Group I self-splicing ribozyme for RNA (Ribonucleic Acid) in-vitro cyclization and application of Group I self-splicing ribozyme

Abstract

The invention relates to a Group I type self-splicing ribozyme for RNA (Ribose Nucleic Acid) in-vitro cyclization. The sequence composition of the Group I type self-splicing ribozyme is shown as SEQ ID NO. 28 and SEQ ID NO. 24. The invention also provides a cyclization skeleton sequence and a corresponding plasmid, wherein the 3'intron-exon fragment and the 5 'intron-exon fragment are respectively a sequence composed of a sequence formed by dividing the Group I type self-splicing ribozyme as shown in SEQ ID NO.28 and SEQ ID NO.24 into two parts at a rearrangement site, wherein the Group I type self-splicing ribozyme is shown in SEQ ID NO.28 and SEQ ID NO.24. The efficiency of a cyclization skeleton sequence formed by a sequence obtained by dividing the Group I self-splicing ribozyme SEQ ID NO.28 used for RNA in-vitro cyclization into two at a rearrangement site of a fifth or sixth structural domain is superior to that of a self-splicing ribozyme derived from anabaena and a self-splicing ribozyme derived from T4 bacteriophage. The new self-splicing ribozyme sequence screened by the reverse side provides a new cyclization arm for the preparation of the circRNA, and provides more and better choices for the preparation of the circRNA.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Group I self-splicing ribozyme for efficient RNA cyclization in vitro and an application thereof. Background Art

[0002] In recent years, research on the design and delivery of nucleic acid drugs, including messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotides (ASO), and ribozymes, has made important progress. Among them, mRNA is favored because of its special status in the "central dogma". mRNA drugs have the advantages of safety, high efficiency, simple process, and short R&D cycle. The development speed and clinical performance are far beyond expectations. mRNA drugs have become another new breakthrough point in the field of biomedicine after protein macromolecule drugs. However, due to the poor stability of mRNA itself and the rapid cell metabolism, more research and improvement are still needed.

[0003] Circular RNA (circRNA) is a new type of RNA molecule that forms a closed loop with phosphodiester bonds at the head and tail. Therefore, it has a stronger stability advantage both inside and outside the cell than linear RNA molecules. At the same time, due to its structural characteristics of carrying internal ribosome entry site (IRES) and coding sequence (CDS), circRNA can also play a protein coding function in vivo. At the production level, since circRNA does not require a 5' cap and PolyA, the relative production cost and process difficulty will be lower than that of mRNA. At present, circRNA technology still needs to break through many technical bottlenecks at the application level, such as in vitro circRNA circularization technology, preparation and purification process, protein expression backbone design, etc. At present, the ribozymes commonly used for in vitro circRNA circularization preparation are divided into type I (group I) and type II (group II) according to their splicing reaction principles. Type I has higher splicing activity than type II.

[0004] Generally speaking, in order to achieve efficient cyclization activity in vitro, people will artificially modify the self-splicing ribozyme sequence. At present, the most effective modification method is the permuted intron-exon (PIE) rearrangement modification. Specifically, the intron sequence with part of the original gene exon left at both ends is first divided into two at the appropriate position; then the 3' intron-exon fragment is designed to the 5' end of the RNA molecule to be cyclized, and the 5' intron-exon fragment is designed to the 3' end of the RNA molecule to be cyclized. Commonly used for the above modification are the two type I self-splicing ribozymes, the leucine transfer RNA (tRNA) intron from Anabaena and the thymidylate synthase (thymidylate synthase) intron from T4 phage. The above two self-splicing ribozyme sequences have high cyclization activity after PIE modification. However, the number of exon bases retained by the self-splicing ribozymes from Anabaena often exceeds 60, which may cause strong immunogenicity and be detrimental to the stability and expression of circRNA. In addition, although the self-splicing ribozymes from T4 phage have fewer exon residues, and can even achieve no residues through optimized design, the self-splicing reaction is not sufficient, and it is easy to produce more nickedRNA, which brings great trouble to the further separation and purification of circRNA. Therefore, it is very important to find more type I self-splicing ribozymes with cyclization activity. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a Group I self-splicing ribozyme and its application with similar or even higher cyclization efficiency than the existing ones.

