Method for synthesizing circular RNA (Ribonucleic Acid) by utilizing click reaction

Abstract

The invention discloses a method for synthesizing circular RNA (Ribonucleic Acid) by utilizing click reaction. The invention provides a method for synthesizing circular RNA (Ribonucleic Acid), which comprises the following steps: 1) synthesizing RNA by taking linear DNA (Deoxyribonucleic Acid) to be cyclized as a template, and adding alkynyl modification and azide modification at two ends of the RNA to obtain RNA with two ends modified by alkynyl and azide; and 2) carrying out a click reaction on the RNA with both ends modified with alkynyl and azide under the catalysis of Cu ions, and collecting a click reaction product to obtain the circular RNA. According to the method, through click chemistry, after the reaction is finished, a solid-phase azide or alkynyl marker is added, so that the purification process can be greatly simplified, and the purification efficiency can be improved; according to the invention, through click chemistry, the reaction can be carried out only through sequence complementation and end-to-end drawing, the conditions are controllable, and the reaction efficiency is high. Therefore, the cyclization efficiency can be greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for synthesizing circular RNA using click reaction. Background Art

[0002] With the deepening of people's understanding of the physiological functions of natural circRNAs, there is an increasing interest in developing circRNA synthesis technologies and exploring the application of synthetic circRNAs in disease treatment. So far, synthetic circRNAs have been used not only for treatment, such as replacing therapeutic proteins and polypeptides as well as vaccines, but also as biosensors. At the same time, in order to optimize the therapeutic effect while reducing side effects, various methods have been tried to synthesize circRNA.

[0003] Currently, there are three main methods for constructing circular RNA: circular RNA construction based on Alu sequence, circular RNA construction based on PIE, and circular RNA construction based on enzyme catalysis.

[0004] Alu element is a type of mobile genetic element (MGE), with more than 1 million copies in the human genome (accounting for 11% of the human genome), and is the main factor for generating genetic diversity and non-allelic recombination events. Therefore, by utilizing the splicing property of Alu element, inserting foreign genes between two Alu elements can generate engineered circular RNA in cells. This method of constructing circular RNA mimics the process of natural circular RNA, and the length of the inserted fragment is not limited. However, it has the disadvantages of complex purification process and the need for endogenous proteins to participate in the circularization process.

[0005] Group I intron is a type of self-splicing nuclease that catalyzes the excision of its own sequence from the precursor RNA sequence to produce mature mRNA, tRNA, and rRNA. Puttaraju and Been (1992) first reported that by introducing group I intron sequences at both ends of the target gene to form a PIE (permuted intron–exon) structure, the target gene can be covalently linked head to tail to form circular RNA. Wesselhoeft and colleagues (2018) reported that the PIE structure constructed using the modified Anabaena pre-tRNAintrons sequence can significantly increase the yield of circular RNA. Although the method of constructing circular RNA based on PIE has high circularization efficiency and simple and controllable preparation process, it has low circularization efficiency for long fragments and has redundant sequences.

[0006] T4 RNA ligase 2 has been successfully used to implement the RNA cyclization construction strategy, which can simultaneously ligate the RNA strand breaks in single-stranded or double-stranded RNAs. However, the enzyme-catalyzed RNA cyclization construction strategy is suitable for the cyclization of short fragments. When performing long-fragment cyclization, the cyclization efficiency is low.

[0007] Currently, the circular RNA construction strategy is mainly based on the PIE process. During the preparation process, there will be three forms: precursor, nicked, and circular RNA. The three forms of RNA have little difference in molecular weight and biological characteristics. Especially for nicked and circular RNA, they only differ in the closed-loop and linear forms. Therefore, the purification work is difficult and inefficient. The PIE-based cyclization process is greatly affected by the sequence and reaction conditions, seriously affecting the cyclization efficiency. For example, the cyclization efficiency of fragments around 2.7k is <50%. Other methods for synthesizing circular RNA cannot perform base modification. As is well known, incorporating modified bases, such as pseudouridine, into mRNA can greatly reduce its immunogenicity and improve the protein expression level. However, experiments have shown that the cyclization method based on the PIE strategy cannot perform base modification. And the efficiency of T4 RNA ligase is low and cannot meet the requirements of large-scale synthesis. For the construction of circular RNA based on the PIE or Alu strategy, strict sequence design and verification are required, otherwise the cyclization efficiency will be affected.

