Preparation method and application of circular RNA (Ribonucleic Acid)

Abstract

The invention provides a preparation method and application of circular RNA, the ring opening rate of the circular RNA is reduced through preparation of the circular RNA, the loss of the circular RNA is reduced, meanwhile, the purity of a product is improved, and RNA molecules after in-vitro cyclization can be effectively transfected into cells. The invention also provides a preparation method and application of the circular RNA. In some embodiments, compared with existing RNA cyclization methods, the method provided by the invention has the advantages that the product purity is improved, and the ring opening efficiency is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and bioengineering, and in particular, relates to a vector for preparing circular RNA and application thereof in protein expression. Background Art

[0002] Circular RNA (circRNA) is a type of covalently closed circular molecule formed by reverse splicing and alternative splicing of precursor mRNA, with no free ends. Unlike linear mRNA, the structural specificity of circular RNA gives it a unique advantage in the development of vaccines and nucleic acid drugs, and can be used to design and produce stable forms of mRNA. The protein translation of circRNA must use a non-classical initiation mechanism, that is, translation initiation that does not rely on the 5'-cap. This initiation pathway is often driven by IRES (internal ribosome entry site), a short RNA fragment with a special secondary structure.

[0003] IRES was first identified in the late 1980s in the 5′-UTR of the poliovirus (PV) and encephalomyocarditis virus (EMCV) genomes, both of which belong to the picornavirus family. Since most viruses have limited resources for encoding proteins in their genomes and lack the ability to replicate independently, they rely on hijacking the host cell machinery for replication and survival. Picornaviruses, on the other hand, obtain the prerequisites for efficient protein synthesis through sequences called their own unique IRES. At present, at least five different types of IRES have been found in picornaviruses, each of which is characterized by a unique secondary structure and eIF / ITAF requirements. Type I IRES-containing viruses are assisted by various initiation factors and host cell ITAFs, and utilize ribosome scanning mechanisms to identify the start codon; Type II IRES ribosomes are recruited through the second start codon with the assistance of various initiation factors and ITAFs; Type III IRES exists in hepatitis A virus, and translation initiation requires initiation factors, ITAFs, and complete eIF4E; Type IV IRES is found in flaviviruses, and the uniqueness of this type of IRES is that it does not require any initiation factors and ITAFs to initiate its translation; Type V IRES exists in Aichi virus A of the Picornaviridae family, and effective translation initiation requires the DExH-box protein DHX29. It is worth noting that, even if the primary structures of the same type of IRES are different, their secondary structures are basically conserved between viral species, and these structures are very important for whether IRES can initiate translation.

[0004] Among the five types, the most widely verified and highly active viral IRESs all belong to type I IRES, such as EV-A71, EV-29, CVB3 and HRV. This type of IRES is mainly found in enteroviruses and rhinoviruses. The structure of type I IRES is mainly divided into 6 domains (I to VI), and different domains have their own unique functions; domain I forms a unique cloverleaf structure and participates in the replication and translation of the viral genome, while domains II to VI are responsible for the activity of IRES. Domain III is required for IRES activity and is responsible for interacting with ITAF. Domain IV has multiple important regions, including C-rich motifs, which are key essential regions for binding to the trans-acting factor poly(rC) binding protein 2 (PCBP2); it also contains a conserved GNRA tetraloop, which is considered to be a primer for long-distance RNA-RNA interactions during the formation of the tertiary structure and is the main regulator of type I IRES activity, but its role in type I IRESs has yet to be fully studied. eIF4G bound by domain V and the conserved AUG in domain VI mediate the attachment of the 43S ribosome preinitiation complex, followed by the ribosome scanning for the start codon and initiation of translation.

[0005] At present, the activity of IRES is related to the secondary / tertiary structure after its RNA folding, and a large amount of data confirms that type I / II IRES mainly exist in enterovirus and rhinovirus in the Picornaviridae family. Therefore, a large number of viruses in the Picornaviridae family were found and summarized, and then by comparing with the type I IRES model structure and the secondary structure of the confirmed and highly active IRES, a series of viral IRES with similar structures were found and their activities were verified, providing a reference for our subsequent research on the activity of type I IRES.

