Method for purifying circular RNA based on SDVB reversed-phase column

Abstract

The method for purifying the circular RNA is provided on the basis of the SDVB reversed-phase column, and the method can be used for industrially producing and purifying the circular RNA on a large scale. The method suitable for separating the circular RNA is obtained by exploring gradient changes of different organic phases, so that the circular RNA in the IVT product can be effectively separated. The invention establishes a method for reversely purifying circular RNA (Ribonucleic Acid) based on an SDVB (Sodium Dodecyl Vinyl Benzene) chromatographic column. The circular RNA purified and prepared by the method has relatively high purity and high cyclization rate, keeps relatively high in-vitro catalytic activity, and is suitable for large-scale analysis and purification of circular RNA molecules.

Description

Technical Field

[0001] The invention relates to the field of nucleic acid purification, and in particular to a method for purifying circular RNA based on an SDVB reverse phase column. Background Art

[0002] Reversed-phase high performance liquid chromatography is a liquid chromatography system composed of a non-polar stationary phase and a polar mobile phase, which is exactly the same as the liquid chromatography system composed of a polar stationary phase and a weakly polar mobile phase.

[0003] The typical stationary phase of RP-HPLC is octadecyl bonded silica gel, and the typical mobile phase is methanol and acetonitrile. RP-HPLC is the most important separation mode of liquid chromatography today, and can be used to separate almost all organic substances that can be dissolved in polar or weakly polar solvents. Reversed phase chromatography is suitable for separating non-polar, polar or ionic compounds, and most analytical tasks are completed by reverse phase chromatography.

[0004] mRNA vaccines introduce mRNA containing antigenic proteins into the human body, directly translate them, and form corresponding antigenic proteins, thereby inducing the body to produce specific immune responses and achieve the effect of preventive immunity. Circular RNA (circRNAs) is a new type of single-stranded, closed-loop RNA. Unlike traditional linear RNA (linearRNA, containing 5' and 3' ends), circRNA molecules have a closed ring structure, are not affected by RNA exonucleases, are more stably expressed, and are not easily degraded.

[0005] There are some published methods for purifying circular RNA. CN101563457A (published on October 21, 2009) discloses a method for preparing RNA purification, which is characterized by using a porous reverse phase as a stationary phase to purify RNA using HPLC. An important factor of the invention is the use of a porous reverse phase. CN112399860A discloses a method for purifying circular RNA, which includes passing RNA through a size exclusion column in a Tris-EDTA or citrate buffer in a high-performance liquid chromatography (HPLC) system to remove one or more of the following: intron fragments, linear RNA with cuts, linear and circular connections, and impurities produced by in vitro transcription and splicing reactions. CN115774075A (published on March 10, 2023) discloses a method for analyzing circRNA, a component of an in vitro transcription product, based on RP-HPLC. The invention uses RP-HPLC to separate circRNA, nickedRNA, and pre-mRNA linear RNA, and calculates the proportion of circRNA, i.e., the circularization rate, as a percentage of the peak area.

[0006] Monolithic columns are also known as the fourth generation of chromatography technology, which has the characteristics of high loading capacity, high flow rate and high resolution. Sartorius monolithic columns have been widely used in the CGT field. Sartorius CIM multus TM SDVB uses reversed-phase chromatography based on styrene-divinylbenzene (SDVB) to separate circular RNA from a mixture of truncated RNA, ssRNA and circular RNA.

[0007] Although HPLC is a conventional method for isolating RNA in the art, it is not suitable for large-scale industrial production. In order to improve the yield and conduct large-scale analysis and purification, the art urgently needs to provide a purification method that can be used in large-scale production. Summary of the invention

[0008] One of the purposes of the present invention is to provide a circular RNA purification method with good separation effect and large-scale application. The present invention explores the gradient changes of different organic phases to obtain a method suitable for circular RNA separation, thereby effectively separating circular RNA in IVT products.