[0006] The technical solutions for achieving the above-mentioned purpose of the present invention include the following.

[0007] The first object of the present invention is to provide a Group I self-splicing ribozyme for RNA in vitro cyclization, the sequence composition of which is shown in SEQ ID NO.28;

[0008] Or a Group I self-splicing ribozyme for RNA in vitro cyclization, whose sequence composition is shown in SEQ ID NO.24.

[0009] The second object of the present invention is to provide a cyclization backbone sequence for in vitro cyclization of RNA, which comprises the following modular elements from 5' to 3': a 5' terminal homologous fragment, a 3' intron-exon fragment, a 5' internal homologous fragment, a target fragment, a 3' internal homologous fragment, a 5' intron-exon fragment, and a 3' terminal homologous fragment; the 3' intron-exon fragment and the 5' intron-exon fragment are sequences obtained by splitting a Group I type self-splicing ribozyme into two at a rearrangement site, respectively, as shown in SEQ ID NO.28 or SEQ ID NO.24.

[0010] In some embodiments, the rearrangement site is selected from the 5th or 6th domain of the secondary structure of the Group I type self-splicing ribozyme.

[0011] In some embodiments, the 5' terminal homologous fragment and the 3' terminal homologous fragment in the cyclization backbone sequence are artificially designed base complementary pairing elements, containing 10-50 nucleotides. The purpose of introducing the above sequences is to bring the linear pre-RNA head and tail closer to each other by base complementary pairing so as to form an active cyclization arm structure.

[0012] In some embodiments, the 5' internal homologous segment and the 3' internal homologous segment are artificially designed base complementary pairing elements that are not completely paired, and preferably have lengths of 19 nucleotides and 21 nucleotides, respectively.

[0013] The third object of the present invention is to provide a plasmid for RNA in vitro circularization, which is a vector into which any of the above-mentioned circularization backbone sequences is inserted.

[0014] In some embodiments, the vector is a pcDNA3.1 vector plasmid.

[0015] The fourth objective of the present invention is to provide the use of any of the above-mentioned Group I self-splicing ribozymes, circularization backbone sequences or plasmids in the preparation of circular RNA in vitro.

[0016] The fifth object of the present invention is to provide a method for preparing circular RNA in vitro, which uses the circularization backbone sequence or a plasmid containing the circularization backbone sequence as a template to obtain a circular RNA molecule through in vitro circularization.

[0017] In some of the embodiments, the preparation method includes obtaining a linearized plasmid: (1) preparing an enzyme digestion reaction system: 6 μg of plasmid, 2 μL of endonuclease, 2 μL of 10×rCutSmart buffer, and filling up to 20 μL with enzyme-free water; (2) reacting the enzyme digestion system at 37° C. overnight (>12 hours); and (3) purifying and recovering the plasmid using VAHTS DNA Clean Beads.

[0018] In some of the embodiments, the preparation method includes obtaining linear RNA: (1) preparing an IVT reaction system: 0.5 μg of linearized plasmid, 2 μL of T7 RNA Polymerase Mix, 2 μL of 10×Transcription buffer, 2 μL of ATP (100 mM), 2 μL of UTP (100 mM), 2 μL of CTP (100 mM), 2 μL of GTP (100 mM), 1 μL of RNase inhibitor, and finally filling up to 20 μL with enzyme-free water; (2) reacting the IVT reaction system at 37° C. for 2 hours; (3) purifying and recovering RNA using VAHTS RNA CleanBeads to obtain an IVT reactant.

