material is preferably (0.0005-0.01):1, such as 0.0005:1, 0.0008:1, 0.001:1, 0.002:1, 0.005:1, 0.008:1, 0.01:1 or any value therebetween.

[0056] In the present application, the Rnase R digestion process comprises providing a mixture I containing sample I containing circular RNA and Rnase R and buffer solution I, and separating the purified product I containing circular RNA after incubation of the mixture I. The separation method can be various existing RNA enrichment methods, such as LiCl precipitation, isopropanol precipitation, sodium acetate precipitation, nucleic acid purification magnetic beads, nucleic acid purification columns, gel recovery, etc. Preferably, the separation method can be a LiCl precipitation method to concentrate the incubated mixture I to enrich the purified product, i.e. to obtain a purified product I containing circular RNA. The specific embodiments of the LiCl precipitation method can be as described above.

[0057] In the Rnase R digestion process, the time of the incubation is preferably 5-60 min, more preferably 10-30 min, such as 10 min, 15 min, 20 min, 25 min, 30 min, or any value therebetween. The concentration of the sample I containing circular RNA in the mixture I is preferably 0.02-2.5 mg / mL, such as 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.4 mg / mL, 2.5 mg / mL, or any value therebetween. The concentration of Rnase R in the mixture I is preferably 0.1-10 IU / mL, such as 0.1 IU / mL, 0.2 IU / mL, 0.4 IU / mL, 0.8 IU / mL, 1 IU / mL, 2 IU / mL, 3 IU / mL, 4 IU / mL, 5 IU / mL, 6 IU / mL, 8 IU / mL, 10 IU / mL, or any value therebetween.

[0058] The buffer solution I preferably comprises a salt, magnesium chloride, and Tris. The concentration of the salt in the buffer solution I is preferably 0.1-1 M, such as 0.1 M, 0.2 M, 0.5 M, 0.8 M, 1 M, or any value therebetween. The concentration of magnesium chloride in the buffer solution I is preferably 0.1-2 mM, such as 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, or any value therebetween. The concentration of Tris in the buffer solution I is preferably 10-100 mM, such as 10 mM, 20 mM, 50 mM, 80 mM, 100 mM, or any value therebetween. The salt in the buffer solution I is preferably at least one selected from the group consisting of sodium chloride, lithium chloride, and potassium chloride.

[0059] In the present application, the alkaline phosphatase treatment comprises providing a mixture III containing the sample III containing circular RNA and the buffer solution III, and then isolating a purified product III containing circular RNA after incubating the mixture III. The isolation method can be any of the existing methods for enriching RNA, such as LiCl precipitation, isopropanol precipitation, sodium acetate precipitation, nucleic acid purification magnetic beads, nucleic acid purification columns, gel recovery, etc. Preferably, the isolation method can be a LiCl precipitation method for concentrating the incubated mixture III to enrich the purified product, i.e., obtaining the purified product III containing circular RNA. The specific embodiments of the LiCl precipitation method can be as described above.

[0060] The time of the incubation in the alkaline phosphatase treatment is preferably 15-120 min, more preferably 30-60 min, such as 30 min, 40 min, 50 min, 60 min, or any value in between. The concentration of the sample III containing circular RNA in the mixture III is preferably 0.02-2.5 mg / mL, such as 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.4 mg / mL, 2.5 mg / mL, or any value in between. The concentration of the alkaline phosphatase in the mixture III is preferably 50-500 IU / mL, such as 50 IU / mL, 100 IU / mL, 150 IU / mL, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, or any value in between.

[0061] The buffer solution III preferably comprises potassium acetate, magnesium acetate, and Tris. The concentration of the potassium acetate in the buffer solution III is preferably 10-200 mM, such as 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, or any value in between. The concentration of the magnesium acetate in the buffer solution III is preferably 1-10 mM, such as 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, or any value in between. The concentration of the Tris in the buffer solution III is preferably 10-100 mM, such as 10 mM, 20 mM, 50 mM, 80 mM, 100 mM, or any value in between.