[0006] Although linear mRNA can achieve mRNA-mediated protein expression, its expression duration is short and the protein expression is insufficient, so it is necessary to develop new mRNA technologies with the ability to express proteins persistently. The target protein can be translated by circular RNA, and the protein expression of the target protein translated by circular RNA can be increased to a certain extent.

[0007] During the preparation of circular RNA, circular RNA is easily affected by reaction temperature and metal ions (mainly Mg2+) to produce open-circular RNA (nick). This type of RNA greatly reduces the high stability of circular RNA and the high expression of subsequent proteins. Summary of the invention

[0008] In order to reduce the nicking rate, we try to avoid the nicking of some circular RNAs produced during transcription during the subsequent cyclization process, and reduce the different IRES activities caused by inconsistent nicking rates during IRES screening, thereby affecting the judgment of high and low IRES activity. Therefore, we optimized the transcription system and purification method. Transcription is a process of synthesizing RNA under the catalysis of RNA polymerase using linearized DNA plasmid as a template and four nucleoside triphosphates, ATP, CTP, GTP, and UTP, as raw materials. During the entire reaction process, the residual amount of NTP is related to the demand for the four bases of RNA. After all the raw materials are almost consumed, the previously generated precursor RNA may be affected by the residual GTP and Mg in the transcription reaction buffer. 2+ Therefore, in order to reduce the cyclization of precursor RNA during transcription, we optimized the NTP dosage and transcription time in the transcription system to reduce the nick rate.

[0009] The purpose of circular RNA purification is to remove some uncircularized linear RNA and byproducts of intron splicing, thereby enriching CircRNA. So far, the widely used size exclusion chromatography (SEC) can separate RNA according to the molecular weight of nucleic acid due to the unique physical properties of its column filler. However, the existing purification conditions (mobile phase components, pH value, flow rate during elution) will cause more circular RNA to open during the purification process. In order to solve this problem, we set different purification conditions for verification, and finally improved the purification effect.

[0010] The circular RNA preparation of the present invention reduces the opening rate of the circular RNA, reduces the loss of the circular RNA, and simultaneously improves the purity of the product by changing the purification method, and the RNA molecules after in vitro circularization can be effectively transfected into cells.

[0011] The purpose of the present invention is to provide a method for preparing circular RNA.

[0012] The specific steps include: preparing precursor RNA by in vitro transcription through a vector, synthesizing the precursor RNA using NTP with a working concentration of 5mM to 12.5mM, and cyclizing the precursor RNA molecule to obtain circular RNA;

[0013] The vector comprises: a T7 promoter, a 5' external homology arm, a 3' intron, a 3' splice site, a 5' internal homology arm, a 5' spacer, a BbsI restriction enzyme site, a 3' internal homology arm, a 5' splice site, a 5' intron, a 3' external homology arm, and a restriction enzyme site HindIII for plasmid linearization, and an internal ribosome entry site (IRES).

[0014] As a preferred embodiment of the present invention, the IRES comprises any one of SEQ ID NOs. 1-66.

[0015] As a preferred embodiment of the present invention, the feasibility of the technical solution of the present invention is verified by using NTPs with working concentrations of 7.5mmol / L, 10mmol / L, and 12.5mmol / L to synthesize the precursor RNA. Further preferably, the IRES preferably includes SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.28, SEQ ID NO.48, SEQ ID NO.49, or SEQ ID NO.61.

[0016] As a preferred embodiment of the present invention, circular RNA purification is also included.

[0017] As a preferred embodiment of the present invention, the pH value of the mobile phase for circular RNA purification ranges from 6 to 8, and the mobile phase includes EDTA.

[0018] As a preferred embodiment of the present invention, the pH of the mobile phase is 6.0, 7.0, 7.4 or 7.5.

[0019] As a preferred embodiment of the present invention, the pH of the mobile phase is 7.5.

[0020] As a preferred embodiment of the present invention, the mobile phase contains EDTA.