[0009] In one aspect of the present invention, a method for purifying circular RNA based on SDVB reverse phase column is provided, characterized in that it comprises the following steps:

[0010] (1) Sample preparation: mRNA is prepared by IVT reaction, and circularized and purified to obtain circularized and purified circular RNA, circular RNA digested with RNaseR, and circular RNA digested with RNaseR and purified;

[0011] (2) Preparation of mobile phase:

[0012] Formula 1: Mobile phase A: 10-200mM neutral mixed solution of glacial acetic acid + triethylamine

[0013] (TEAA); Mobile phase B: 10-200mM neutral mixed solution of glacial acetic acid + triethylamine (TEAA)

[0014] +10-25% acetonitrile

[0015] Formulation 2: Mobile phase A: 5-25 mM N-diisopropylethylamine (DIPEA) + 10-100 mM hexafluoroisopropanol (HFIP); Mobile phase B: 5-25 mM N-diisopropylethylamine (DIPEA) + 10-100 mM hexafluoroisopropanol (HFIP) + 10-25% acetonitrile

[0016] Formula 3: Mobile phase A: 5-25mM TAE + 300-500mM hexafluoroisopropanol (HFIP); Mobile phase B: 5-25mM TAE + 300-500mM hexafluoroisopropanol (HFIP) + 10-25% acetonitrile; Formula 4: Mobile phase A: 50-250mM hexylamine acetate solution (HAA); Mobile phase B: 50-250mM hexylamine acetate solution (HAA) + 10-25% acetonitrile

[0017] (3) Chromatographic separation: set the gradient to 40-60%, 25.0-100.0CV, during which the main peak was collected according to the UV260 response value, and different peaks were collected separately;

[0018] (4) Collect the target chromatographic peaks of circular RNA and impurities respectively.

[0019] (5) Ultrafiltration of target peak: The ultrafiltration method is to replace the ultrafiltration tube and membrane package with twice the volume of the liquid, and replace 8-11 times.

[0020] Furthermore, in step (1), the IVT transcription time is 1-8 h, the cyclization temperature is 37-55° C., and the cyclization time is 5-60 min.

[0021] Furthermore, the purification method of step (1) includes but is not limited to LiCl precipitation and hydrophobic chromatography.

[0022] Furthermore, the in vitro transcription product in step (1) is any mRNA in vitro transcription product.

[0023] Furthermore, the method further comprises step (3) wherein the sample loading amount is 0.2-2.0 mg / ml.

[0024] Furthermore, the method also comprises step (3) of obtaining a chromatogram.

[0025] Furthermore, step (5) comprises: characterizing the ultrafiltered circular RNA by gel electrophoresis and analyzing it by a bioanalyzer.

[0026] Furthermore, step (5) comprises: performing long-term expression of the purified circular RNA in vitro.

[0027] The present invention discloses a method for purifying circular RNA based on SDVB reverse phase, comprising the following steps:

[0028] 1) Sample preparation: RNA products are obtained by transcription and then cyclized;

[0029] 2) Prepare the mobile phase: the mobile phase A is glacial acetic acid-triethylamine solution, and the mobile phase B is a mixed solution of glacial acetic acid-triethylamine solution and acetonitrile; or, the mobile phase A is glacial acetic acid-triethylamine solution or N-diisopropylethylamine (DIPEA)-hexafluoroisopropanol (HFIP) solution, and the mobile phase B is a mixed solution of glacial acetic acid-triethylamine solution and acetonitrile or a mixed solution of NN-diisopropylethylamine (DIPEA)-hexafluoroisopropanol (HFIP) solution and acetonitrile; or the mobile phase A is TAE-hexafluoroisopropanol (HFIP) solution, and the mobile phase B is a mixed solution of TAE-hexafluoroisopropanol (HFIP) solution and acetonitrile; or the mobile phase A is hexylamine acetate solution (HAA), and the mobile phase B is a mixed solution of hexylamine acetate solution (HAA) and acetonitrile. ;

[0030] 3) Loading: injecting the sample into a sample injection loop, using a styrene-divinylbenzene (SDVB) chromatography column;

[0031] 4) Elution: Set the gradient and elute at 25.0-100.0CV;

[0032] 5) Sample collection and ultrafiltration: The main peak eluate was collected according to the UV260 response value and concentrated by ultrafiltration.

[0033] Preferably, the mobile phase A in step 2) is 10-100 mM glacial acetic acid-triethylamine solution, and the mobile phase B is 10-100 mM glacial acetic acid-triethylamine solution + 10-25% acetonitrile.

[0034] Preferably, the loading amount in step 3) is 0.5-2 mg / mL.

[0035] Preferably, in step 2), the pH value of mobile phase A is 7.0±0.5, and the pH value of mobile phase B is 7.5.

[0036] Preferably, the preparation method of the mobile phase A is as follows: 5.6 ml of glacial acetic acid, 13.86 ml of triethylamine, add RNase-free water to make up to 1000 ml, and adjust the pH to 7.0±0.5. The volume configuration can be expanded.