[0062] The application will now be described in detail by way of specific examples.

[0063] Preparation of a preparation containing circular RNA

[0064] A circular RNA comprising a Coxsackievirus B3 internal ribosome entry site (CVB3 IRES) and a Nanoluciferase coding region, denoted Circ-NL, was synthesized in vitro.

[0065] The preparation process is as follows: (i) configuring the mixture of in vitro transcription synthesis reaction at room temperature, sequentially adding each component according to the order and volume in Table 1, after mixing, placing the mixed solution on a vortex shaker for 2s to ensure that the solution is mixed, and then reacting at 37℃ for 1h to complete the in vitro transcription synthesis reaction; (ii) adding 20μL of ultrapure water (DNase / RNase free), 3μL of cyclization buffer solution (500mM of Tris-HCl, 100mM of MgCl2, 10mM of DTT) to the reaction system, and reacting at 55℃ for 10min to complete the cyclization reaction; (iii) pre-precipitating the cyclization product in step (ii) by the LiCl precipitation method: adding 1 / 3 volume of 8M LiCl solution to the reaction system, cooling at-80℃ for 30min, then taking out the sample and centrifuging in a high-speed refrigerated centrifuge at 16000rpm for 20min, after centrifugation, a white sheet-shaped precipitate can be seen at the bottom of the test tube, carefully avoiding the white precipitate and removing the supernatant, then adding 400μL of pre-cooled 70% ethanol, centrifuging at 16000rpm for 20min at 4℃, removing the supernatant after centrifugation, and blowing dry the white precipitate in a nuclease-free environment until the precipitate becomes transparent to obtain the preparation containing Circ-NL.

[0066] Table 1

[0067] The reaction system in Table 1 can be proportionally expanded or reduced according to the specific circumstances of the experiment, and in this preparation example, the volume is according to the following table.

[0068] The content of each component in the preparation containing Circ-NL prepared by the above method was tested by capillary electrophoresis method, and the results are shown in Figure 1. The content level of the uncyclized linear RNA precursor in each impurity component is the highest. According to the test results, the purification treatment steps of the preparation can be selected as Rnase R digestion treatment, cellulose chromatography treatment, and alkaline phosphatase treatment.

[0069] The gene sequence of the DNA template (including the Nanoluciferase coding region) is SEQ ID NO: 1, and the gene sequence of Circ-NL is SEQ ID NO: 2.

[0070] Example 1 RR-WMC-Q purification treatment

[0071] (1) Rnase R digestion treatment (RR): 120 μg of the preparation containing circular Circ-NL, 1 μL of Rnase R (20 IU / mL, Haigene), 5 μL of 10x buffer solution I (0.5 M of sodium chloride, 1 mM of magnesium chloride, 50 mM of Tris) and appropriate amount of ultrapure water (DNase / RNase free) were mixed to form a 50 μL reaction system (the concentration of the preparation containing circular Circ-NL was 2.4 mg / mL, and the concentration of Rnase R was 0.4 IU / mL), and the reaction was incubated at 37°C for 40 min; the mixture after the incubation reaction was concentrated to enrich the purified product according to the LiCl precipitation method of step (iii) in Preparation Example 1, to obtain the purified product I-1 containing circular Circ-NL;

[0072] (2) Cellulose chromatography treatment (WMC): it was realized by fast protein liquid chromatography (FPLC), and the treatment process was shown in Figure 3. 0.4 g of cellulose material of basswood was loaded into a 1 mL chromatographic column, the chromatographic column was washed with 0.1 mM NaOH until the baseline was stable, the system was equilibrated with buffer solution II (16% (v / v) ethanol, 125 mM NaCl, 10 mM HEPES) to the baseline, 100 μg of the purified product I-1 containing circular Circ-NL was loaded by using a sample loop, the eluent containing circular RNA was collected, and the eluent was concentrated to enrich the purified product according to the LiCl precipitation method of step (iii) in Preparation Example 1, to obtain the purified product II-1 containing circular Circ-NL.

