Modification method for improving stability and translation efficiency of circular RNA and application of modification method

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a modification method for improving RNA stability and translation efficiency and application of the modification method. The invention provides a method for improving RNA (Ribonucleic Acid) stability and RNA translation efficiency, which comprises the following steps: reacting imidazole or a derivative thereof, an adduct and RNA, and optimizing modification conditions, selection and concentration of a modification compound. The modified RNA provided by the invention not only has good hydrolysis resistance, but also has beneficial translation efficiency. In the condition of the same amount of RNA at the same time, the modified RNA can generate more proteins, which is more beneficial to induction of proposal immune response. Meanwhile, due to the fact that the modified RNA has good stability, the modified RNA has the potential of being matched with non-LNP delivery.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a modification method for improving the stability and translation efficiency of circular RNA and an application thereof. Background Art

[0002] RNA vaccines play an important protective role. Its short research and development cycle, high biosafety and ability to induce strong specific immunity make RNA vaccines one of the current mainstream emerging vaccine types. According to incomplete statistics, about 400 different types of RNA vaccines have entered clinical trials, covering infectious diseases and tumor vaccines. Existing RNA vaccines mainly use lipid nanoparticles (LNP) as packaging and delivery systems, because LNP can provide delivery for RNA, and more importantly, isolate external substances from degrading RNA to ensure the stability and biological activity of RNA. However, LNP has poor targeting and is prone to off-target effects, inducing inflammation and organ toxicity; if it is to be compatible with other non-LNP delivery systems for precise delivery, it is necessary to ensure that RNA remains relatively stable under complex physiological conditions.

[0003] In 2023, Eric Kool's team published an article in Nature Chemistry titled Reversible 2′-OH acylation enhances RNA stability, which explained that acetylation of the hydroxyl group at the ribose position of RNA can enhance RNA's resistance to hydrolysis. However, this article also mentioned that the modification method did not promote RNA translation; on the contrary, some modified compounds inhibited RNA translation.

[0004] Due to the instability of RNA molecules, they are easily degraded, and the above method is difficult to adapt to delivery systems other than lipid nanoparticles; and existing RNA vaccines only involve optimizing the translation efficiency of RNA sequences, but usually these optimizations are only targeted at a certain species and cannot achieve broad-spectrum translation efficiency enhancement of translatable RNA. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes a modification prevention method with stronger stability, higher translation efficiency and wider application range of RNA, which is as follows:

[0006] The first aspect of the present invention aims to provide a method for RNA modification.

[0007] The second aspect of the present invention aims to provide an RNA molecule.

[0008] The purpose of the third aspect of the present invention is to provide a method for preparing the RNA molecule of the second aspect of the present invention.

[0009] The purpose of the fourth aspect of the present invention is to provide the method of the first aspect of the present invention and the application of the RNA molecule of the second aspect of the present invention.

[0010] The fifth aspect of the present invention aims to provide a vaccine.

[0011] The sixth aspect of the present invention is to provide a kit.

[0012] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides any one of the methods a1) to a2),

[0014] a1) Methods for improving RNA stability;

[0015] a2) Methods for improving RNA translation efficiency;

[0016] The method comprises the following steps:

[0017] reacting imidazole or its derivative, adduct, and RNA;

[0018] The adduct includes N,N'-carbonyldiimidazole.

[0019] Preferably, the RNA comprises at least one of circular RNA, linear RNA, double-stranded RNA, and stem-loop RNA.

[0020] More preferably, it is a circular RNA.

[0021] Preferably, the imidazole or its derivative comprises at least one of imidazole, histidine and ethynyl imidazole.

[0022] Preferably, the derivatives include pharmaceutically acceptable salts and pharmaceutically acceptable chemical modifications.

[0023] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0024] Preferably, the pharmaceutically acceptable modification comprises at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

[0025] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0026] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0027] Preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0028] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0029] Preferably, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine.

[0030] Preferably, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0031] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0032] Preferably, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine and ornithine.

[0033] Preferably, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0034] Preferably, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation. For example, lipophilicity can be enhanced by introducing fluorine atoms (fluoroethyl) to improve blood-brain barrier permeability. The corresponding modification method can be selected according to the actual use.

[0035] Preferably, the ratio of imidazole or its derivatives, adducts and RNA is (175-225 mM): (800-1200 mM): 1 g / L.