[0008] Therefore, finding a new method for synthesizing circular RNA has become a research direction. Summary of the Invention

[0009] The object of the present invention is to provide a method for synthesizing circular RNA using click reaction.

[0010] In the first aspect, the present invention provides a method for synthesizing circular RNA, including the following steps:

[0011] 1) Synthesize RNA using the linear DNA to be cyclized as a template, and add alkyne modification and azide modification to both ends of the RNA to obtain RNA with alkyne and azide modifications at both ends;

[0012] 2) Perform click reaction on the RNA with alkyne and azide modifications at both ends under the catalysis of Cu ions, and collect the click reaction product, that is, circular RNA is obtained.

[0013] In the above method, the RNA with alkyne and azide modifications at both ends is RNA with alkyne modification at the 5' end and azide modification at the 3' end.

[0014] In the above method, step 1) includes the following steps:

[0015] 1)-a) Synthesize RNA using the linear DNA to be cyclized as a template, and introduce 5'-terminal alkynyl-modified GTP into the RNA synthesis system to obtain RNA with a 5'-terminal alkynyl modification.

[0016] The chemical structural formula of the 5'-terminal alkynyl-modified GTP is shown in Formula 1 below:

[0017]

[0018] 1-b) Perform 3'-azide modification on the RNA with a 5'-terminal alkynyl modification to obtain RNA with a 5'-terminal alkynyl modification and a 3'-terminal azide modification.

[0019] Furthermore, the 3'-azide modification is achieved by introducing 3'-azide-modified ddATP in the tailing reaction.

[0020] The RNA synthesis system described above includes: the linear DNA to be cyclized, dNTP, 5'-alkynyl-modified GTP, and RNA polymerase, as shown in Table 1 specifically. In the examples of the present invention, the RNA polymerase is T7 RNA polymerase, and the synthesis condition is incubation at 37 °C for 2 hours.

[0021] The reaction system of the tailing reaction described above includes: the RNA with a 5'-terminal alkynyl modification, Poly(A) Polymerase, and 3'-azide-modified ddATP. In the examples of the present invention, Poly(A) Polymerase is E. coli Poly(A) Polymerase. As shown in Table 6 specifically, the condition of the tailing reaction is incubation at 37 °C for 0.5 hours.

[0022] In the above text, the click reaction system includes CuSO4, THPTA, Na-Ascorbate, and RNA with a 5'-terminal alkynyl modification and a 3'-terminal azide modification, as shown in Table 7 specifically. The reaction condition is reaction at room temperature for 0.5 hours under argon protection.

[0023] In the method described above,

[0024] The method further includes the step of purifying circular RNA from the click reaction product, and the steps are as follows:

[0025] Use a solid-phase alkynyl label and a solid-phase azide label to capture the uncyclized RNA from the click reaction product to obtain circular RNA.

[0026] In the method described above, the solid-phase alkynyl label is a short-chain DNA molecule with a 3'-terminal azide modification;

[0027] The solid-phase azide label is a short-chain DNA molecule with a 5'-terminal alkynyl modification;

[0028] In an embodiment of the present invention, the short chain consists of 25 A's and can also be replaced with other sequences or chemical molecules, etc.

[0029] The specific steps for obtaining purified circular RNA by fishing uncircularized RNA with a solid-phase alkyne label and a solid-phase azide label are as follows:

[0030] 1) Add the solid-phase alkyne label 5’-Alkyne-A25-3’biotin to the click reaction product and react (react at room temperature for 0.5 hours), then add the reaction product to streptavidin-labeled magnetic beads and react (react at room temperature for 0.5 hours) to obtain a magnetic bead reaction product;

[0031] 2) Collect the supernatant in the magnetic bead reaction product, then add the solid-phase azide label 5’-Azido-A25-3’biotin and continue to react (react at room temperature for 0.5 hours), and collect the supernatant;

[0032] 3) Purify the supernatant using an RNA purification and concentration kit, collect the purified product to obtain circular RNA.

