Preparation and purification method of circular RNA (Ribonucleic Acid)

Abstract

The invention provides a preparation and purification method of circular RNA (Ribonucleic Acid), which comprises the following steps: on the basis of an Ana-PIE cyclization strategy, additionally adding a section of ployA into a cyclization element, and carrying out A-gt on 5-Spacer and 3-Spacer in the cyclization element; and C, traversing mutation, selecting a mutation site which does not influence the structure and function of the cyclization element, constructing a vector inserted into the cyclization element, carrying out in-vitro transcription and cyclization reaction by taking the vector subjected to enzyme digestion as a template, and carrying out Oligo dT affinity purification on the obtained product. The circular RNA prepared by the method disclosed by the invention is high in purity, cellular immunity can be reduced, in-vivo experiments prove that the circular RNA prepared by the method disclosed by the invention can be efficiently expressed in vivo and is long in duration, and the method disclosed by the invention is simple in purification step, efficient, high in recovery rate, low in cost and easy for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of circular RNA technology, and specifically relates to a method for preparing and purifying circular RNA. Background Technology

[0002] Circular RNA (circRNA) was first discovered in 1979. Due to unclear formation mechanisms and limitations in sequencing methods, it wasn't until 2012 that it was demonstrated that circRNAs might play specific functions in regulating biological processes. In 2018, Daniel Anderson et al. first demonstrated that engineered circRNAs could stably and efficiently express proteins in eukaryotic cells, pioneering a new application of exogenous circRNAs for protein expression in eukaryotic cells and proving that circRNAs are an effective alternative to linear mRNAs.

[0003] Currently, there are three main methods for in vitro circularization of circular RNA: chemical methods, ligase methods, and ribozyme methods. Chemical methods produce unstable products and are prone to intermolecular reactions that generate polymers, so most research does not favor this technique. Ligase methods are achieved using RNA ligases, such as T4 RNA ligase I and T4 RNA ligase II. Similar to chemical methods, although the T4 RNA ligase method shows minimal immunogenicity, the sensitive nature of phosphodiester bonds often leads to intermolecular linkages and the generation of numerous polymer byproducts. Ribozyme ligation is currently the most common circularization method. It is achieved through a continuous transesterification reaction mediated by the PIE (Permuted Intron-Exon) system. Type I introns require a free G-OH group to attack the 5' splice site of the intron, generating a 3-OH group at the end of the exon. This 3-OH group can then attack the 5' splice site of the intron, causing the two exons to link together and the intron to be cleaved. However, this method is limited by RNA length and structure, with a circularizable length of only 300-500 bp, which greatly hinders its industrial application. In 2018, Daniel Anderson and Alexander Wesselhoeft modified PIE to propose Ana-PIE. This scheme adds homology arms to the 5' and 3' ends of the precursor RNA, bringing the splice sites closer together spatially. This increased the circularization efficiency from 16% to 48% while extending the circularizable RNA length to 5 kb. Furthermore, considering the proximity of the 3-intron splice site to the IRES (translation initiation element) and the fact that both sequences are highly structured and might spatially interfere with each other, a spacer sequence was added to preserve the secondary structure of the intron. The addition of the spacer significantly improved the circularization efficiency.

[0004] After optimizing the cyclization method, purification has become a major obstacle to the pharmaceutical development of circRNA. Cyclated products inevitably contain impurities such as linear precursor RNA, free introns, nicked RNA (the open-ring isoform of circular RNA), and NTPs. These impurities are considered strong immunogens and can induce a strong innate immune response. Currently, the conventional purification method is a combination of RNase R and HPLC, where RNase R is a Mg... 2+ The 3-5 exonuclease-dependent method can digest almost all linear precursor RNAs, but it is not good at digesting circular RNAs and lasso RNAs. Therefore, it is often used as the first step in the purification of circular RNAs to remove linear precursor RNA, free introns, and nickeled RNA. Afterwards, HPLC is used for further separation of the circular RNA, truncating the latter half of the peak spectrum to reduce the content of residual precursor and nickel. In addition, alkaline pyrophosphatase (Calf Intestinal, CIP) is added to remove free 5-ppp groups to prevent recognition by RIG-I and Toll-like receptors, thus preventing cellular immunity. Conventional purification methods are relatively cumbersome, have high scale-up costs, are time-consuming, and have relatively low recovery rates.