[0036] Preferably, the reaction is carried out in an acidic buffer.

[0037] Preferably, the pH of the acidic buffer solution is 3.0-5.0.

[0038] More preferably, the pH of the acidic buffer solution is 3.0 to 3.6.

[0039] Preferably, the reaction time is 2 to 12 hours.

[0040] Preferably, the reaction temperature is 4°C to 37°C.

[0041] Preferably, the acidic buffer comprises at least one of glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-citric acid buffer, and acetic acid-sodium acetate buffer.

[0042] Preferably, the reaction further includes a purification step.

[0043] The second aspect of the present invention provides an RNA molecule having a modification as shown in the following formula:

[0044]

[0045] The third aspect of the present invention provides a method for preparing the RNA molecule of the second aspect of the present invention, which is prepared using the method described in the first aspect of the present invention.

[0046] Specifically, the reaction process is shown in the following formula:

[0047]

[0048] The fourth aspect of the present invention provides the use of the method of the first aspect of the present invention and the RNA molecule of the second aspect in preparing a product.

[0049] Preferably, the product comprises at least one of a drug, a vaccine, a reagent, and a kit.

[0050] Preferably, the product comprises pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0051] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0052] Preferably, the dosage form of the product includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0053] Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0054] Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0055] The drugs described in the present invention are in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, and cataplasms, that is, pharmaceutical preparations include tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches, gels, cataplasms and the like, but are not limited to the above forms.

[0056] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and as inactive ingredients of medicaments. A compilation of pharmaceutically acceptable excipients can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994); Pharmacopoeia of the People's Republic of China - Catalogue of Pharmaceutical Excipients.

[0057] The fifth aspect of the present invention provides a vaccine comprising the RNA molecule described in the second aspect of the present invention.

[0058] Preferably, the vaccine may be formulated or administered in combination with one or more other components. For example, the immunogenic composition may include other components, including but not limited to adjuvants.

[0059] Preferably, the vaccine is an mRNA vaccine.

[0060] In the fifth aspect of the present invention, mRNA vaccines are mRNAs encoding viral antigens that are introduced into the body and expressed in the body to produce antigens, which then stimulates an immune response in the body. Since mRNA only exists in the cytoplasm and will not be integrated into the host genome and can be naturally degraded in the body, it is safer. The mRNA vaccine itself can be used as an adjuvant to induce the body to produce a natural immune effect, and the mRNA vaccine can induce strong humoral immunity and cellular immunity, and generally has a stronger immune effect than traditional inactivated vaccines and subunit protein vaccines. The mRNA vaccine does not need to be used in combination with other vaccine adjuvants, so adverse reactions caused by other substances can be reduced. However, those skilled in the art can also understand that the mRNA vaccine can be used in combination with conventional adjuvants in the art to achieve better technical effects.

[0061] Preferably, the adjuvant includes but is not limited to biological adjuvants (such as interleukins, interferons, etc.), inorganic adjuvants (aluminum hydroxide, alum, aluminum phosphate, etc.), synthetic adjuvants (double-stranded polyinosinic acid, double-stranded polyadenylic acid, etc.), oils (vegetable oils, animal oils, mineral oils, etc.), and Freund's adjuvants (complete Freund's adjuvant, incomplete Freund's adjuvant).

[0062] Preferably, the vaccine may also include a liposome delivery system.

[0063] Preferably, the liposomal delivery system comprises lipid nanoparticles (LNP).

[0064] For example, the RNA (eg, mRNA) disclosed in the present invention can be formulated in lipid nanoparticles (LNPs). Lipid nanoparticles generally comprise ionizable cationic lipids, non-cationic lipids, sterols and PEG lipid components and target nucleic acid cargo. The lipid nanoparticles of the present disclosure can be produced using components, compositions and methods generally known in the art, see, for example, CN116535381B, TW202340136A, CN116178193B, CN116178193B, CN115784921B, CN115745820B, CN115677518B, CN114957027B, CN114044741B, CN116854754A, CN116785265A, CN116789764A, CN116396178A, CN116375592A, CN116082275A, all of which are incorporated herein by reference in their entirety.

[0065] Preferably, "cationic lipid" refers to a lipid with a positive charge at a selected pH value. Cationic liposomes are easily combined with negatively charged nucleic acids, that is, they interact with negatively charged phosphate groups present in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNP is one of the current mainstream delivery vectors.