[0037] Preferably, the preparation method of the mobile phase B is as follows: 5.6 ml of glacial acetic acid, 13.86 ml of triethylamine, 250 ml of acetonitrile, RNase-free water is added to make up to 1000 ml, and the pH is adjusted to 7. The volume configuration can be enlarged.

[0038] Preferably, the elution gradient in step 4) is 0-40% 12.5CV; 40-60% 75.0CV; 60-100% 12.5CV.

[0039] Preferably, the method further comprises the step of detecting the prepared concentrated circular RNA sample.

[0040] Preferably, the detection comprises one or more of gel electrophoresis, circularization rate analysis, and circular RNA activity detection.

[0041] Preferably, the circular RNA is human erythropoietin circular RNA (oRNA-hepo) or luciferase circular RNA (oRNA-luci).

[0042] The present invention provides a method for purifying circular RNA based on an SDVB reverse phase column. A method capable of industrial large-scale production of purified circular RNA is provided. The circular RNA purified and prepared by the method has high purity and high cyclization rate, and maintains high in vitro catalytic activity, thereby solving the problems of low purity and low activity of circular RNA in the prior art. The present invention is suitable for large-scale analysis and purification of circular RNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 a. SDVB chromatogram of oRNA-luci.

[0044] Figure 1 b. SDVB chromatogram of oRNA-hepo.

[0045] Figure 2 a. Circular luci gel electrophoresis diagram, where ORNA is circular RNA before column purification, and ORNA+SDVB is circular RNA after column purification.

[0046] Figure 2 b. Circular hepo gel electrophoresis diagram, where ORNA is circular RNA before column purification, and ORNA+SDVB is circular RNA after column purification.

[0047] Figure 3 a. Circular Luci 2100 bioanalyzer data graph.

[0048] Figure 3 b. Cyclic hepo 2100 bioanalyzer data plot.

[0049] Figure 4 a. SEC data of oRNA-luci.

[0050] Figure 4 b. SEC data of oRNA-hepo.

[0051] Figure 5 a. qPCR detection of Luci molecule circularization efficiency.

[0052] Figure 5b. qPCR detection of hepo molecule circularization efficiency.

[0053] Figure 6 a. In vitro activity data of oRNA-luci molecules, where liner is linear mRNA and ORNA+SDVB is circular RNA after column purification.

[0054] Figure 6 b. In vitro activity data of oRNA-hepo molecules, where liner is linear mRNA and ORNA+SDVB is circular RNA after column purification. DETAILED DESCRIPTION

[0055] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0056] Preparation of circular RNA:

[0057] Add enzyme-free water, buffer, NTP and enzyme to the EP tube in the following reaction system, and react at 37℃ for 1-4h. Add DNaseI to the transcription product at the end of the reaction and digest at 37℃. The reaction system can be scaled up in equal proportions.

[0058] Ingredients Volume(ul) 10xtranscriptionbuffer 2 ATP 2 UTP 2 CTP 2 GTP 2 T7RNAP 0.8 PPase 0.04 RRI 0.5 Template (1mg / ml) 1 Water for injection To 20

[0059] Cyclization process:

[0060] Add buffer and GTP buffer, mix well and react at 55℃ for 5-15min.

[0061] RNA precipitation: LiCl precipitation method was used to obtain purified RNA.

[0062] Preparation of circular RNA after RNaseR digestion:

[0063] RNA was digested according to the RNaseR instruction manual.

[0064] Or add 10ul Buffer and 1ul RNaseR to the transcription product after the reaction is completed, and react at 37℃ for 20-60min;

[0065] Preparation of circular RNA after RNaseR digestion and purification:

[0066] The circular RNA digested with RNaseR is recovered by LiCl precipitation to obtain the circular RNA digested with RNaseR and purified.

[0067] E-gel electrophoresis analysis experiment

[0068] 1. Perform denaturation pretreatment on RNA according to the equipment instruction manual.

[0069] 2. Place E-Gel TM EX agarose gel (2%) was placed in E-Gel TM PowerSnap electrophoresis instrument.

[0070] 3. Add the denatured mRNA to the electrophoresis instrument, start electrophoresis, and take pictures.

[0071] 2100 Bioanalyzer Analysis Experiment

[0072] 1. Pre-treat the samples and ladder according to the 2100 Bioanalyzer operating instructions and the Agilent RNA6000Nano kit instructions.

[0073] 2. Pre-treat samples and load ladder according to experimental settings.

[0074] 3. Bioanalizer test: Open the 2100export software, close the cover of the test chamber, and the corresponding chip number will be displayed; instum-assay-total RNAnano II. Select the corresponding program to start the test.