[0033] In a second aspect, the present invention provides a compound as shown in Formula 1:

[0034]

[0035] In a third aspect, the present invention provides the use of the compound described in the second aspect in the preparation of 5’-terminal alkyne-modified RNA;

[0036] Or the present invention provides the use of the compound described in the second aspect in the synthesis of circular RNA;

[0037] Or the present invention provides the use of the compound described in the second aspect in the synthesis of circular RNA from linear DNA molecules.

[0038] In a fourth aspect, the present invention provides a kit for synthesizing circular RNA, including the following:

[0039] 1) dNTP;

[0040] 2) The compound described in the second aspect;

[0041] 3) RNA polymerase;

[0042] 4) Poly(A) Polymerase;

[0043] 5) The 3’-azide-modified ddATP described in the first aspect;

[0044] 6) CuSO4;

[0045] 7) THPTA;

[0046] 8) Sodium Ascorbate

[0047] In a fifth aspect, the present invention provides the use of the kit described in the fourth aspect in the synthesis of circular RNA;

[0048] Alternatively, the present invention provides the use of the kit described in the fourth aspect in the synthesis of circular RNA from linear DNA molecules.

[0049] In the present invention, through click chemistry, after the reaction is completed, adding a solid-phase azide or alkyne label can greatly simplify the purification process and improve the purification efficiency; through click chemistry, the reaction can be carried out only by bringing the head and tail closer through sequence complementarity, with controllable conditions and high reaction efficiency. Therefore, the cyclization efficiency can be greatly improved.

[0050] Based on the click chemistry cyclization strategy, the present invention can well avoid the deficiency of inability to perform base modification. By means of a segmented method, modification (CDS region) or non-modification (IRES region) is carried out separately to construct a chimeric circular RNA, simultaneously meeting conditions such as high efficiency and low immunogenicity.

[0051] The circular RNA based on click chemistry in the present invention can theoretically simulate natural circular RNA products and does not require a complex sequence design and screening process. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a schematic diagram of the synthesis of circular RNA by click reaction.

[0053] Figure 2 It is the synthesis route of 5'-Alkyne GTP.

[0054] Figure 3 It is a reference diagram of the reaction formula for the solid-phase synthesis of 5'-Alkyne GTP.

[0055] Figure 4 It is a reference diagram of the ammonolysis process reaction formula.

[0056] Figure 5 It is the detection result of circular RNA expression.

[0057] Figure 6 It is the detection principle of the cyclization effect of the product containing circular RNA and the detection result of the control linear RNA.

[0058] Figure 7 It is the detection result of the product containing circular RNA and the control linear RNA.

[0059] Figure 8 It is the reaction diagram of intermediate 4.

[0060] Figure 9 Reaction diagram for intermediate 5

[0061] Figure 10 Reaction diagram for intermediate 6

[0062] Figure 11 Reaction formula for ammonolysis process Specific implementation manners

[0063] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods

[0064] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources

[0065] Example 1. Establishment of a method for synthesizing circular RNA using click reaction

[0066] Schematic diagram of synthesizing circular RNA by click reaction is as Figure 1 shown

[0067] I. Synthesis of 5'-Alkyne-modified RNA

[0068] 1. Synthesis of 5'-Alkyne-modified (alkynyl-modified) RNA

[0069] Using the linear DNA to be cyclized as a template, incubate at 37 °C for 2 hours according to the system shown in Table 1 below to synthesize RNA while performing 5'-Alkyne modification, obtaining a reaction product containing 5'-Alkyne-modified RNA

[0070] Table 1 is the synthesis system

[0071] Component Dosage DNA template 1 μg ATP (100 mM) 2 μl CTP (100 mM) 2 μl UTP (100 mM) 2 μl GTP (100 mM) 1.11 μl 5'-Alkyne GTP (100 mM) 0.89 μl 5x Buffer 4 μl T7 RNA polymerase (20 U / μl) 2 μl RNase-free ddH2O Up to 20 μl

[0072] T7 RNA polymerase in the above table is Thermo T7 RNA polymerase, purchased from ThermoFisher, product number: EP0112

[0073] The chemical structural formula of 5'-Alkyne GTP (hereinafter simply referred to as compound AG) in the above table is as follows

[0074]

[0075] 2. Purify to obtain 5'-Alkyne-modified RNA

[0076] The reaction product obtained in 1 above is subjected to Purify with the RNA Cleanup Kit (product number: T2040L), collect the purified product, which is a 5'-Alkyne modified RNA solution (solvent is DEPC dH2O, concentration is 1 μg / μl).