[0005] Therefore, in order to promote the application and development of circular RNA in in vivo drugs, it is necessary to design simpler, more efficient, easier to scale up and lower cost purification methods based on the Ana-PIE circularization strategy, and to develop purification methods more suitable for industrial production of high-purity circular RNA. Summary of the Invention

[0006] The purpose of this invention is to provide a new method for preparing and purifying circular RNA. This method can simultaneously achieve high yield and high purity of circular RNA, and its purification steps are simpler, more efficient, and easier to scale up for industrial production.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing and purifying circular RNA, which is based on the Ana-PIE circularization strategy and Oligo dT affinity purification.

[0009] Specifically, based on the Ana-PIE cyclization strategy, an additional polyA segment was added to the cyclization element, and A->C traversal mutations were performed on the 5-Spacer and 3-Spacer of the cyclization element. Mutation sites that do not affect the structure and function of the cyclization element were selected, and a vector for inserting the cyclization element was constructed. The digested vector was used as a template for in vitro transcription and cyclization reactions, and the resulting product was purified using Oligo dT affinity.

[0010] In this embodiment of the invention, the cyclization element sequentially includes the following sequence: 5-external homologous arm, 3-I type intron, E2 exon, 5-internal homologous arm, 5-Spacer, IRES, CDS, 3-Spacer, 3-internal homologous arm, E1 exon, 5-intron, 3-external homologous arm, and polyA, wherein the 5-Spacer and 3-Spacer are composed of A and C respectively, and the 5-Spacer and 3-Spacer do not affect the structure and function of the 3-I type intron, E2 exon, IRES, CDS, E1 exon, and 5-intron.

[0011] In this embodiment of the invention, the 5-intron is preferably a type I intron.

[0012] In this embodiment of the invention, the lengths of the 5-Spacer and 3-Spacer are independently 10 to 80 nucleotides, for example, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, and 80 nucleotides.

[0013] More preferably, the 5-Spacer is 30 to 80 nucleotides in length.

[0014] More preferably, the 5-Spacer has a length of 40 to 60 nucleotides, for example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.

[0015] More preferably, the 3-Spacer is 10 to 30 nucleotides in length.

[0016] More preferably, the length of the 3-Spacer is 15 to 25 nucleotides, for example, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides.

[0017] In this embodiment of the invention, the number of A in the 5-Spacer and 3-Spacer is 2 to 4 times the number of C.

[0018] More preferably, the number of A in the 5-Spacer is 2 to 3 times the number of C.

[0019] More preferably, the number of A in the 3-Spacer is 3 to 4 times the number of C.

[0020] In this embodiment of the invention, the number of consecutive A's in the 5-Spacer and 3-Spacer does not exceed 5.

[0021] More preferably, the number of consecutive A's in the 5-Spacer is at most 4.

[0022] More preferably, the number of consecutive A's in the 3-Spacer is at most 5.

[0023] According to some embodiments of the present invention, the amino acid sequence of the 5-Spacer of the Ana-PIE cyclization strategy includes any one of SEQ ID NO: 1 to 5.

[0024] According to some embodiments of the present invention, the amino acid sequence of the 3-Spacer includes any one of SEQ ID NO: 6 to 10.

[0025] In this embodiment of the invention, the length of polyA is 60 to 80 nucleotides.

[0026] More preferably, the length of the polyA is 65 to 75 nucleotides, for example, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, or 75 nucleotides.

[0027] The nucleotide sequences of the cyclization elements of this invention, except for the 5-Spacer and 3-Spacer, are not strictly limited. Theoretically, 5-external homologous arms, 3-type I introns, E2 exons, 5-internal homologous arms, IRES, CDS, 3-internal homologous arms, E1 exons, 5-introns, and 3-external homologous arms designed according to the Ana-PIE cyclization strategy and conventional techniques in the field can all be used in this invention.

[0028] According to some embodiments of the present invention, the sequence of the 5-exohomologous arm is as shown in SEQ ID NO: 11 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0029] According to some embodiments of the present invention, the sequence of the 3-I type intron is as shown in SEQ ID NO: 12 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0030] According to some embodiments of the present invention, the sequence of the E2 exon is as shown in SEQ ID NO: 13 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0031] According to some embodiments of the present invention, the sequence of the 5-inner homologous arm is as shown in SEQ ID NO: 14 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0032] According to some embodiments of the present invention, the sequence of the IRES is as shown in SEQ ID NO: 15 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0033] According to some embodiments of the present invention, the sequence of the 3-inner homologous arm is as shown in SEQ ID NO: 17 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0034] According to some embodiments of the present invention, the sequence of the E1 exon is as shown in SEQ ID NO: 18 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0035] According to some embodiments of the present invention, the sequence of the 5-intron is as shown in SEQ ID NO: 19 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0036] According to some embodiments of the present invention, the sequence of the 3-exohomologous arm is as shown in SEQ ID NO: 20 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

[0037] In this embodiment of the invention, the in vitro transcription uses unmodified rNTPs as raw materials.