[0075] SEC-HPLC analysis of cyclization rate

[0076] Take an appropriate amount of circular RNA, refer to the detection SOP, and analyze the RNA purity using a molecular exclusion chromatography column (SEC-HPLC). Finally, the proportion of circular RNA, i.e., the circularization rate, is calculated as the percentage of the peak area.

[0077] qPCR detection of circularization efficiency

[0078] 1 μg of circular RNA was reverse transcribed using the Super-Script II Reverse Transcriptase Kit (Thermo Fisher). qRT-PCR was performed for 40 cycles using the Power SYBR Green Master Mix (Thermo Fisher). Relative expression was calculated using the △△CT method, and the circular sequence was normalized to the backbone sequence.

[0079] RNA transfection experiments

[0080] 1. Take out the cultured Cos7 cells, dilute to 1E5 / mL after digestion, add 1 ml to each well of a 24-well plate, and culture in an incubator overnight.

[0081] 2. Prepare the mRNA / circular RNA tube and transfection reagent tube according to the table below, mix well and place at room temperature for 5 minutes.

[0082] name mRNA / Lipo2000 Opti-MEM mRNA tube (luci / epo) 1ug To50ul Transfection reagent tube 2.5ul To50ul

[0083] 3. Add Lipo2000 dilution into the mRNA tube, mix gently and let stand for 15 minutes.

[0084] 4. Take out the 24-well plate, remove the supernatant, add 500ul of fresh Opti-MEM medium to each well, add 100ul of transfection complex to each well, shake gently to mix, and place in the incubator for continued culture.

[0085] Hepo activity detection:

[0086] The supernatant was collected at 24 and 48 h after hepo transfection and used for detecting the hepo expression level using ELISA kit (Abcam).

[0087] LucI activity detection

[0088] 1. 24 hours after transfection, remove the plate, discard the supernatant, add 500ul PBS buffer to rinse once, and discard the supernatant.

[0089] 2. Add 105ul of pre-diluted lysis buffer to cover the cells and leave at room temperature for 15 minutes.

[0090] 3. Transfer the lysate to an EP tube and centrifuge at 13,000 rpm for 10 min.

[0091] 4. Add the supernatant to a 96-well plate, 30ul per well, repeat three wells for each sample, add 30ul of luciferin substrate in a dark place, and read the plate with an ELISA reader.

[0092] 5. NanoDrop A205 was used to measure protein concentration and normalize the concentration of cell lysate.

[0093] Example 1 Preparation of oRNA-luci after cyclization and purification

[0094] 1. Prepare oRNA-luci as a sample by in vitro transcription according to the specific implementation method;

[0095] 2. Mobile phase preparation: Mobile phase A preparation: Prepare a mixed solution of 50 mM glacial acetic acid + triethylamine (TEAA) and adjust the pH to 7.0 ± 0.5;

[0096] 3. Preparation of mobile phase B: Prepare a mixed solution of 50 mM glacial acetic acid + triethylamine (TEAA), add acetonitrile to a final concentration of 15%, and adjust the pH to 7.5.

[0097] 4. Chromatography: The chromatographic column used is a styrene-divinylbenzene (SDVB) chromatography column.

[0098] (1) Set the flow rate to 2 ml / min, use mobile phase A to equilibrate the instrument and equipment, and flush until the baseline is stable;

[0099] (2) Inject the sample into the injection loop and switch to inject;

[0100] (3) Continue to use mobile phase A to equilibrate the instrument and equipment, and flush until the baseline is stable;

[0101] (4) The gradient was set to 0-40% 12.5CV; 40-60% 75.0CV; 60-100% 12.5CV, during which different main peaks were collected according to the UV260 response value, and then the collected liquid of the circular RNA peak was ultrafiltered to obtain the oRNA-luci purified sample.

[0102] 5. The obtained samples are used for inspection or activity experiments.

[0103] 6. Test results Figure 1 a- Figure 6 a. Among them, Figure 1 a shows the SDVB chromatography, showing the effective separation of circular RNA; Figure 2 a is a diagram of annular luci glue; Figure 3 a is a graph of the circular luci 2100 bioanalyzer data; Figure 4 a is luci SEC data; Figure 5 a qPCR detection of luci molecule circularization efficiency; Figure 6 a is the in vitro activity data of luci molecule.