[0066] Preferably, "neutral lipids" refer to lipids that are uncharged or exist in the form of zwitterions at a selected pH value and play an auxiliary role. The neutral lipids may adjust the fluidity of the nanoparticles to a lipid bilayer structure and improve the efficiency by promoting lipid phase transition, and may also affect the specificity of the target organ.

[0067] Preferably, "structural lipids" refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids. The structural lipids can be selected from, but not limited to, the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha-tocopherol, corticosteroids, and mixtures thereof.

[0068] A sixth aspect of the present invention provides a kit.

[0069] Preferably, the kit includes vaccine use-related instruments, including but not limited to syringes, containers, etc.

[0070] Preferably, the kit includes instructions, which record the usage and precautions of the kit.

[0071] The beneficial effects of the present invention are:

[0072] The present invention provides a method for improving RNA stability and RNA translation efficiency, wherein imidazole or its derivatives, adducts, and RNA are reacted, and the modification conditions, the selection of the modification compound, and the concentration are optimized. The modified RNA in the present invention not only has good hydrolysis resistance, but also has a beneficial translation efficiency. This plays a very important role in the research of RNA vaccines: under the same time and the same amount of RNA conditions, the modified RNA in the present invention can produce more protein, which is more conducive to inducing a provocative immune response. At the same time, because the modified RNA has good stability, it has the potential to be suitable for non-LNP delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0074] Figure 1 Results for pH optimization of modification reactions.

[0075] Figure 2 Optimize results for modification reaction time.

[0076] Figure 3 This is the result of the RNA degradation experiment of the present invention.

[0077] Figure 4Fluorescence microscope images and overall brightness comparison statistics of RNA expressing mCherry and modified RNA (IM-RNA) of the present invention after HEK293T transfection and expression for 72 hours.

[0078] Figure 5 The luciferase luminescence reading results were measured after 72 hours of transfection of luciferase-expressing RNA and modified RNA (IM-RNA) in HEK293T cells.

[0079] Figure 6 The expression levels of OVA and RBD were detected by immunoblotting 72 hours after the RNA and modified RNA (IM-RNA) expressing OVA and RBD were transfected into HEK293T cells. RBD is a secreted protein, so the RBD protein content in the supernatant and cell lysate of the cell culture medium was detected simultaneously. OVA is a cell membrane surface protein, so the OVA protein content in the cell lysate was detected. Actin is a protein constantly expressed in each cell and was used as an internal protein reference in the experiment to ensure that the total amount of protein in each sample was consistent.

[0080] Figure 7 Flow cytometry was used to detect the percentage of BMDC antigen delivery cells: OVA-protein was performed by directly incubating BMDC with OVA protein for 24 hours; OVA-RNA and OVA-IM-RNA were performed by transfecting BMDC with equal amounts of RNA expressing OVA and modified RNA, and then detecting the percentage of delivery cells 24 hours later.

[0081] Figure 8 for Figure 7 The statistical analysis results were analyzed using the One-way Anova statistical analysis method: ns, p>0.05; ****, p<0.0001.

[0082] Fig. 9 Schematic diagram of the chemical groups modified by the modification method of the present invention on small molecule RNA and the modification status verified by MADEL-TOF MS.

[0083] Fig.10 It is a schematic diagram of the structure of the RNA template of the present invention.

[0084] Fig.11 This is the validation result of the macromolecular RNA modification of the present invention.

[0085] Fig.12 These are the verification results of different concentrations of CDI and IM of the present invention.

[0086] Fig.13 These are the verification results of different imidazole derivatives.

[0087] Fig.14 The validation results of different adducts. DETAILED DESCRIPTION

[0088] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0089] The chemical reagents used in the present invention include: imidazole (CAS 288-32-4), N, N'-carbonyl diimidazole (1,1'-carbonyl diimidazole, CDI, CAS 530-62-1), 0.5M citric acid-sodium citrate buffer solution (pH = 3.0), 3M sodium acetate solution (pH = 5.0), and ethanol (CAS 64-17-5).