[0073] (3) Alkaline phosphatase treatment (Qip): 20 μg of the purified product II-1 containing circular Circ-NL, 1 μL of alkaline phosphatase (5000 IU / mL, NEB) and 2 μL of 10x buffer solution III (100 mM potassium acetate, 5 mM magnesium acetate, 60 mM Tris) and appropriate amount of ultrapure water (DNase / RNase free) were mixed to form a 20 μL reaction system (the concentration of the purified product II-1 containing circular Circ-NL was 1 mg / mL, and the concentration of alkaline phosphatase was 250 IU / mL), and the reaction was incubated at 37°C for 20 min; the mixture after the incubation reaction was concentrated to enrich the purified product according to the LiCl precipitation method of step (iii) in Preparation Example 1, to obtain the purified product III-1 containing circular Circ-NL.

[0074] As can be seen from the capillary electrophoresis results of FIG. 1 and FIG. 2, the peak of the uncircularized linear RNA precursor disappeared after RNase R digestion, and the proportion of high molecular weight RNA impurity components decreased from 7% to 1.5%, indicating that the purity of the circular RNA was significantly improved, from 64% to 89%, and the proportion of small intron RNA fragments (Intron) did not change significantly.

[0075] In the FPLC chromatogram of FIG. 4, the elution peak (0-3 min) is the purified product after treatment with linden cellulose chromatography, and the ratio of the absorption peak intensity at 260 nm to the absorption peak intensity at 280 nm is about 2, indicating that the product is a pure nucleic acid. As can be seen from the lateral chromatography strip assay (LFSA) test results of FIG. 5, the purified product II-1 after linden cellulose chromatography purification treatment does not indeed have double-stranded RNA components, indicating that cellulose chromatography treatment can efficiently remove double-stranded RNA impurity components. As can be seen from the capillary electrophoresis results of the purified product III-1 (FIG. 6), the purity of the circular RNA was improved from 89% to 96.3% after cellulose chromatography treatment and alkaline phosphatase treatment.

[0076] Example 2 RR-WMC-Q purification treatment

[0077] The purification of the circular RNA was carried out according to the method in Example 1, except that the cellulose chromatography treatment in step (2) was carried out by microseparation column method, and the treatment process is shown in FIG. 7: 0.0088 g of linden cellulose was weighed into a microseparation column, 700 μL of buffer solution II (16% (v / v) ethanol, 125 mM NaCl, 10 mM HEPES) was added, and shaken vigorously for 10 min. After centrifugation at 7000 rpm for 10 s, the supernatant was removed, 500 μL of buffer solution II was added, and shaken vigorously for 5 min. After centrifugation at 7000 rpm for 10 s, the supernatant was removed, and the washing operation was repeated 5 times. After centrifugation at 7000 rpm for 10 s, the eluent was removed, and the washing of the linden cellulose microseparation column was completed. 50 μg of the purified product I-2 containing circular Circ-NL and 210 μL of buffer solution II were added to the washed linden cellulose microseparation column, shaken vigorously for 3 min, and then centrifuged at 7000 rpm for 10 s. The eluent was collected. The eluent was added to another washed linden cellulose microseparation column, shaken vigorously for 3 min, and then centrifuged at 7000 rpm for 10 s. The collected eluent was the purified product of the linden cellulose microseparation column chromatography. The eluent was concentrated to enrich the purified product according to the LiCl precipitation method of step (iii) in Preparation Example 1, and the purified product containing circular Circ-NL was obtained, i.e. the purified product II-2 containing circular Circ-NL. The other conditions were the same as in Example 1, and the purified product III-2 containing circular Circ-NL was obtained.

[0078] From the capillary electrophoresis results of the preparation before purification and the purified product III-2 (Fig. 1 and Fig. 8), it can be seen that after the RR-WMC-Q purification treatment, the purity of the circular RNA is increased from 64% to 95.6%.