[0038] In this embodiment of the invention, Mg is used. 2+ The in vitro transcription product was subjected to the cyclization reaction with rGTP.

[0039] In this embodiment of the invention, the Oligo dT affinity purification is performed by loading the sample through Tris-HCl containing sodium chloride at a pH of 7.2–7.8, followed by elution with H2O, and collecting the flow-through and eluent.

[0040] According to some embodiments of the present invention, the preparation and purification method includes the following steps:

[0041] (1) Based on the Ana-PIE cyclization strategy, design cyclization elements and construct plasmids;

[0042] (2) Linearized plasmids were obtained by digesting the plasmids from step (1) with restriction endonucleases.

[0043] (3) Use the linearized plasmid from step (2) as a template for in vitro transcription;

[0044] (4) Using Mg 2+ cyclization of in vitro transcription products with rGTP;

[0045] (5) The product obtained in step (4) was purified by Oligo DT affinity to obtain the circular RNA.

[0046] Furthermore, in step (3), unmodified rNTPs are used as raw materials for in vitro transcription and self-circularization.

[0047] Furthermore, the cyclization reaction is carried out in a reaction system containing in vitro transcription products precipitated with LiCl, rGTP, MgCl2, DTT, and Tris-HCl.

[0048] Further, in step (4), Tris-HCl containing NaCl with a pH of 7.2–7.8 is used for loading, and H2O is used to elute the circular RNA bound to the Oligo dT packing material.

[0049] Compared with the prior art, the present invention has the following advantages:

[0050] This invention develops a novel method for preparing and purifying circular RNA based on the Ana-PIE circularization strategy combined with Oligo dT affinity purification. The circular RNA prepared and purified by this method has high purity and can reduce cellular immunity. In vivo experiments have confirmed that the circular RNA prepared and purified by this method can be expressed efficiently in vivo for a long duration. The purification steps in this method are simple, efficient, have a high recovery rate, low cost, and are easy to scale up for production. Attached Figure Description

[0051] Figure 1 A schematic diagram of Ana-PIE cyclization;

[0052] Figure 2 Map of rd_Fluc_C001 plasmid;

[0053] Figure 3 This is a predicted diagram of the secondary structure of circular RNA from Example 1.

[0054] Figure 4 This is a predicted diagram of the secondary structure of the circular RNA in Comparative Example 1.

[0055] Figure 5 The images show the results of nucleic acid electrophoresis detection during the production of CircRNA. A: Identification by single enzyme digestion of the rd_Fluc_C001 plasmid (M: 15000bp Maker, lane1: plasmid before digestion, lane2: plasmid after digestion); B: Concentration and recovery of the rd_Fluc_C001 plasmid after digestion (M: 15000bp Maker, lane1: plasmid before digestion, lane2: concentrated recovery of plasmid after single enzyme digestion); C: In vitro transcription of Fluc CircRNA; D: Circulation of Fluc CircRNA.

[0056] Figure 6 The purity of the product after the cyclization reaction in Example 1 was determined by HPLC.

[0057] Figure 7The chromatogram of Fluc CircRNA affinity purification in Example 1 (Peak A: flow-through, Peak B: H2O elution);

[0058] Figure 8 Electrophoresis diagram for identifying Fluc CircRNA components in Example 1 (lane1: before affinity purification, lane2: PeakA elution, lane3 and lane4: H2O elution);

[0059] Figure 9 The chromatogram of Fluc CircRNA affinity purification in Comparative Example 1 is shown (Peak A: flow-through, Peak B: elution with 30 mM Tris-HCl, Peak C: elution with H2O).