[0021] The present invention also provides a method for preparing circular RNA, the steps comprising: preparing precursor RNA by in vitro transcription through a vector, cyclizing the obtained precursor RNA molecule, and purifying the obtained circular RNA, wherein the pH value of the mobile phase during the purification of the circular RNA is in the range of 6 to 8;

[0022] The vector includes a T7 promoter, a 5' external homology arm, a 3' intron, a 3' splice site, a 5' internal homology arm, a 5' spacer, a BbsI restriction enzyme site, a 3' internal homology arm, a 5' splice site, a 5' intron, a 3' external homology arm, and a restriction enzyme site HindIII for plasmid linearization, and an internal ribosome entry site (IRES).

[0023] As a preferred embodiment of the present invention, NTP with a working concentration of 5 mmol / L to 12.5 mmol / L is used to synthesize the precursor RNA.

[0024] As a preferred embodiment of the present invention, the mobile phase includes EDTA.

[0025] The present invention also provides a circular RNA prepared according to the above technical solution.

[0026] The circular RNA described in the present invention can encode hFIX, SPB, VEGF-A, human methylmalonyl CoA variant enzyme, CFTR, cancer autoantigens, gene editing enzymes, HIV antibodies, CD19 antibodies, CD22 antibodies, CD3 antibodies, CLDN6 antibodies, Luc2, GLuc, Fluc, eGFP, hEPO, Cas9 nuclease, new coronavirus S protein or tumor suppressor factors.

[0027] As a preferred embodiment of the present invention, the circular RNA encodes Luc2 or Fluc.

[0028] As a preferred embodiment of the present invention, the protein coding region or non-coding region is the Fluc gene, which verifies the feasibility of the technical solution of the present invention.

[0029] A method for expressing a protein in a cell of the present invention comprises transfecting a circular RNA into the cell.

[0030] Preferably, the cell is a eukaryotic cell.

[0031] In the present invention, the ring-opening rate of the circular RNA is reduced during the preparation process of the circular RNA, while the purity of the product is increased, and the translation level of the circular RNA is improved. And the RNA molecules after the in vitro cyclization are effectively transfected into the cell, and compared with the prior art, the present invention has the following beneficial effects: in order to reduce the cyclization of the precursor RNA during the transcription process, we optimize the NTP dosage and transcription time in the transcription system, and reduce the ring-opening (nick) rate. The purpose of circular RNA purification is to remove some uncircularized linear RNA and the byproducts after intron splicing, so as to enrich the circular RNA. The present invention optimizes the purification conditions, and finally enriches more circular RNA, increasing the yield of RNA. And the circularized RNA shows a good transfection effect in the cell, and the application value is extremely extensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the secondary structure diagram of the viral IRES sequence;

[0033] Figure 2 It is a linear relationship diagram between NTP concentration and precursor RNA yield;

[0034] Figure 3 This is a picture of circular RNA in the product detected by agarose gel electrophoresis;

[0035] Figure 4 To detect the cyclization rate of precursor RNA by RP-HPLC;

[0036] Figure 5 is the ring opening rate of circular RNA in different mobile phases;

[0037] Figure 6 Schematic diagram of the construction process of circular RNA plasmid;

[0038] Figure 7 This is a graph showing the luciferase activity detection of viral IRES in 293T cells;

[0039] Figure 8 This is a graph showing the luciferase activity detection of viral IRES in HepG2 cells;

[0040] Fig. 9 This is a graph showing the luciferase activity detection of viral IRES in A549 cells;

[0041] Fig.10 It is the ratio diagram of luciferase activity mediated by viral IRES and CVB3;

[0042] Fig.11 Figure 1 is a diagram of circular RNA purification;

[0043] Fig.12 This is the fluorescence expression diagram of circular RNA in vivo;

[0044] Fig.13 This is a fluorescence expression diagram of circular RNA in organs in vivo. DETAILED DESCRIPTION

[0045] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0046] Example 1: IRES sequence search

[0047] By consulting the virus database (VIPR) and screening out viruses of the genus Picornaviridae, their secondary structures were analyzed using RNA folding Form Version 2.3, and compared with the verified high-activity IRES (CVB3), viral IRES sequences with similar secondary structures, low free energy, and more stable structures were selected to verify their activity. The structures are shown in Figure 1 .