[0077] Then store the above 5'-Alkyne modified RNA solution at -80 °C for later use to obtain 5'-Alkyne modified RNA.

[0078] 3. Preparation of 5'-Alkyne GTP

[0079] 5'-Alkyne GTP (hereinafter referred to as compound AG) can be synthesized, and the synthesis route is as Figure 2 , and the whole synthesis process includes the following steps:

[0080] 1). Solid-phase synthesis, the reaction formula is referred to Figure 3 , and the required reactants are shown in Table 3.

[0081] Note: Figure 3 The abbreviations or English names in the figure are shown in Table 2 below.

[0082] Table 2 is for abbreviations

[0083] Abbreviation or English name Chinese name TCA Trichloroacetic acid DMT 4,4'-Dimethoxytriphenylmethyl ETT 5-Mercaptotetrazole Py Pyridine <![CDATA[Ac2O]]> Acetic anhydride DEA Diethylamine NH4OH Ammonia water

[0084] Table 3 is the reactants required for solid-phase synthesis

[0085]

[0086] Specific steps:

[0087] (1) Check the target sequence to be synthesized, analyze the auxiliary reagents and monomer usage amounts required, dissolve the monomers separately with acetonitrile to prepare a 0.2 M solution concentration (the monomer preparation can be 10% in excess, and there is AmiditePurge in the program design), and supplement the auxiliary reagents to a sufficient amount. Weigh 2 g of the solid-phase carrier Primer Support 5G UnyLinker 350 into a 24 ml column.

[0088] (2) Use an AKTA 100 synthesizer for synthesis.

[0089] (3) The specific solid-phase synthesis method is as follows:

[0090] This solid-phase synthesis reaction will have three cyclic reactions, namely the reaction of Primer Support 5G UnyLinker350 with rA-Phosphoramidite, the reaction of the obtained intermediate with rG-Phosphoramidite, and the reaction of the obtained intermediate with 5’-Hexynyl Phosphoramidite finally. Each cycle can be further divided into 4 reaction steps: Detritylation Step, Coupling step, Oxidation step, and Capping step, which are specifically as follows:

[0091] Primer Support 5G UnyLinker 350 reacts with the Deblock reagent to remove DMT, and then the obtained intermediate 1 reacts with rA-Phosphoramidite in the presence of the Activator reagent to undergo a condensation reaction to obtain intermediate 2. The molar ratio of intermediate 1 to rA-Phosphoramidite is 1:3. Intermediate 2 is oxidized to intermediate 3 under the condition of the oxidation reagent. If intermediate 1 does not react in the Coupling step, it will react with the CAP reagent (CAP-A / CAP-B reagent) after the oxidation reaction to obtain intermediate 4, as Figure 8 shown.

[0092] Intermediate 3 will also undergo the above four reaction steps and react with rG-Phosphoramidite to obtain intermediate 5, as Figure 9 shown.

[0093] Intermediate 5 will also undergo the above four reaction steps and react with 5’-Hexynyl Phosphoramidite to obtain intermediate 6, as Figure 10 shown.

[0094] 2) Ammonolysis process (cleavage and deprotection)

[0095] Figure 4 For the reaction formula of the ammonolysis process, refer to the figure, and the reaction formula is referred to as Figure 11 .

[0096] The material ratio used in the reaction is shown in Table 4 below.