[0060] Figure 10 Electrophoresis diagram for identifying Fluc CircRNA components in Comparative Example 1 (lane1: before affinity purification, lane2: Peak A elution, lane3 and lane4: Peak B elution, lane5: H2O elution);

[0061] Figure 11 Electrophoresis images of Fluc CircRNA treated with RNase R for different reaction times in Comparative Example 2 (C: no RNase R treatment, 0–18: reaction time at 37°C after adding RNase R);

[0062] Figure 12 Electrophoresis images of Fluc CircRNA before and after RNase R treatment in Comparative Example 2 (lane1: before treatment, lane2: after RNase R treatment);

[0063] Figure 13 The chromatogram of HPLC purification of Fluc CircRNA is shown in Comparative Example 2.

[0064] Figure 14 Figure 1 shows the identification and in vivo results of LNP-Fluc CircRNA in Example 1. In the figure, A: LNP-Fluc CircRNA gel retardation experiment (lane1: LNP-Fluc CircRNA, lane2: LNP-Fluc CircRNA after membrane perforation using Triton X-100), B: Particle size distribution of LNP-Fluc CircRNA, C: Observation of Fluc CircRNA expression in mice 3 h after injection, and D: Observation of changes in Fluc CircRNA expression intensity in mice at different time points. Detailed Implementation

[0065] Existing methods for preparing and purifying circular RNA products have limitations in terms of purity. Conventional methods involve RNase R digestion and HPLC separation after circularization, which are cumbersome, costly, time-consuming, and have relatively low recovery rates, making them difficult to scale up industrially. Therefore, the inventors of this application, through extensive research and experimental verification, propose a simpler, more convenient, lower-cost, and easily scalable method for preparing and purifying circular RNA that simultaneously achieves both high purity and high recovery rates.

[0066] Specifically, based on the Ana-PIE circularization strategy, the inventors of this application added an extra polyA segment outside the 3-external homologous arm, thus simplifying the purification steps using oligo dT affinity column chromatography. However, because the spacer also contains a polyA fragment, the circRNA and the predictor are still difficult to separate. Therefore, using RNAFold, A->C mutation scanning was performed on the 5-Spacer and 3-Spacer sequences. Suitable mutation sites were identified without affecting spacer function. Furthermore, based on the secondary structure predicted by RNAFold, the mutated polyAC does not affect the structure and function of separating IRES and exons. Experiments have demonstrated that the Fluc CircRNA prepared and purified using the method of this invention has high purity and, after LNP delivery, can be normally expressed in mice for a long duration.

[0067] Based on research and experimental verification, the specific nucleotide sequences of the mutated 5-Spacer that can be used are shown in Table 1, and the specific nucleotide sequences of the mutated 3-Spacer that can be used are shown in Table 2. The 5-Spacers in Table 1 and the 3-Spacers in Table 2 can be used in random combinations and have universal applicability, and can be used for different target gene circularization systems.

[0068] Table 1

[0069]

[0070]

[0071] Table 2

[0072] Sequence1 AAAAACCCAAAAACACAA SEQ ID NO: 6 Sequence2 AAACAACCAAAAACACAA SEQ ID NO: 7 Sequence3 AAACAACCAAACAACAAA SEQ ID NO: 8 Sequence4 AACAAACCAAACAACAAA SEQ ID NO: 9 Sequence5 AACAAACCAACAAACAAA SEQ ID NO: 10

[0073] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0075] Firefly luciferase (Fluc), a 61 kDa monomeric protein, produces enzymatic activity without post-translational processing. The following examples and comparative examples are based on the Ana-PIE cyclization strategy and OligoDT affinity purification. The cyclization element CDS expresses the Fluc protein (hereinafter referred to as Fluc CDS). Circular RNA is generated through in vitro transcription. After purification, the circRNA can be delivered to mice by LNP, and the expression of the Fluc protein can be examined at different time points.

[0076] Example 1

[0077] This embodiment provides a preferred implementation of the method for preparing and purifying CircRNA according to the present invention, as detailed below:

[0078] 1. Construction of rd_Fluc_C001 plasmid

[0079] like Figure 1 As shown, in Mg 2+ In the presence of GTP, precursor circularization forms circRNA and introns. This embodiment uses an Ana-PIE circularization strategy to design circularization elements, the original sequences of which sequentially include: a 5-external homologous arm, a 3-anabaena intron, an E2 exon, a 5-internal homologous arm, a 5-spacer, a CVB3 IRES, Fluc CDS, a 3-spacer, a 3-internal homologous arm, an E1 exon, a 5-intron, a 3-external homologous arm, and polyA. The Fluc CDS region was sequence-optimized and synthesized by GenScript Biotech Inc., with a puc57-kana-mini backbone and a T7 promoter. The final sequence was named rd_Fluc_C001, and the plasmid map is shown below. Figure 2 As shown, the nucleotide sequences of the elements involved are shown in Table 3.