[0104] Example 2 Preparation of oRNA-hepo after RNaseR digestion and purification

[0105] 1. Prepare oRNA-hepo as a sample by in vitro transcription according to the specific implementation method;

[0106] 2. Mobile phase preparation: Mobile phase A preparation: Prepare a mixed solution of 50 mM glacial acetic acid + triethylamine (TEAA) and adjust the pH to 7.0 ± 0.5;

[0107] 3. Preparation of mobile phase B: Prepare a mixed solution of 50 mM glacial acetic acid + triethylamine (TEAA), add acetonitrile to a final concentration of 15%, and adjust the pH to 7.0±0.5.

[0108] 4. Chromatography: The chromatographic column used is a styrene-divinylbenzene (SDVB) chromatography column.

[0109] (1) Set the flow rate to 2 ml / min, use mobile phase A to equilibrate the instrument and equipment, and flush until the baseline is stable;

[0110] (2) Inject the sample into the injection loop and switch to inject;

[0111] (3) Continue to use mobile phase A to equilibrate the instrument and equipment, and flush until the baseline is stable;

[0112] (4) The gradient was set to 0-40% 12.5CV; 40-60% 75.0CV; 60-100% 12.5CV, during which different main peaks were collected according to the UV260 response value, and the collected liquid of the circular RNA peak was ultrafiltered to obtain the oRNA-epo purified sample.

[0113] 5. The obtained samples are used for inspection or activity experiments.

[0114] 6. Test results Figure 1 b- Figure 6 b. Among them, Figure 1 b shows the SDVB chromatography, showing the effective separation of circular RNA; Figure 2 b is a picture of annular hepo gel; Figure 3 b is the data graph of cyclic hepo 2100 bioanalyzer; Figure 4 b is hepo SEC data; Figure 5 b qPCR detection of hepo molecule circularization efficiency; Figure 6 b is the in vitro activity data of hepo molecule.

[0115] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for purifying circular RNA based on SDVB reverse phase, characterized in that: The steps include: 1) Sample preparation: RNA products are obtained by transcription and then cyclized; 2) preparing a mobile phase: wherein the mobile phase A is a glacial acetic acid-triethylamine solution, and the mobile phase B is a mixed solution of a glacial acetic acid-triethylamine solution and acetonitrile; or, the mobile phase A is a glacial acetic acid-triethylamine solution or a N-diisopropylethylamine (DIPEA)-hexafluoroisopropanol (HFIP) solution, and the mobile phase B is a mixed solution of a glacial acetic acid-triethylamine solution and acetonitrile or a mixed solution of a N-diisopropylethylamine (DIPEA)-hexafluoroisopropanol (HFIP) solution and acetonitrile; or the mobile phase A is a TAE-hexafluoroisopropanol (HFIP) solution, and the mobile phase B is a mixed solution of a TAE-hexafluoroisopropanol (HFIP) solution and acetonitrile; or the mobile phase A is an acetic acid hexylamine solution (HAA), and the mobile phase B is an acetic acid hexylamine solution (HAA) and acetonitrile. 3) Loading: injecting the sample into a sample injection loop, using a styrene-divinylbenzene (SDVB) chromatography column; 4) Elution: Set the gradient and elute at 25.0-100.0CV; 5) Sample collection and ultrafiltration: The main peak eluate was collected according to the UV260 response value and concentrated by ultrafiltration.

2. The method according to claim 1, characterized in that: The mobile phase A in step 2) is 10-100 mM glacial acetic acid-triethylamine solution or N-diisopropylethylamine-hexafluoroisopropanol solution or TAE-hexafluoroisopropanol (HFIP) solution or hexylamine acetate solution (HAA), and the mobile phase B is 10-100 mM glacial acetic acid-triethylamine solution or N-diisopropylethylamine-hexafluoroisopropanol solution or TAE-hexafluoroisopropanol (HFIP) solution or hexylamine acetate solution (HAA) and acetonitrile mixed solution.

3. The method according to claim 1, characterized in that: The loading amount in step 3) is 0.5-2 mg / mL.

4. The method according to claim 2, characterized in that: The pH value of mobile phase A in step 2) is 7.0±0.5, and the pH value of mobile phase B is 7.

5.

5. The method according to claim 1, characterized in that: The elution gradient in step 4) was 0-40% in 12.5CV; 40-60% in 75.0CV; 60-100% in 12.5CV.

6. The method according to claim 1, characterized in that: The method further comprises the step of detecting the prepared concentrated circular RNA sample.

7. The method according to claim 6, characterized in that: The detection includes one or more of gel electrophoresis, cyclization rate analysis, and circular RNA activity detection.

8. The method according to claim 1, characterized in that: The circular RNA is human erythropoietin circular RNA (oRNA-hepo) or luciferase circular RNA (oRNA-luci).

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