[0048] Example 2: In vitro transcription

[0049] Using the linearized plasmid as a template, the T7 transcription kit (T7 High Yield RNA Transcription Kit) was used to synthesize the precursor RNA using different working concentrations of NTP (7.5mM, 10mM, 12.5mM). Where mM is mmol / L, and NTP in this embodiment includes ATP, CTP, GTP and UTP. The reaction system is shown in Tables 1-3 below. After the reaction is completed, the linearized template is digested with DNase I (37°C-15min). Wherein, DNase I is deoxyribonuclease I.

[0050] Table 1

[0051]

[0052]

[0053] Table 2

[0054]

[0055] Table 3

[0056]

[0057] Example 3: Detection of cyclization rate in precursor RNA

[0058] Add DEPC water to the above reaction system, mix well, add 8 mol LiCl, place at -80°C for 20-30 min, then place at 4°C, centrifuge at 12000 rpm for 20 min, discard the supernatant, wash the precipitate twice with 75% ethanol, discard the supernatant, dry the precipitate thoroughly, and finally add 100uL DEPC water to re-dissolve. Take 1uL sample for Nanodrop One C (Thermo) to determine RNA concentration and calculate RNA yield, take 500ngRNA for agarose gel electrophoresis, the results are shown in Figure 2 and Figure 3 , as shown in the figure: high concentration of NTP can reduce the production of circular RNA without affecting the production of precursor RNA, and can effectively control the subsequent circular RNA opening rate.

[0059] RP-HPLC detection of circular RNA in the transcription product: DNAPac-RP column (Thermo Fisher, 4μm-1500A., 2.1x100mm) was used to detect circular RNA, where mobile phase A was 0.1M TEAA (PH7.0), mobile phase B was 0.1M TEAA and 20% CAN, elution gradient was 20-80%, flow rate was 0.2-0.6mL / min. The results are shown in Figure 2. Figure 4As shown, in the process of circular RNA preparation, the transcription system in Table 3 is optimal and can produce the least circular RNA without affecting the RNA yield.

[0060] The above results show that the residual amount of NTP in the entire in vitro transcription reaction is related to the actual demand for the four bases of RNA. When all the raw materials are almost consumed, the presence of residual GTP and divalent magnesium ions in the transcription reaction buffer causes the previously generated precursor RNA to partially cyclize. By optimizing the amount of NTP and transcription time in the transcription system, the cyclization of precursor RNA during transcription can be effectively reduced, and it also has a certain reference significance for the occurrence and quantification of the subsequent circular RNA opening rate.

[0061] Example 4. Optimization of circular RNA purification method

[0062] Different mobile phases were configured. The specific composition formula of the mobile phase is shown in Table 4.

[0063] Table 4

[0064] Mobile phase Components PH 1 Na2HPO4.12H2O, KH2PO4.2H2O 7.0 2 Tris-HCL, EDTA, PB 7.4 3 Tris-HCL, EDTA 6.0 4 Tris-HCL, EDTA 7.5

[0065] 1. Use SRT SEC-1000 (Saifen Technology, 5μm-1000A°-300mm x 21.2mm, NO: 215950-10030) purification column to separate and purify with different mobile phases and flow rates.

[0066] 2. RP-HPLC detection of ring-opening rate: A DNAPac-RP column (Thermo Fisher, 4 μm-1500A°, 2.1x100 mm) was used to detect circular RNA, wherein mobile phase A was 0.1 M TEAA (PH7.0), mobile phase B was 0.1 M TEAA and 20% CAN, the elution gradient was 20-80%, and the flow rate was 0.2-0.6 mL / min.

[0067] The existing purification methods for circular RNA (mobile phase components, pH value, flow rate during elution) will cause more circular RNA to open during the purification process. In order to solve this problem, we set different purification conditions for verification. Figure 5 As shown, at the same flow rate (2 mL / min), after SEC purification using mobile phase 1, mobile phase 2, and mobile phase 3, the ring-opening rate of circular RNA was much higher than that of unpurified circular RNA; while after SEC purification using mobile phase 4, the ring-opening rate of circular RNA was basically the same as that before purification.