[0080] Table 3

[0081]

[0082]

[0083]

[0084] The rd_Fluc_C001 plasmid is 4818 bp in length, and the transcribed precursor RNA is 3034 bp in length, including a circular region of 2579 bp, a 3-intron of 164 bp, a 5-intron of 221 bp, and a poly A region of 70 bp. Secondary structure prediction of Fluc CircRNA can be found in [link to relevant documentation]. Figure 3 It does not affect the cyclic element or the CDS region, and meets the expected requirements.

[0085] 2. CircRNA Production

[0086] 2.1 Plasmid single enzyme digestion

[0087] The rd_Fluc_C001 plasmid was digested with BspQ I (Novazia, GMP4103PC-01) at 50°C for 1 hour. The digestion efficiency was assessed using TAE electrophoresis. Figure 5 As shown in Figure A, after reacting at 50℃ for 1 hour, the rd_Fluc_C001 plasmid was almost completely linearized. The fully digested plasmid was then purified and concentrated using a plasmid recovery kit (Novizan, FastPure Gel DNA extraction Kit). To prevent problems during the recovery process, the concentrated and recovered product was identified by nucleic acid electrophoresis, as shown below. Figure 5 As shown in Figure B, the size and purity of the concentrated and recovered product meet expectations.

[0088] 2.2 CircRNA IVT Transcription

[0089] In vitro transcription (IVT) was performed using purified rd_Fluc_C001 as a template. Unlike conventional linear RNA, circRNA transcription is affected by modified bases that prevent circRNA from forming a circular structure. Therefore, conventional rNTPs were used as the raw material for in vitro transcription. rUTPs were used instead of N1-Me-pseudoU, and no capping analogue was added (IVT transcription reagent was purchased from Novizan). After incubation at 37°C for 3 hours, the transcription products were precipitated with LiCl and detected by TBE electrophoresis. Figure 5 The nucleic acid electrophoresis in Figure C shows that the in vitro transcribed circRNA contains three distinct bands: the precursor, the circRNA, and the introns. This demonstrates that the precursor underwent some self-circularization during the in vitro transcription process.

[0090] 2.3 CircRNA cyclization reaction

[0091] To achieve higher cyclization efficiency, Mg was used. 2+An additional cyclization step was performed on the in vitro transcription products using rGTP. The LiCl-precipitated circRNA was diluted to a concentration of 1000 ng / μl, heated to 70°C for 5 min, and then immediately placed on ice for 3 min. rGTP was added sequentially to a final concentration of 2 mM, along with a self-made 10× cyclization buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT). The reaction system was incubated at 55°C for 15 min. Samples were then taken for nucleic acid electrophoresis detection. The results are shown below. Figure 5 Figure D shows the HPLC identification of the product after the cyclization reaction. Figure 6 As shown, the peaks from left to right represent the precursor, circRNA, introns, and residual peaks of enzymes and rNTPs present in the IVT reaction. After the cyclization reaction, the cyclization efficiency of Fluc circRNA increased from 82% to 92%.

[0092] 3. CircRNA purification

[0093] Flux CircRNA was purified using a chromatography column (NanoGel dt20). The sample was loaded and flow-throughd using 100 mM Tris-HCl, 0.5 M NaCl, pH 7.5, followed by elution with H2O to the RNA molecules bound to the Oligo DT packing material. Figure 7 As shown, Peak A is the flow-through peak, and Peak B is the elution peak. Figure 8 The flow-through and elution peaks showed that the only macromolecules contained were CircRNA. The flow-through and elution buffers were collected, concentrated and replaced according to standard procedures, and then stored for later use.

[0094] Comparative Example 1

[0095] The preparation and purification method of the circular RNA used in this comparative example is basically the same as that in Example 1, except that the 5-Spacer and 3-Spacer are different. The nucleotide sequence of the 5-Spacer used in this comparative example is: AAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACA (SEQ ID NO: 23), and the nucleotide sequence of the 3-Spacer used in this comparative example is: AAAAAACAAAAAACAAAA (SEQ ID NO: 24). Figure 4 The predicted secondary structure of the circular RNA in this comparative example showed no significant difference from that in Example 1.