[0019] In some of the embodiments, the preparation method includes obtaining circular RNA: (1) preparing a cyclization reaction system: 10 μL of the IVT reactant, 2 μL of 10×Transcription buffer, 0.4 μL of GTP (100 mM), 1 μL of RNase inhibitor, and finally filling up to 20 μL with enzyme-free water; (2) reacting the cyclization reaction system at 55° C. for 20-30 minutes; (3) adding 1 μL of DNase I to the above cyclization system, reacting at 37° C. for 15 minutes to remove the DNA template; and (4) purifying and recovering the RNA using VAHTS RNA Clean Beads.

[0020] The sequence composition of the present invention screened from numerous sequences is shown in SEQ ID NO.28 or SEQ ID NO.24 as Group I self-splicing ribozymes. These Group I self-splicing ribozymes are firstly proved to be used for RNA in vitro cyclization after "PIE" modification, especially the efficiency of the cyclization backbone sequence composed of the sequence obtained by splitting the rearrangement site of the 5th or 6th domain of these Group I self-splicing ribozymes is better than that of the self-splicing ribozymes derived from Anabaena and T4 phage. The new self-splicing ribozyme sequence screened in the present invention provides a new cyclization arm for the preparation of circRNA, and provides more and better choices for the preparation of circRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the cyclization result of Group I self-splicing ribozymes numbered 29-56 in Table 1.

[0022] Figure 2 It is a schematic diagram of the comparison of the circularization efficiency of self-splicing ribozymes code 52, 56 (SEQ ID NO.24 and SEQ ID NO.28) and existing Ana and T4td.

[0023] Figure 3 This is a schematic diagram of the effects of different rearrangement sites of Group I self-splicing ribozymes codenamed 52 and 56 on the circularization efficiency.

[0024] Figure 4 This is a schematic diagram of the domain region of the Group I self-splicing ribozyme code-named 56.

[0025] Figure 5 It is the cellular expression effect of Group I self-splicing ribozymes code-named 52 and 56. DETAILED DESCRIPTION

[0026] For ease of understanding of the present invention, the present invention will be described more fully below with reference to the embodiments, and preferred embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should be understood that the experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. The various commonly used reagents used in the embodiments are all commercially available products.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0028] The present invention is further described below through specific examples, but is not intended to limit the protection scope of the present invention.

[0029] The reagents and instruments used in the examples are listed in the following table:

[0030]

[0031]

[0032] Embodiment 1

[0033] The Group I self-splicing ribozyme sequences (including exon sequences, shown in lowercase letters in the table) code-named 29-58 used in this example were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The specific sequences are shown in Table 1 below.

[0034] Table 1: Group I self-splicing ribozyme sequences

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] The circularization skeleton constructed for the verification of the circularization ability of the self-splicing ribozyme comprises the following element modules from 5' to 3': (1) 5' terminal homologous fragment; (2) 3' intron-exon fragment; (3) 5' internal homologous fragment; (4) target fragment; (5) 3' internal homologous fragment; (6) 5' intron-exon fragment; (7) 3' terminal homologous fragment. The 5' terminal homologous fragment and the 3' terminal homologous fragment are artificially designed base complementary pairing elements, containing 20 nucleotides, and the 5' internal homologous fragment and the 3' internal homologous fragment are artificially designed base complementary pairing elements, containing 20 nucleotides.

[0044] The target fragment is a protein coding or non-coding sequence according to actual needs. The sequence used in this embodiment is the commonly used eGFP fluorescent protein coding sequence as an example.

[0045] The 3' intron-exon fragment and the 5' intron-exon fragment are sequences obtained by splitting the Group I self-splicing ribozymes in Table 1 into two at appropriate positions. The appropriate positions are selected from the 5th and 6th domain regions of the Group I self-splicing ribozymes.

[0046] The Group I self-splicing ribozymes include Group I self-splicing ribozymes coded 29-56 in Table 1, of which 31, 36, 39, 40, 41, 50, 52, 53, and 56 are active. The above-mentioned cyclization backbone sequences are inserted into the pcDNA3.1 vector plasmid for amplification.