[0068] EDTA is a complex that can bind to some divalent metal ions (Mg2+), which can prevent metal ions from activating proteases and reduce the impact of metal ions on nucleic acid quality. We speculate that adding a certain amount of EDTA to the mobile phase of SEC purification may have a certain inhibitory effect on ring opening. The above results confirm our idea that during SEC purification, the mobile phase components and pH value are the main factors causing circular RNA ring opening. Mobile phases with high pH and EDTA in the components can effectively inhibit circular RNA ring opening.

[0069] Example 5, IRES screening

[0070] Figure 6 Schematic diagram of the IRES screening process

[0071] 1. Plasmid construction

[0072] A circular RNA empty vector for screening IRES was constructed, which included a T7 promoter, a 5' external homology arm, a 3' intron, a 3' splice site, a 5' internal homology arm, a 5' spacer, a BbsI restriction enzyme site, a 3' internal homology arm, a 5' splice site, a 5' intron, a 3' external homology arm, and a restriction enzyme site HindIII for plasmid linearization. Table 5 shows the circular RNA vector backbone information.

[0073] Table 5

[0074]

[0075]

[0076] 2. Construct a circular RNA vector containing an IRES sequence, and insert the IRES sequence to be screened into the BbsI restriction site of the circular RNA empty vector. Table 6 shows the IRES sequence information.

[0077] Table 6

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] 3. Plasmid linearization-transcription template preparation

[0097] HindIII restriction endonuclease linearizes the circular RNA plasmid containing the IRES sequence. The linearized plasmid is purified by a large number of DNA gel recovery kits and then used for in vitro transcription. The reaction system is shown in Table 7.

[0098] Table 7

[0099]

[0100] IV. Preparation of Precursor RNA by In Vitro Transcription

[0101] The linearized plasmid was used as a template and the precursor RNA was synthesized using the T7 High Yield RNA Transcription Kit. The specific reaction system is shown in Table 3. After the reaction, the linearized template was digested with DNase I (37°C-15min). The precursor RNA was then purified.

[0102] 5. Precursor RNA Circularization

[0103] The cyclization reaction buffer was configured according to Table 8, and the cyclization reaction system was configured according to Table 9. After the reaction was completed, 500 ng was taken for agarose gel electrophoresis to detect whether cyclization was achieved.

[0104] Table 8

[0105] name Components 10× cyclization buffer 500mM Tris-HcL (PH7.5), 100mM MgCL2, 10mM DTT

[0106] Table 9

[0107]

[0108] 6. Ultrafiltration Concentration

[0109] Circular RNA was concentrated by ultrafiltration using a 1.5 mL 100KD ultrafiltration tube.

[0110] 7. Circular RNA Purification

[0111] The concentrated circular RNA was purified using the above-mentioned improved SEC purification column. During the purification process, 1X TE (pH 7.5) was used as the mobile phase and the sample was eluted at a flow rate of 2mL / min. The circular RNA was ultrafiltered and concentrated using a 1.5mL 100KD ultrafiltration tube. Finally, 1uL of the sample was used to determine the RNA concentration using Nanodrop One C (Thermo), and 500ng was used for agarose gel electrophoresis (1.5% agarose, 1XTBE, 1 / 10000GelGreen) to detect the integrity of the circular RNA.

[0112] Example 6. Expression of circular RNA in cells

[0113] Before transfection, 293T cells, HepG2 cells and A549 cells were cultured at 3×10 4 The cells were inoculated at a density of cells / well in a 96-well white plate and cultured in an incubator (37°C, 5% CO2). When the cell confluence was about 75%, the cells were transfected with 200ng mRNA / well using Lipofectamine 3000 (Invitrogen) reagent. The specific operations were carried out according to the reagent instructions. F-Luc fluorescence detection, 18 hours after cell transfection, the cells were collected, and the fluorescence expression of the cells was detected using the Bright-LiteTM Luciferase Assay System kit. The data showed that the activity of the four sequences (Rabovirus A / SDS9 / 01, Enterovirus J-CHN / BJ / 2018-2J, Bopivirus sp.-bovine / TV-9682 / 2019-HUN and SAFV-3_NL1999-590) in the three cells was higher than that in the control group. See the results. Figure 7-10 (Control group: CVB3).