[0096] In this comparative example, Fluc CircRNA was purified using a chromatography column (NanoGel dt20). Figure 9 and Figure 10As shown, the sample was loaded and flow-through using 100mM Tris-HCl, 0.5M NaCl, and pH=7.5. Peak A was the flow-through peak. Nucleic acid electrophoresis showed that there were virtually no large molecules, indicating that the flow-through solution contained NTPs, incompletely transcribed RNA, linearized template plasmids, and various enzymes used in in vitro transcription (IVT). Then, RNA molecules bound to Oligo DT were eluted using 30mM Tris-HCl at pH=7.5. Peak B was the Tris-HCl elution peak. Nucleic acid electrophoresis showed that the main component was circRNA, with a small amount of precursor. Afterwards, H2O was used for elution. Peak C was the H2O elution peak. Nucleic acid electrophoresis showed that the peak mainly consisted of precursor and introns, with a small amount of circRNA.

[0097] Comparing the purification results of Example 1 and Comparative Example 1, it can be seen that the circular RNA of Example 1 has higher purity and higher recovery rate.

[0098] Comparative Example 2

[0099] This comparative example is basically the same as Example 1, except for the purification method. This comparative example uses a combination of RNase R and HPLC for purification, and the specific steps are as follows:

[0100] 1. RNase R treatment

[0101] Circulated circRNA was added to a final concentration of 750 ng / μl, followed by RNase R (Wuhan Genoin) at a final concentration of 35 ng / μl. The mixture was incubated at 37°C for 3–18 min to remove precursors and introns. Immediately afterwards, the mixture was incubated at 70°C for 5 min to inactivate RNase R. Samples were then taken for nucleic acid electrophoresis detection. Figure 11 As shown, after 6 min of treatment with RNase R, the digestion of precursor and introns reached equilibrium with the content of circRNA. Subsequently, with prolonged treatment time, circRNA began to degrade significantly. Therefore, subsequent 6 min of RNase R treatment was chosen to maximize the digestion of precursor and introns while preserving circRNA. Figure 12 As shown, the condition of treating with RNase R for 6 min to remove precursor and introns can be scaled up from 20 μl to 1 ml reaction system.

[0102] 2. HPLC purification

[0103] The circular RNA treated with RNase R was purified by HPLC, and the chromatogram is shown below. Figure 13As shown in the figure, the column (SRT SEC-1000) extracts both the fore-end and tail-end segments when circular RNA elutes, with the tail-end segment containing a lower concentration of Nicked. (Comparison) Figure 13 and Figure 6 It was found that after treatment with RNase R, the number of precursors and introns was significantly reduced, but the enzyme / NTP peak remained unchanged. It was also found that after treatment with RNase R, the circRNA underwent a certain degree of degradation (the main peak was elongated). The product shown in red dotted line was collected.

[0104] The purification method based on RNase R and HPLC used in this comparative example, while removing precursors and introns, has a certain impact on the content and purity of circRNA. In addition, it is costly, slow, and difficult to scale up for production.

[0105] Application Examples

[0106] Purified Fluc CircRNA from Example 1 was prepared by encapsulation using SM-102 gel electrophoresis, and the resulting sample was labeled LNP-FlucCircRNA. Encapsulation efficiency was assessed using gel retardation assays. Figure 14 As shown in Figure A, the encapsulation efficiency is greater than 95%. The average particle size and polydispersity index of LNP-Fluc CircRNA were determined using a nanoparticle size analyzer, as shown in Figure A. Figure 14 As shown in Figure B, the average particle size was 89.95 nm, and the PDI was 0.078. BALB / c mice were immunized with LNP-Fluc CircRNA intramuscularly for 6-8 weeks, as shown in Figure B. Figure 14 As shown in Figure C, high expression of Fluc could be detected 3 hours after injection. Subsequent measurements at different time points, such as... Figure 14 As shown in Figure D, Fluc CircRNA expression can last for up to 7 days, which is an advantage in terms of expression duration compared to the 2-3 days of conventional linear RNA expression. The purified CircRNA prepared in Example 1 has an advantage in expression duration.

[0107] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing and purifying circular RNA, characterized in that, Based on the Ana-PIE cyclization strategy, an additional polyA segment was added to the cyclization element, and A->C traversal mutations were performed on the 5-Spacer and 3-Spacer of the cyclization element. Mutation sites that do not affect the structure and function of the cyclization element were selected, and a vector for inserting the cyclization element was constructed. The digested vector was used as a template for in vitro transcription and cyclization reactions, and the resulting product was purified using Oligo dT affinity.