[0079] Example 3 RR-WMC-Q purification treatment

[0080] The purification of the circular RNA was carried out according to the method in Example 1, except that the composition and concentration of the reaction system in the Rnase R digestion treatment, the cellulose chromatography treatment and the alkaline phosphatase treatment were different, specifically: in step (1), 20 μg of the preparation containing circular Circ-NL, 1 μL of Rnase R (100 IU / mL, Haigene), 5 μL of buffer solution I (0.2 M lithium chloride, 0.5 mM magnesium chloride, 10 mM Tris) and an appropriate amount of ultrapure water (DNase / RNase free) were mixed to prepare a 50 μL reaction system (the concentration of the preparation containing circular Circ-NL was 0.4 mg / mL, and the concentration of Rnase R was 2 IU / mL); in step (2), buffer solution II (8% (v / v) of n-propanol, 120 mM lithium chloride, 5 mM HEPES) was used, and microcrystalline cellulose material was used; in step (3), 10 μg of the purified product II-3 containing circular Circ-NL, 1 μL of alkaline phosphatase (2000 IU / mL, NEB) and 2 μL of buffer solution III (10 mM potassium acetate, 2 mM magnesium acetate, 10 mM Tris) and an appropriate amount of ultrapure water (DNase / RNase free) were mixed to prepare a 20 μL reaction system (the concentration of the purified product II-3 containing circular Circ-NL was 0.5 mg / mL, and the concentration of alkaline phosphatase was 100 IU / mL), and the rest of the conditions were the same as in Example 1, i.e. to obtain the purified product III-3 containing circular Circ-NL.

[0081] From the capillary electrophoresis results of the preparation before purification and the purified product III-3 (Fig. 1 and Fig. 9), it can be seen that after the RR-WMC-Q purification treatment, the purity of the circular RNA is increased from 64% to 90.0%.

[0082] Example 4 RR-WMC-Q purification treatment

[0083] The purification of the circular RNA was performed according to the method in Example 1, except that the composition and concentration of the reaction system in the Rnase R digestion treatment, the cellulose chromatography treatment, and the alkaline phosphatase treatment were different, specifically: in step (1), 50 μg of the preparation containing the circular Circ-NL, 1 μL of Rnase R (250 IU / mL, Haigene), 5 μL of buffer solution I (0.8 M potassium chloride, 1.8 mM magnesium chloride, 100 mM Tris), and an appropriate amount of ultrapure water (DNase / RNase free) were mixed to form a 50 μL reaction system (the concentration of the preparation containing the circular Circ-NL was 1 mg / mL, and the concentration of Rnase R was 5 IU / mL); in step (2), buffer solution II (25% (v / v) isopropyl alcohol, 250 mM potassium chloride, 20 mM HEPES), and a carboxymethyl cellulose material were used; in step (3), 40 μg of the purified product II-4 containing the circular Circ-NL, 1 μL of alkaline phosphatase (8000 IU / mL, NEB), 2 μL of buffer solution III (200 mM potassium acetate, 10 mM magnesium acetate, 100 mM Tris), and an appropriate amount of ultrapure water (DNase / RNase free) were mixed to form a 20 μL reaction system (the concentration of the purified product II-4 containing the circular Circ-NL was 2 mg / mL, and the concentration of alkaline phosphatase was 400 IU / mL), and the remaining conditions were the same as in Example 1, i.e., the purified product III-4 containing the circular Circ-NL was obtained.

[0084] From the capillary electrophoresis results of the preparation before purification and the purified product III-4 (Fig. 1 and Fig. 10), it can be seen that after the RR-WMC-Q purification treatment, the purity of the circular RNA increased from 64% to 90.1%.