[0047] The cyclization sequences including the cyclization arms used in this example are respectively the above-mentioned introns and are shown in Table 2:

[0048] Table 2: Cyclization sequences

[0049]

[0050]

[0051] The experimental methods and processes used in this example are as follows:

[0052] 1. Prepare linearized plasmid according to conventional methods

[0053] 1.1 Plasmid digestion

[0054] 1.1.1 Configure the reaction system according to the table below

[0055] The pcDNA3.1 vector plasmid with the above-mentioned circularized backbone sequence inserted 6.0 μg XOt 2.0μL 10×rCutSmart buffer 2.0μL <![CDATA[DEPC H2O]]> Up to 20μL

[0056] 1.1.2 Incubate at 37℃ overnight (>12h).

[0057] 1.2 Agarose gel electrophoresis

[0058] 1.2.1 Prepare 1% agarose gel: weigh 1g agarose gel powder, add 100mL 1×TAE buffer (50×TAE buffer diluted 50 times with ultrapure water), heat in a microwave oven at high temperature for 3 minutes to completely dissolve the agarose powder, then pour into a gelatin plate, insert a 25-hole comb, wait for it to completely cool and solidify, remove the comb and set aside;

[0059] 1.2.2 Take 1 μL of the enzyme digestion reaction solution and mix it with an equal volume of 1× DNA loading to prepare the loading solution;

[0060] 1.2.3 Add the sample solution to the gel loading well for electrophoresis (constant voltage 120 volts, 30 minutes);

[0061] 1.2.4 After electrophoresis, use a gel imaging system to image the plasmid to ensure that it is completely linear.

[0062] 1.3 Linearized plasmid recovery (magnetic bead method)

[0063] 1.3.1 The temperature of the magnetic bead solution is equilibrated to room temperature 30 minutes in advance;

[0064] 1.3.2 Invert repeatedly to mix the magnetic bead solution thoroughly, pipette 10 μL of magnetic bead solution and add it to 20 μL of enzyme digestion sample, and gently pipette 10 times;

[0065] 1.3.3 Incubate at room temperature for 10 minutes to allow the plasmid to fully bind to the magnetic beads;

[0066] 1.3.4 Place the sample on the magnetic rack for 10 minutes and carefully remove the supernatant;

[0067] 1.3.5 Keep the sample on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant; repeat the wash once and remove the supernatant;

[0068] 1.3.6 Centrifuge at low speed (2,500 rpm, 20 seconds) to concentrate the residual liquid at the bottom of the tube, place the centrifuge tube back on the magnetic stand, remove the residual liquid with a 10 μL pipette, and dry it at room temperature with the lid open for 5 minutes;

[0069] (7) Remove the sample from the magnetic rack, add 30 μL of enzyme-free water, mix thoroughly by pipetting, and let stand at room temperature for 10 minutes;

[0070] (8) Let the mixture stand on the magnetic rack for 5 minutes. After the solution becomes clear, carefully pipette 27 μL of the supernatant into a new enzyme-free centrifuge tube.

[0071] (9) Determine the concentration and record the concentration, A260 / 280, and A260 / 230.

[0072] 2. RNA preparation in vitro

[0073] 2.1 RNA synthesis

[0074] 2.1.1 Prepare the reaction system according to the following table:

[0075] DNA Template 0.5-1.0 μg T7 RNA Polymerase Mix 2μL 10×Transcription buffer 2μL ATP(100mM) 2μL CTP (100 mM) 2μL GTP (100 mM) 2μL UTP(100mM) 2μL RNase inhibitor 1μL <![CDATA[DEPC H2O]]> Up to 20μL

[0076] 2.1.2 Incubate at 37℃ for 2 hours.

[0077] 2.2 DNA template elimination

[0078] 2.2.1 Add 1 μL DNase I to the system after the above cyclization reaction;

[0079] 2.2.2 Incubate at 37°C for 15 minutes.