2. The method for preparing and purifying circular RNA according to claim 1, characterized in that, The cyclization element comprises the following sequence in sequence: 5-external homologous arm, 3-type I intron, E2 exon, 5-internal homologous arm, 5-Spacer, IRES, CDS, 3-Spacer, 3-internal homologous arm, E1 exon, 5-intron, 3-external homologous arm and polyA, wherein the 5-Spacer and 3-Spacer are composed of A and C respectively, and do not affect the structure and function of the 3-type I intron, E2 exon, IRES, CDS, E1 exon and 5-intron.

3. The method for preparing and purifying circular RNA according to claim 1, characterized in that, The lengths of the 5-Spacer and 3-Spacer are independently 10 to 80 nucleotides; And / or, the number of A in the 5-Spacer and 3-Spacer is 2 to 4 times the number of C; And / or, the number of consecutive A's in the 5-Spacer and 3-Spacer does not exceed 5.

4. The method for preparing and purifying circular RNA according to claim 3, characterized in that, The 5-Spacer has a length of 30 to 80 nucleotides, preferably 40 to 60 nucleotides; And / or 4, the length of the 3-Spacer is 10 to 30 nucleotides, preferably 15 to 25 nucleotides; And / or, the number of A in the 5-Spacer is 2 to 3 times the number of C; And / or, the number of consecutive A's in the 5-Spacer is at most 4; And / or, the number of A in the 3-Spacer is 3 to 4 times the number of C; And / or, the number of consecutive A's in the 3-Spacer is at most 5.

5. The method for preparing and purifying circular RNA according to claim 1, characterized in that, The amino acid sequence of the 5-Spacer includes any one of SEQ ID NO: 1 to 5, and / or the amino acid sequence of the 3-Spacer includes any one of SEQ ID NO: 6 to 10.

6. The method for preparing and purifying circular RNA according to claim 1, characterized in that, The length of polyA is 60 to 80 nucleotides, preferably 65 to 75 nucleotides.

7. The method for preparing and purifying circular RNA according to claim 2, characterized in that, The sequence of the 5-external homologous arm is as shown in SEQ ID NO: 11 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the type 3-I intron is as shown in SEQ ID NO: 12 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the E2 exon is as shown in SEQ ID NO: 13 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the 5-internal homologous arm is as shown in SEQ ID NO: 14 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the IRES is as shown in SEQ ID NO: 15 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the 3-internal homologous arm is as shown in SEQ ID NO: 17 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the E1 exon is as shown in SEQ ID NO: 18 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the 5-intron is as shown in SEQ ID NO: 19 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown. And / or, the sequence of the 3-exohomologous arm is as shown in SEQ ID NO: 20 or has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown.

8. The method for preparing and purifying circular RNA according to claim 1, characterized in that, The in vitro transcription used unmodified rNTPs as raw materials; And / or, using Mg 2+ The in vitro transcription product was subjected to the cyclization reaction with rGTP; Afterwards, the Oligo dT affinity purification was performed by loading the sample through Tris-HCl containing sodium chloride at a pH of 7.2–7.8, followed by elution with H2O, and collecting the flow-through and eluent.

9. The method for preparing and purifying circular RNA according to any one of claims 1 to 8, characterized in that, The preparation and purification method includes the following steps: (1) Based on the Ana-PIE cyclization strategy, design cyclization elements and construct plasmids; (2) Linearized plasmids were obtained by digesting the plasmids from step (1) with restriction endonucleases. (3) Use the linearized plasmid from step (2) as a template for in vitro transcription; (4) Using Mg 2+ cyclization of in vitro transcription products with rGTP; (5) The product obtained in step (4) was purified by Oligo dT affinity to obtain the circular RNA.

10. The method for preparing and purifying circular RNA according to claim 9, characterized in that, In step (3), in vitro transcription is performed using unmodified rNTPs as raw materials; And / or, the cyclization reaction is carried out in a reaction system containing in vitro transcription products precipitated with LiCl, rGTP, MgCl2, DTT, and Tris-HCl; And / or, the reaction temperature of the cyclization reaction is 50–60°C; And / or, in step (4), Tris-HCl containing NaCl with a pH of 7.2–7.8 is used for loading, and circular RNA bound to Oligo dT packing material is eluted with H2O.

Images