[0085] Comparative Example 1 RR-SEC-Q purification treatment

[0086] The purification of the circular RNA was performed according to the method in Example 1, except that the cellulose chromatography treatment in step (2) was achieved by molecular sieve chromatography (SEC), and the treatment process was as follows: after the system was cleaned with 0.1 mM NaOH until the baseline was stable, the system was equilibrated with 50 mM potassium phosphate buffer until the baseline was stable, a molecular sieve chromatography column (product number 215980P-4630) purchased from Sepax Technologies was connected to the system, 400 μg of the purified product I-1' containing the circular Circ-NL was loaded using a loading ring, and the purified product II-1' was collected according to the literature by collecting the second half of the elution peak 5 (as shown in Fig. 11). The remaining conditions were the same as in Example 1, and the purified product III-1' containing the circular Circ-NL was finally obtained.

[0087] Comparative Example 2 WMC treatment

[0088] Purification of circular RNA was performed only by cellulose chromatography treatment (WMC), which involved the same steps and conditions as the cellulose chromatography treatment in Example 1, to obtain purified product II-2' containing circular Circ-NL.

[0089] Comparative Example 3 Qip treatment

[0090] Purification of circular RNA was performed only by alkaline phosphatase treatment (Qip), which involved the same steps and conditions as the alkaline phosphatase treatment in Example 1, to obtain purified product III-3' containing circular Circ-NL.

[0091] Comparative Example 4 WMC-Q purification treatment

[0092] Purification of circular RNA was performed according to the method of Example 1, except that step (1) was not included, and the other conditions were the same.

[0093] Comparative Example 5 RR-Q purification treatment

[0094] Purification of circular RNA was performed according to the method of Example 1, except that step (2) was not included, and the other conditions were the same.

[0095] Test Example

[0096] (1) The recovery rate of circular RNA was tested using different purification methods (cellulose chromatography, cellulose micro-separation column method, and molecular sieve chromatography) in step (2), and the results are shown in Table 2.

[0097] Table 2

[0098] (2) Cell evaluation of circular RNA before and after purification

[0099] The cell function of the circular RNA purified product obtained by different purification methods in the above examples and comparative examples was tested using PMA-differentiated THP-1 cells and Hela cells, and the results are shown in Figure 12, and the specific operation is as follows:

[0100] (i) Under sterile conditions, HELA cells were inoculated in a 48-well plate at a concentration of 1 x 10 5 cells / mL, and after the cells were attached for 12 h, the circular RNA purified product solution obtained by different purification methods was transfected at a concentration of 200 ng / well; under sterile conditions, THP-1 cells were inoculated in a 48-well plate at a concentration of 5 x 10 5The cells were inoculated at 2 x 105cells / mL in a 48-well plate, and cultured with 100 ng / mL PMA for 24 h to differentiate the cells and adhere to the wall. The circular RNA purification product solutions obtained by different purification methods were transfected at a concentration of 100 ng / well.

[0101] (ii) After the transfected cells were cultured in a 37°C, 5% CO2 incubator for 24 h, they were taken out, placed at room temperature for 5-10 min, balanced to room temperature, and 100 ng / well of the circular RNA purification product solution was added to each well. The luciferase detection reagent (purchased from Promega Company, item number N1110) was mixed uniformly and centrifuged, and the luminescence signal was detected at room temperature for more than 3 min. The luminescence signal value of the unpurified sample was taken as 1, and the luminescence signal value of the other samples was calculated as the multiple of the luminescence signal value of the unpurified sample, thereby obtaining FIG. 12.

[0102] As can be seen from FIG. 12, the circular RNA (RR-WMC-Q) obtained by Rnase R digestion treatment, beech cellulose chromatography treatment, and alkaline phosphatase digestion has an expression effect comparable to that of the circular RNA (SEC-WMC-Q) obtained by Rnase R digestion treatment, molecular sieve chromatography treatment, and alkaline phosphatase digestion, and is superior to other single or two combinations of purification methods, fully proving that the purification method has a 1+1+1>3 effect in terms of biological efficacy.