[0080] 2.3 RNA purification

[0081] 2.3.1 Place the RNA magnetic beads at room temperature 30 minutes in advance and mix thoroughly before use;

[0082] 2.3.2 Add 36 μL RNA magnetic bead suspension (1.8× sample volume) to the cyclization reaction system, mix thoroughly and let stand at room temperature for 5 minutes;

[0083] 2.3.3 Place the sample on a magnetic rack and let it stand for 5 minutes before removing the supernatant;

[0084] 2.3.4 Keep the sample on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 1 minute and carefully remove the liquid;

[0085] 2.3.5 Repeat the washing with 80% ethanol once to fully remove the liquid (use a 10 μL pipette tip to remove after the last instant centrifugation);

[0086] 2.3.6 Keep the sample on the magnetic rack and allow the magnetic beads to dry naturally for 5-10 minutes;

[0087] 2.3.7 Resuspend the magnetic beads with 23 μL enzyme-free water, let stand at room temperature for 5 minutes, then place the sample on the magnetic rack, let stand for 5 minutes, then carefully pipette 20 μL of supernatant into a new enzyme-free centrifuge tube;

[0088] 2.3.8 Determine the RNA concentration, record relevant data (including concentration, A260 / 280, A260 / 230), and obtain linear RNA.

[0089] 3. RNA cyclization and detection

[0090] 3.1 RNA circularization

[0091] 3.1.1 Prepare the cyclization reaction system according to the following table:

[0092] Linear RNA 10μL (15-20μg) 10×Transcription buffer 2μL GTP (100 mM) 0.4μL RNase Inhibitor (40U / μL) 1μL <![CDATA[DEPC H2O]]> Up to 20μL

[0093] 3.1.2 Incubate at 55°C for 20-30 minutes to obtain circular RNA.

[0094] 3.2 RNA purification

[0095] 3.2.1 Place the RNA magnetic beads at room temperature 30 minutes in advance and mix thoroughly before use;

[0096] 3.2.2 Add 36 μL RNA magnetic bead suspension (1.8× sample volume) to the cyclization reaction system, mix thoroughly and let stand at room temperature for 5 minutes;

[0097] 3.2.3 Place the sample on a magnetic rack and let it stand for 5 minutes before removing the supernatant;

[0098] 3.2.4 Keep the sample on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 1 minute and carefully remove the liquid;

[0099] 3.2.5 Repeat the washing with 80% ethanol once to fully remove the liquid (use a 10μL pipette tip to remove after the last instant centrifugation);

[0100] 3.2.6 Keep the sample on the magnetic rack and allow the magnetic beads to dry naturally for 5-10 minutes;

[0101] 3.2.7 Resuspend the magnetic beads with 33 μL enzyme-free water, let stand at room temperature for 5 minutes, then place the sample on the magnetic rack, let stand for 5 minutes, then carefully pipette 30 μL of supernatant into a new enzyme-free centrifuge tube;

[0102] 3.2.8 Determine RNA concentration and record relevant data (including concentration, A260 / 280, A260 / 230);

[0103] 3.3 Cyclization efficiency detection

[0104] 3.3.1 Prepare 2% agarose gel (1M Urea-TBE) and wait until it is completely solidified;

[0105] 3.3.2 Take 300 ng RNA sample (corresponding to IVT product and cyclization product) and mix with nucleic acid dye, then add an equal volume of 2× RNA loading buffer and mix well before loading;

[0106] 3.3.3 Electrophoresis at 120V constant voltage for 30 minutes;

[0107] 3.3.4 After electrophoresis is completed, use a gel imaging system to image and save the image;

[0108] 3.3.5 ImageJ grayscale analysis was used to calculate the RNA circularization rate, and the calculation formula was “circRNA band grayscale value / (circRNA band grayscale value+pre-RNA band grayscale value+nicked RNA band grayscale value)”.

[0109] Experimental Results

[0110] 1. Detection of the cyclization ability of Group I self-splicing ribozyme No. 29-56

[0111] In this experiment, the self-splicing ribozyme rearrangement sites are all in the 6th domain (the domain of the secondary structure can be referred to Nucleic Acids Res. 2008 Jan; 36 (Database issue): D31-7) loop region. Figure 1 The Group I self-splicing ribozymes numbered 29-56 in Table 1 were incubated at 55°C for 20 minutes to obtain the circularization results of circular RNA. The ribozymes numbered 31, 36, 39, 40, 41, 50, 52, 53, and 56 in Table 1 can achieve RNA circularization in vitro, and the better results are 52 and 56, which are 84.1% and 91.8%, respectively.