[0114] Example 7: Expression of circular RNA in animals

[0115] Circular RNA was prepared according to the above Example 5, and the electrophoresis results after purification were shown in Fig.11. SM102-LNP was used to encapsulate circular RNA. 100ul of 5ug h circular RNA was drawn up with an insulin syringe each time, and the drug was injected into the tail vein of the mouse by intravenous injection. 6 hours after administration, 100ul of a mixture of 30mg / ml D-luciferin potassium salt and 5% anesthetic was injected intraperitoneally. After 5 minutes, the mice were dissected, and the liver and spleen were taken for organ imaging. The results of in vivo and organ (liver / spleen) imaging showed that compared with the control group (control group 1: CVB3; control group 2: EV29), 3-4 of the six sequences mediated higher Luci expression levels, among which the IRES sequence named Rabovirus A / SDS9 / 01d had the highest activity, followed by SAFV-3_NL1999-590. Results are shown in Figure 12-13 (Control group: CVB3).

[0116] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing circular RNA, characterized in that: The steps include: Precursor RNA is prepared by in vitro transcription of a vector, the precursor RNA is synthesized using NTP at a working concentration of 5 mmol / L to 12.5 mmol / L, and the obtained precursor RNA molecule is cyclized to obtain circular RNA; The vector includes a T7 promoter, a 5' external homology arm, a 3' intron, a 3' splice site, a 5' internal homology arm, a 5' spacer, a BbsI restriction enzyme site, a 3' internal homology arm, a 5' splice site, a 5' intron, a 3' external homology arm, and a restriction enzyme site HindIII for plasmid linearization, and an internal ribosome entry site (IRES).

2. The method for preparing circular RNA according to claim 1, characterized in that: The IRES comprises any one of SEQ ID NOs. 1-66.

3. The method for preparing circular RNA according to claim 1, characterized in that: Also included is circular RNA purification.

4. The method for preparing circular RNA according to claim 3, characterized in that: The pH value of the mobile phase for circular RNA purification ranges from 6 to 8, and the mobile phase includes EDTA.

5. A method for preparing circular RNA, characterized in that: The steps include: Precursor RNA is prepared by in vitro transcription of a vector, the obtained precursor RNA molecule is cyclized, and the obtained circular RNA is purified. The pH value of the mobile phase during circular RNA purification is in the range of 6 to 8; The vector includes a T7 promoter, a 5' external homology arm, a 3' intron, a 3' splice site, a 5' internal homology arm, a 5' spacer, a BbsI restriction enzyme site, a 3' internal homology arm, a 5' splice site, a 5' intron, a 3' external homology arm, and a restriction enzyme site HindIII for plasmid linearization, and an internal ribosome entry site (IRES).

6. The method for preparing circular RNA according to claim 5, characterized in that: Use NTPs with a working concentration of 5 mmol / L to 12.5 mmol / L to synthesize precursor RNA.

7. The method for preparing circular RNA according to claim 5, characterized in that: The mobile phase consisted of EDTA.

8. A circular RNA prepared according to the preparation method according to any one of claims 1 to 6.

9. The circular RNA prepared according to claim 8, characterized in that The circular RNA can encode hFIX, SPB, VEGF-A, human methylmalonyl CoA variant enzyme, CFTR, cancer autoantigens, gene editing enzymes, HIV antibodies, CD19 antibodies, CD22 antibodies, CD3 antibodies, CLDN6 antibodies, Luc2, GLuc, Fluc, eGFP, hEPO, Cas9 nuclease, new coronavirus S protein or tumor suppressor factors.

10. A method for expressing a protein in a cell, comprising transfecting the circular RNA obtained according to claim 8 into the cell.

11. A method for expressing a protein in a cell according to claim 10, characterized in that: in The cell is a eukaryotic cell.

Images