[0097] Table 4 shows the material ratio used

[0098]

[0099] Specific steps:

[0100] Remove the solid support after the reaction from the synthesizer, add it to an explosion-proof bottle, add 100 ml of ammonia water, and ammonolyze it for 2 h at 65 - 80 °C using an ammonolysis instrument. Let it cool to room temperature naturally, centrifuge and filter to obtain the supernatant, freeze-dry it, add 25 ml of DMSO to assist dissolution, then add 50 ml of TEA·3HF to dissolve the crude product, react at 80 °C for 1.5 h until the reaction is complete, add 3M NaAc solution, and continue to add 200 ml of n-butanol to precipitate the solid. Filter to obtain the crude product of AG.

[0101] 3) Purification process (ion purification)

[0102] The material ratio is as shown in Table 5 below.

[0103] Table 5 is the material ratio

[0104]

[0105]

[0106] Specific steps:

[0107] Prepare the mobile phase: Phase B: 2M NaBr solution; Phase A: 20 mM NaH2PO4 solution (adjust the pH to 7.5 - 7.8 using NaOH);

[0108] Chromatographic column specifications: 30 mm, 170 mm; packing: GE source Q 15.

[0109] Purify using preparative HPLC to obtain the pure product of AG, and remove a large amount of water by rotary evaporation at low temperature (30 °C) to concentrate the purified solution. Use the preparative HPLC UV detector to detect and collect samples at the start / end.

[0110] 4) Desalting process

[0111] Use G25 for desalting to remove small molecule salts such as free sodium bromide to obtain the pure product of AG.

[0112] 5) Freeze-drying process

[0113] Add the pure product of AG to a centrifuge tube and freeze-dry it at -45 °C to obtain 45 mg of dry powder, which is 5'-Alkyne GTP (AG).

[0114] After detection, the chemical structural formula of 5'-Alkyne GTP (AG) is as follows:

[0115]

[0116] II. 3'-azide modification

[0117] 1. Perform 3'-azide modification by adding a tail to RNA

[0118] Using the purified 5'-Alkyne modified RNA (5’-G-ssRNA) in item 1 above as a template, incubate for 0.5 hours at 37 °C according to the system shown in Table 6 below for RNA tailing to obtain a reaction product containing 5’-Alkyne-RNA-3’-Azido.

[0119] Table 6 is the RNA tailing reaction system

[0120]

[0121]

[0122] The E.coli Poly(A)Polymerase in the above table was purchased from Novoprotein, product number: DD4111

[0123] 2. Purify to obtain 5’-Alkyne-RNA-3’-Azido

[0124] Purify the reaction product obtained in item 1 above using RNA Cleanup Kit (product number: T2040L) to collect the purified product, which is a 5’-Alkyne-RNA-3’-Azido solution (the solvent is DEPC dH2O, concentration is 1 μg / μl).

[0125] Then freeze the above 5’-Alkyne-RNA-3’-Azido solution at -80 °C for later use to obtain 5’-Alkyne-RNA-3’-Azid.

[0126] III. Click reaction

[0127] Carry out a click reaction on the 5’-Alkyne-RNA-3’-Azido obtained in item 2 of the above II according to the system shown in Table 7, react at room temperature for 0.5 hours under argon protection, collect the reaction product to obtain a product containing circular RNA (including circular RNA, 5’ppp-G-ssRNA-A-N3 3’, 5’ppp-G-ssRNA-OH 3’).

[0128] Table 7 is the click reaction system

[0129]

[0130] IV. Remove impurities and purify to obtain circular RNA

[0131] 1. Add 1 μmol of 5’-Alkyne-A25-3’biotin (a short chain composed of 25 As, with an alkyne group modified at the 5’ end and biotin modified at the 3’ end) to the 100 μl of the product containing circular RNA obtained above. Add the above (III. Click reaction) reaction system to 150 μl, and react at room temperature for 0.5 hours.

[0132] 2. Add 1 mg of Dynabeads TM MyOne TM Streptavidin C1 (product number: 65002) to the reaction product obtained in 1. Fully suspend the magnetic beads, incubate with rotation at room temperature for 20 minutes, then place them on a magnet. Wait until all the magnetic beads are adsorbed, aspirate the supernatant with a pipette, and transfer it to the centrifuge tube from the previous step. There is a total of 150 μl of solution, and collect the supernatant.