[0112] Figure 1 "L" represents IVT product, and "C" represents cyclization product. Quantification of cyclization efficiency was achieved by grayscale analysis function of ImageJ software.

[0113] 2. Compare the cyclization efficiency of the self-splicing ribozymes coded 52 and 56 with the existing Ana and T4td

[0114] In this experiment, the self-splicing ribozyme rearrangement sites are all in the 6th domain region. Figure 2The circularization efficiency of the self-splicing ribozymes numbered 52 and 56 displayed is compared with the publicly known Ana (Anabaena in Table 1) and T4td (corresponding T4td in Table 1) self-splicing ribozymes.

[0115] from Figure 2 It can be seen that circular RNA was obtained by constant temperature incubation at 55°C for 30 minutes. The cyclization efficiency of the self-splicing ribozyme of sequence 56 reached 92.3%, which is much higher than the cyclization efficiency of Ana (85.3%) and T4td (80.4%) self-splicing ribozymes, while the cyclization efficiency of the self-splicing ribozyme of sequence 52 was comparable to that of Ana and T4td.

[0116] Figure 2 "L" indicates IVT product, and "C" indicates cyclization product. pre-RNA indicates precursor RNA, i.e., IVT product. circRNA indicates circular RNA. nicked RNA indicates incompletely reacted RNA or hydrolyzed circRNA. Quantification of cyclization efficiency was achieved using the grayscale analysis function of ImageJ software.

[0117] 3. Effects of different rearrangement sites of self-splicing ribozymes on circularization efficiency

[0118] Figure 3 The self-splicing ribozyme is shown to have an effect on the cyclization efficiency of the self-splicing ribozyme with different rearrangement sites coded as 56 (SEQ ID NO. 28). Figure 3 D3, D5, D6, and D7 indicate that the rearrangement sites are located in the 3rd, 5th, 6th, and 7th domains of the ribozyme secondary structure, respectively (see Figure 4 ).

[0119] from Figure 3 It can be seen that circular RNA was obtained by constant temperature incubation at 55℃ for 30 minutes. When the rearrangement site of the self-splicing ribozyme code 56 is in the 5th and 6th domains, the cyclization efficiency is high, among which the 6th domain has the highest efficiency, which is 92.2%. When the rearrangement site is in the 5th domain, the cyclization efficiency is 88.5%. When the rearrangement site is in the 3rd and 7th domains, the cyclization efficiency is only 0. From the results, it can be seen that the choice of rearrangement site of the self-splicing ribozyme No. 56 is important for its cyclization activity. We understand that some rearrangement sites may affect the correct folding of the ribozyme, thereby causing the loss of its ribozyme function.

[0120] Figure 3 Pre-RNA means precursor RNA, i.e., IVT product. circRNA means circular RNA. “M” means ssRNAladder. Quantification of circularization efficiency was achieved by grayscale analysis function of ImageJ software.

[0121] Example 2

[0122] The purpose of this example is to characterize the cell expression effect of the prepared circRNA.

[0123] The specific scheme for preparing circRNA is the same as that in Example 1 (incubated at 55°C for 30 minutes, and the self-splicing ribozyme rearrangement sites are all in the 6th domain region), and the remaining materials and methods are as follows.