[0103] (3) Immunological evaluation of circular RNA before and after purification

[0104] The A549 cells were used to compare the immunostimulatory properties of the circular RNA purification products before and after treatment with alkaline phosphatase in the above examples and comparative examples, and the results are shown in FIG. 13. The specific operation is as follows:

[0105] (i) Under sterile conditions, A549 cells were inoculated at 2 x 105cells / mL in a 24-well plate, and after 24 h of cell adhesion, the circular RNA purification product solutions before and after treatment with alkaline phosphatase were transfected at a concentration of 200 ng / well. 5 The cells were inoculated at 2 x 105cells / mL in a 48-well plate, and cultured with 100 ng / mL PMA for 24 h to differentiate the cells and adhere to the wall. The circular RNA purification product solutions obtained by different purification methods were transfected at a concentration of 100 ng / well.

[0106] (ii) 6h after transfection, the cells were lysed and total RNA was isolated using FastPure Cell / Tissue Total RNA Isolation Kit V2 (purchased from Vazyme, Cat No. RC112-01); 200 ng of total RNA was used as reverse transcription template according to the experimental protocol of the manufacturer using qScript cDNA Synthesis Kit (purchased from QuantaBio, Cat No. 733-1174); two-step qRT-PCR with annealing temperature of 58℃ was performed using Luna Universal qPCR Master Mix based on SYBR green technology in QuantStudio 3 and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific); the relative quantification of mRNA abundance was normalized using the average of housekeeping genes U1 small nuclear RNA (snRNA) and B2M, and the primers used are shown in Table 3.

[0107] Table 3

[0108] As can be seen from the test results of FIG. 13, the circular RNA purification product prepared by RR-WMC-Q has less stimulatory effect on natural immune recognition receptors RIG-I and TLR3 / 7 / 8, and has similar immunogenicity to the purification product prepared by RR-SEC-Q.

[0109] In summary, the circular RNA purification product obtained by the method provided by the present application using Rnase R digestion treatment, cellulose chromatography treatment and alkaline phosphatase treatment not only has low immunogenicity and good cell expression effect, but also, compared with the purification method of RR-SEC-Q, increases the recovery rate of circular RNA by more than 6 times while ensuring high purity and good biological efficacy of the circular RNA purification product, and can be applied to micro-separation column or fast protein liquid chromatography, and can realize industrial scale-up application of circular RNA purification.

[0110] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-described embodiments without departing from the principles and spirit of the present application within the scope of the present application.

Claims

1. A method for purifying a circular RNA, characterized by, The purification method includes the following steps: S1. Providing a preparation containing circular RNA; S2. Treating the preparation with RNase R digestion, cellulose chromatography and alkaline phosphatase to obtain purified circular RNA; The cellulose chromatography process includes providing a mixture II of sample II containing circular RNA and buffer solution II, and then contacting the mixture II with cellulose material to separate and obtain a purified product II containing circular RNA. The buffer solution II contains an organic alcohol and a salt, as well as optional complexing agents and buffering substances, at a concentration that allows double-stranded RNA to bind to the cellulose material but does not allow circular RNA to bind to the cellulose material. The cellulose material is selected from at least one of lignocellulose, microcrystalline cellulose, alpha-cellulose, carboxymethyl cellulose, carboxyethyl cellulose, and cellulose acetate.

2. The method of purifying a circular RNA according to claim 1, wherein, The RNase R digestion process includes providing a sample I containing circular RNA and a mixture I of RNase R and buffer solution I, and then incubating the mixture I to obtain a purified product I containing circular RNA.

3. The method of purifying a circular RNA according to claim 2, wherein, The alkaline phosphatase treatment includes providing a mixture III of sample III containing circular RNA and buffer solution III, incubating the mixture III, and then separating the purified product III containing circular RNA.

4. The method of purifying a circular RNA according to claim 2, wherein, In the RNase R digestion process, the concentration of sample I containing circular RNA in mixture I is 0.02-2.5 mg / mL.

5. The method of purifying a circular RNA according to claim 2, wherein, In the Rnase R digestion treatment, the concentration of Rnase R in mixture I is 0.1-10 IU / mL.

6. The method of purifying a circular RNA of claim 2, wherein, In the Rnase R digestion process, the buffer solution I contains salt, magnesium chloride and tris(hydroxymethyl)aminomethane, with the salt concentration being 0.1-1 M, the magnesium chloride concentration being 0.1-2 mM, and the tris(hydroxymethyl)aminomethane concentration being 10-100 mM.