[0133] 3. Aspirate the supernatant from the reaction product obtained in 2, and then add 1 μmol of 5’-Azido-A25-3’biotin (a short chain composed of 25 As, with an azide group modified at the 5’ end and biotin modified at the 3’ end). Add the above (III. Click reaction Clickreaction) reaction system to 200 μl again, and react at room temperature for 0.5 hours.

[0134] 4. Add 1 mg of Dynabeads TM MyOne TM Streptavidin C1 (product number: 65002) to the reaction product obtained in 3. Fully suspend the magnetic beads, incubate with rotation at room temperature for 20 minutes, then place them on a magnet. Wait until all the magnetic beads are adsorbed, aspirate the supernatant with a pipette, and transfer it to the centrifuge tube from the previous step. There is a total of 200 μl of solution, and collect the supernatant.

[0135] 5. Purify the above supernatant using the reference RNA Cleanup Kit (product number: T2040L) to collect the purified product, which is a circular RNA solution (the solvent is DEPC dH2O, and the concentration is 1 μg / μl).

[0136] Example 2. Preparation of Circular GFP by Click Method

[0137] I. Synthesis of 5'-Alkyne-Modified RNA

[0138] The linear DNA to be circularized is the linearized plasmid sequence shown in Sequence 1, which expresses GFP.

[0139] Synthesize according to the methods of 1 and 2 in Example 1 to obtain 5'-Alkyne-modified RNA.

[0140] II. 3'-Azide Modification

[0141] Synthesis was carried out according to the method of Example 1 II to obtain 5'-Alkyne-RNA-3'-Azido.

[0142] III. Click reaction

[0143] The reaction was carried out according to the method of Example 1 III to obtain a product containing circular RNA.

[0144] The product containing circular RNA obtained above was detected as follows:

[0145] 1. Mix 1 μg of the product containing circular RNA with a DNA probe (5‘AGAGUUGCCCGU(TAC)G 3’

[0146] RNA(DNA)) at a molar ratio of 1:5, and supplement with EDPC dH2O to 10 μl;

[0147] 2. After denaturation at 75 °C for 10 minutes, cool to room temperature at room temperature;

[0148] 3. Referring to the RNase H instruction manual (M0297, NEB), add 1.5 μl of 10x Buffer and 0.2 μl (1 U) of RNase H, supplement with EDPC dH2O to 15 μl, and incubate at 37 °C for 15 minutes;

[0149] 4. Add 1 μl of 0.5 M EDTA to terminate the reaction and place it on ice for later use.

[0150] Use linear RNA (the coding nucleic acid is positions 810-1527 of Sequence 1; this RNA is denoted as linear in the figure) as a control.

[0151] Figure 6 It is the detection principle of the cyclization effect of the product containing circular RNA and the detection results of the control linear RNA (the coding nucleic acid is positions 810-1527 of Sequence 1; this RNA is denoted as linear IVT in the figure).

[0152] The detection results of the product containing circular RNA (denoted as scaffold 1 in the figure) and the control linear RNA (denoted as linear in the figure) are as Figure 7As shown, it can be seen that after digestion with RNase H, linear RNA can produce two smaller digestion products without residual precursor RNA, indicating that this method can be used for circularization efficiency detection. Under the same conditions, the product containing circular RNA retains a product with the same size as the precursor after digestion with RNase H, and at the same time produces two smaller digestion products. Through Imagej gray scale analysis, after digestion with RNase H(+), the gray scale of the band with the same size as the precursor is about 68% compared with the gray scale of the precursor without RNase H(-) treatment, indicating that the circularization efficiency is 68%.

[0153] IV. Purification to Remove Impurities to Obtain Circular RNA

[0154] Purification was carried out according to the method in Example 1 IV to obtain circular RNA.

[0155] The purified circular RNA was detected by HPLC. It was correctly loaded onto a Sec 1000A semi-preparative column (Sepax, 10×300mm 5μm 1000A, 215950-10030), and detected under the conditions of mobile phase of 150 mM phosphate buffer (pH 6.0) + 6 M urea and a flow rate of 1.3 mL / min.

[0156] The final product was a single peak (single product), and the purity was greater than 95%.