[0124] The cells used in this example are adherent human renal epithelial cell line 293T (cultured in DMEM medium containing 10% fetal bovine serum) and suspension human leukemia monocytic cell line THP-1 (cultured in 1640 medium containing 10% fetal bovine serum). The circRNA sequence used includes an IRES sequence, a coding sequence for green fluorescent protein, and a spacer sequence, and the specific sequence is as follows:

[0125] >circEGFP

[0126] (SEQ ID NO.36)

[0127] The reagents and instruments used in this example are as follows:

[0128]

[0129]

[0130] Experimental methods and procedures used:

[0131] 1. 293T cells (adherent cells) implementation process:

[0132] 1.1 Cell plating

[0133] 1.1.1 Remove the culture medium from 293T cells when the confluence reaches about 90% and wash with an appropriate amount of PBS;

[0134] 1.1.2 Remove PBS, add 1-3mL 0.25%-EDTA trypsin, and incubate the cells in a 37°C, 5% CO2 cell culture incubator for 2-3 minutes. When the cells are completely detached, immediately add 5mL of serum-containing culture medium for neutralization, mix thoroughly by pipetting, and transfer to a 15mL centrifuge tube;

[0135] 1.1.3 Centrifuge at 800 rpm for 5 minutes, remove the supernatant, resuspend in 1 mL of complete culture medium, pipette thoroughly, add 9 mL of culture medium, and mix thoroughly;

[0136] 1.1.4 Take 10 μL of cell suspension, mix thoroughly with an equal volume of AOPI staining reagent, add to the cell counting chamber, and count using a cell counter;

[0137] 1.1.5 Plate 293T cells in a 24-well plate at a density of 100,000 cells per well and mix thoroughly using the “cross” method;

[0138] 1.1.6 Place the cells in a 37°C, 5% CO2 cell culture incubator for overnight culture.

[0139] 1.2 Cell circRNA transfection

[0140] 1.2.1 Dilute MessengerMAX and circRNA according to the following table and let each stand for 10 minutes:

[0141]

[0142] 1.2.2 Mix the diluted lipid and circRNA in equal volumes and let stand at room temperature for 10 minutes;

[0143] 1.2.3 Remove the old cell culture medium, add 250 μL Opti-MEM Medium, and evenly add 100 μL of the incubated lipid-mRNA transfection complex to the culture medium, and immediately mix thoroughly using the "cross-over" method;

[0144] 1.2.4 After incubating the cells in a 37°C, 5% CO2 cell culture incubator for 24 hours, the green fluorescence expression was observed using a fluorescence microscope.

[0145] 1.3 Flow cytometry

[0146] 1.3.1 Turn on the flow cytometer in advance, clean the pipeline, and keep it on standby;

[0147] 1.3.2 The 293T cells transfected with circRNA were digested with 0.25%-EDTA trypsin, washed and resuspended with PBS to obtain a cell density of 10 6 Single cell suspension of about cells / mL;

[0148] 1.3.3 Flow cytometry was used for detection and analysis to obtain the expression level data of different circRNAs in cells.

[0149] 2. THP-1 cell (suspension cell) implementation process:

[0150] 2.1 Cell plating

[0151] 2.1.1 Transfer the THP-1 cell suspension with good growth status to a 15 mL centrifuge tube;

[0152] 2.1.2 Centrifuge at 800 rpm for 5 minutes, remove the supernatant, resuspend with 1 mL of Opti-MEM Medium, pipette thoroughly, then add 9 mL of Opti-MEM Medium and mix thoroughly;

[0153] 2.1.3 Take 10 μL of cell suspension, mix thoroughly with an equal volume of AOPI staining reagent, add to the cell counting chamber, and count using a cell counter;

[0154] 2.1.4 THP-1 cells were plated in a 24-well plate at a density of 200,000 cells per well and mixed thoroughly by the "cross-cross" method; 2.2 Cell circRNA transfection

[0155] 2.2.1 Dilute MessengerMAX and circRNA according to the following table and let each stand for 10 minutes:

[0156]

[0157] 2.2.2 Mix the diluted lipid and circRNA in equal volumes and let stand at room temperature for 10 min;

[0158] 2.2.3 Evenly add 100 μL of the incubated lipid-mRNA transfection complex to the culture medium and immediately mix thoroughly using the "cross-over" method;

[0159] 2.2.4 After the cells were cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours, the green fluorescence expression was observed using a fluorescence microscope.