7. The method of purifying a circular RNA of claim 1, wherein, In the cellulose chromatography process, the concentration of the organic alcohol in the buffer solution II is 5-40% (v / v), selected from at least one of n-propanol, isopropanol and ethanol.

8. The method of purifying a circular RNA of claim 1, wherein, In the cellulose chromatography process, the salt concentration in the buffer solution II is 100-300 mM, and is selected from at least one of sodium chloride, lithium chloride, and potassium chloride.

9. The method of purifying a circular RNA of claim 1, wherein, The complexing agent in buffer solution II is ethylenediaminetetraacetic acid (EDTA), with a concentration of 0.1-1 mM.

10. The method of purifying a circular RNA of claim 1, wherein, The buffering substance in buffer solution II is 4-hydroxyethylpiperazine ethanesulfonic acid and / or trihydroxymethylaminomethane, with a concentration of 5-20 mM.

11. The method of purifying a circular RNA of claim 1, wherein, The cellulose chromatography process includes (1) contacting a mixture II of sample II containing circular RNA and buffer solution II with cellulose material in a chromatographic column or microseparation column, and (2) applying gravity, centrifugal force, pressure or vacuum to the chromatographic column or microseparation column to separate the liquid phase from the solid phase, collecting the liquid containing circular RNA, and obtaining purified product II containing circular RNA.

12. The method of purification of a circular RNA according to claim 11, wherein, Step (1) involves contacting the mixture II containing the circular RNA sample II and the buffer solution II with the cellulose material under any of the following conditions: flow, shaking, and agitation.

13. The method for purifying circular RNA according to claim 11, characterized in that, In the chromatographic column, the mass ratio of sample II containing circular RNA to cellulose material is (0.00005-0.001):1; in the microseparation column, the mass ratio of sample II containing circular RNA to cellulose material is (0.0005-0.01):

1.

14. The method for purifying circular RNA according to claim 3, characterized in that, In the alkaline phosphatase treatment, the concentration of sample III containing circular RNA in mixture III is 0.02-2.5 mg / mL, and the concentration of alkaline phosphatase in mixture III is 50-500 IU / mL.

15. The method for purifying circular RNA according to claim 3, characterized in that, In the alkaline phosphatase treatment, the buffer solution III contains potassium acetate, magnesium acetate, and tris(hydroxymethyl)aminomethane, wherein the concentration of potassium acetate is 10-200 mM, the concentration of magnesium acetate is 1-10 mM, and the concentration of tris(hydroxymethyl)aminomethane is 10-100 mM.

16. The method for purifying circular RNA according to any one of claims 1 to 3, characterized in that, In step S1, the preparation containing circular RNA is at least one of circular RNA produced in vivo and extracted, circular RNA biosynthesized in vitro, and circular RNA biosynthesized in vitro and pretreated.

17. The method for purifying circular RNA according to claim 3, characterized in that, In the RNase R digestion process, the sample containing circular RNA is at least one of the preparation containing circular RNA provided in step S1, purified product II containing circular RNA obtained by cellulose chromatography, and purified product III containing circular RNA obtained by alkaline phosphatase treatment.

18. The method for purifying circular RNA according to claim 3, characterized in that, In the cellulose chromatography process, the sample containing circular RNA is at least one of the following: the preparation containing circular RNA provided in step S1, the purified product containing circular RNA obtained by RNase R digestion, and the purified product containing circular RNA obtained by alkaline phosphatase treatment.

19. The method for purifying circular RNA according to claim 3, characterized in that, In the alkaline phosphatase treatment, the sample containing circular RNA is at least one of the preparation containing circular RNA provided in step S1, purified product I containing circular RNA obtained by RNase R digestion, and purified product II containing circular RNA obtained by cellulose chromatography.

20. Circular RNA prepared by the purification method according to any one of claims 1 to 19.

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