[0157] V. Detection of Circular RNA Expression

[0158] Transfection process:

[0159] Lipofectamine TM 3000 transfection reagent, product number: L3000015, ThermoFisher

[0160] Opti-MEM TM Medium, product number: 31985070, ThermoFisher

[0161] Taking a 96-well plate as an example

[0162] 1. Seed 293 cells on the first day and perform transfection when the cells reach about 70–90% confluence on the second day;

[0163] 2. Dilute 0.1 μg of RNA to be transfected with 25 μl of Opti-MEM TM medium to prepare an RNA premix, and mix well;

[0164] 3. Dilute 0.1 μl of Lipofectamine TM 3000 with 25 μl of Opti-MEM TM medium to prepare a premix, and mix well;

[0165] 4. Add the diluted RNA (at a 1:1 ratio) to each tube of the diluted Lipofectamine TM 3000 reagent;

[0166] 5. Incubate at room temperature for 10 - 15 minutes to obtain the RNA-lipid complex;

[0167] 6. Add the RNA-lipid complex obtained in step 5 to the cells obtained in step 1.

[0168] 7. Observe under a fluorescence microscope 24 hours after transfection.

[0169] In the above, the RNA to be transfected is the circular RNA (denoted as click-GFP (circular) in the figure) and the linear RNA (denoted as M0023 (linear) in the figure; the coding nucleic acid is positions 810 - 1527 of sequence 1) obtained in the above four.

[0170] The results are as Figure 5 shown. It can be seen that the circular RNA obtained by Click is expressed in 293 cells.

Claims

1. A method for synthesizing circular RNA, comprising the following steps: 1) Synthesize RNA using the linear DNA to be circularized as a template, and add alkynyl modification and azide modification to both ends of the RNA to obtain RNA with alkynyl and azide modifications at both ends; 2) Perform a click reaction on the RNA with alkynyl and azide modifications at both ends under the catalysis of Cu ions, and collect the click reaction product to obtain circular RNA.

2. The method according to claim 1, wherein: The RNA with alkynyl and azide modifications at both ends is RNA with an alkynyl modification at the 5' end and an azide modification at the 3' end.

3. The method according to claim 1 or 2, wherein: Step 1) comprises the following steps: 1)-a. Synthesize RNA using the linear DNA to be circularized as a template, and introduce 5'-terminal alkynyl-modified GTP into the RNA synthesis system to obtain RNA with an alkynyl modification at the 5' end; The chemical structural formula of the 5'-terminal alkynyl-modified GTP is as shown in Formula 1 below: 1-b) Perform 3'-azide modification on the RNA with an alkynyl modification at the 5' end to obtain RNA with an alkynyl modification at the 5' end and an azide modification at the 3'.

4. The method according to claim 3, wherein: The 3'-azide modification is achieved by introducing 3'-azide-modified ddATP in the tailing reaction.

5. The method according to any one of claims 1-4, wherein: The method further comprises the step of purifying circular RNA from the click reaction product, and the steps are as follows: Use a solid-phase alkynyl marker and a solid-phase azide marker to capture the uncircularized RNA from the click reaction product to obtain circular RNA.

6. The method according to claim 5, wherein: The solid-phase alkynyl marker is a short-chain DNA molecule with an azide modification at the 3' end; The solid-phase azide marker is a short-chain DNA molecule with an alkynyl modification at the 5' end.

7. A compound, as shown in Formula 1:

8. Use of the compound shown in claim 7 in the preparation of RNA with an alkynyl modification at the 5' end; or use of the compound shown in claim 7 in the synthesis of circular RNA; Or use of the compound shown in claim 7 in the synthesis of circular RNA from a linear DNA molecule.

9. A kit for synthesizing circular RNA, comprising the following: 1) dNTP; 2) The compound shown in claim 7; 3) RNA polymerase; 4) Poly(A) Polymerase; 5) The 3'-azide-modified ddATP described in claim 4; 6) CuSO4; 7) THPTA; 8) Na-Ascorbate.

10. Use of the kit described in claim 9 in the synthesis of circular RNA; Or, use of the kit described in claim 9 in the synthesis of circular RNA from a linear DNA molecule.

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