[0160] 2.3 Flow cytometry

[0161] 2.3.1 Turn on the flow cytometer in advance, clean the pipeline, and keep it on standby;

[0162] 2.3.2 The circRNA-transfected THP-1 cells were centrifuged, washed and resuspended with PBS to obtain a single-cell suspension with a cell density of about 106 cells / mL;

[0163] 2.3.3 Flow cytometry was used for detection and analysis to obtain the expression level data of different circRNAs in cells.

[0164] Figure 5 The expression of circRNA of Group I self-splicing ribozymes of different SEQ ID NO.28 and SEQ ID NO.24 in 293T cells and THP-1 cells after 24 hours of transfection is shown. Among them, NC represents cells that are not transfected with circRNA, and different digital numbers represent cells transfected with circRNA molecules expressing green fluorescent protein corresponding to circEGFP obtained by circulation of the circulant arm. From the results observed under a fluorescence microscope, it can be seen that compared with the negative control, a certain proportion of cells transfected with circEGFP produced by self-splicing ribozymes of SEQ ID NO.28 and SEQ ID NO.24 all emit green fluorescence (293T cells have a relatively high expression, and THP-1 cells have a relatively weak expression). The results of the flow cytometry test also confirm the observation results of the fluorescence microscope. The circEGFP obtained by circulation of the circulant arm corresponding to the two Group I self-splicing ribozymes of SEQ ID NO.28 and SEQ ID NO.24 has achieved efficient expression in the cells.

[0165] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A Group I self-splicing ribozyme for RNA cyclization in vitro, characterized in that: The sequence composition of the Group I self-splicing ribozyme is shown in SEQ ID NO.

28.

2. A Group I self-splicing ribozyme for RNA cyclization in vitro, characterized in that: The sequence composition of the Group I self-splicing ribozyme is shown in SEQ ID NO.

24.

3. A cyclization backbone sequence for RNA in vitro cyclization, characterized in that: The cyclization backbone sequence comprises the following modular elements from 5' to 3': a 5' terminal homologous fragment, a 3' intron-exon fragment, a 5' internal homologous fragment, a target fragment, a 3' internal homologous fragment, a 5' intron-exon fragment, and a 3' terminal homologous fragment; the 3' intron-exon fragment and the 5' intron-exon fragment are respectively sequences obtained by splitting the Group I type self-splicing ribozyme described in claim 1 or 2 into two at the rearrangement site.

4. The cyclization backbone sequence according to claim 3, characterized in that The rearrangement site is selected from the 5th or 6th domain of the secondary structure of the Group I type self-splicing ribozyme.

5. The cyclization backbone sequence according to claim 3, characterized in that The 5' terminal homologous segment and the 3' terminal homologous segment in the circularized backbone sequence are artificially designed base complementary pairing elements; and / or the 5' internal homologous segment and the 3' internal homologous segment are artificially designed base complementary pairing elements.

6. A plasmid for RNA in vitro cyclization, characterized in that The vector is inserted with the cyclization backbone sequence described in any one of claims 3 to 5.

7. The plasmid according to claim 6, characterized in that The vector is a pcDNA3.1 vector plasmid.

8. Use of the Group I self-splicing ribozyme according to claim 1 or 2, the cyclization backbone sequence according to any one of claims 3 to 5, or the plasmid according to any one of claims 6 to 7 in preparing circular RNA in vitro.

9. A method for preparing circular RNA in vitro, characterized in that: The method comprises using the cyclization backbone sequence of any one of claims 3 to 5 or the plasmid of any one of claims 6 to 7 as a template, and obtaining a circular RNA molecule by in vitro cyclization.

10. The method for preparing circular RNA in vitro according to claim 9, characterized in that: The method comprises: S1. Using the circularized backbone sequence according to any one of claims 2 to 5 or the plasmid according to any one of claims 6 to 7 as a template, preparing a linearized plasmid, S2. Prepare linear RNA using linearized plasmid to obtain IVT reaction product; S3. Prepare circular RNA by cyclizing the IVT reactants at 55°C for 20-30 minutes.

Images