[0090] The kit used in the present invention includes: T7 RNA synthesis kit (NEB, cat. E2040S), RNA purification kit (NEB, cat. T2050L), EcoRI restriction endonuclease kit (NEB, cat. R3101S), DNA enzyme kit (ThermoScientific, cat. EN0529), and RNA enzyme R kit (Beyotime, cat. R7092S).

[0091] Example 1 RNA modification process

[0092] 1. Experimental methods

[0093] 1) Preparation of RNA template

[0094] The circular RNA template design was based on Engineering circular RNA for potent and stable translation in eukaryotic cells published by Wesselhoeft et al. in Nature Communications in 2018: the T7 promoter, cyclization element and gene insertion site were designed and then fully synthesized. The internal ribosome entry site (IRES) and the expressed protein sequence were inserted between the cyclization elements through molecular cloning. After completion, the template was amplified using engineered Escherichia coli, and then linearized using restriction endonucleases. The schematic diagram of the RNA template is shown in the figure below. Fig.10 shown.

[0095] The protein expression models used in the study are mCherry red fluorescent protein (GenBank: MZ027319.1), chicken ovalbumin (OVA, Gene ID: 396058), new coronavirus spike protein binding domain (receptor binding domain, RBD, Gene ID: 43740568) and firefly luciferase protein (firefly luciferase, F-Luc, Gene ID: 116160065).

[0096] 2) RNA synthesis and cyclization

[0097] After obtaining the linearized template, the T7 RNA synthesis kit was used to synthesize the circular RNA precursor. After the synthesis was completed, 5mM GTP was added and the reaction was carried out at 55 degrees Celsius for 20 minutes for cyclization. After the cyclization was completed, it was cooled to room temperature (25 degrees Celsius), and DNA enzyme (DNase I, 20U / 50μL reaction system) and RNase R (RNase R, 20U / 100μL reaction system) were added to react at 37 degrees Celsius for 1 hour to remove the linearized template and the uncyclized RNA. The obtained product was finally purified with an RNA purification kit, and the RNA was quantified by 260nm spectral absorption in NanoDrop.

[0098] 3) RNA modification

[0099] The following materials were added to the reaction system (calculated based on the final concentration in the system): 0.1 mg / mL RNA, 0.02 M citric acid-sodium citrate buffer solution (pH 3.4), 0.1 M N, N'-carbonyldiimidazole and 0.02 M imidazole, all dissolved in water. The reaction was placed at room temperature with vigorous shaking for 6 hours to complete the modification.

[0100] The above reaction conditions are the final parameters after optimization, and the optimized conditions include pH and reaction time. The pH was adjusted using different proportions of 0.02M citric acid and 0.02M sodium citrate solutions to obtain buffer solutions of pH = 3.4, 4.5, 6.0, 7.4 and 8.6, respectively; subsequently, it was applied to the reaction system (0.1mg / ml RNA, 0.1MN, N'-carbonyldiimidazole and 0.02M imidazole), purified and then transfected with cells to verify its translation effect. The reaction time was under the reaction conditions of 0.1mg / mlRNA, 0.02M citric acid-sodium citrate buffer solution (pH = 3.4), 0.1MN, N'-carbonyldiimidazole and 0.02M imidazole, and the reaction solution was collected after 1, 2, 3, 4, 5, and 6 hours, purified and then transfected with cells to verify its translation effect.

[0101] 4) RNA purification and storage after modification

[0102] First, add 1 / 10 of the reaction system volume of 3M sodium acetate solution, then add 3 times the reaction system volume of ethanol, mix well and let stand at -20 degrees Celsius for 1 hour to reduce the overall temperature to -20°C. Precool the centrifuge to -20°C, centrifuge at 16000g for 30 minutes, remove the supernatant, and obtain a translucent oily precipitate. Resuspend with an equal volume of 70v / v% ethanol solution (70% ethanol, 30% water), precool the centrifuge to -20 degrees Celsius, centrifuge at 16000g for 10 minutes, remove the supernatant, and obtain a translucent oily precipitate. Air-dry the precipitate at room temperature for 10 minutes to remove the remaining ethanol, and then dissolve it with an appropriate amount of water to the required concentration. The product is stored at -20°C.

[0103] 2. Experimental results

[0104] 1) Optimization of pH conditions

[0105] In this experiment, circular RNA expressing mCherry fluorescent protein was modified under different pH conditions. 0.5 μg of modified and purified RNA was transfected into 2×10 5 Human embryonic kidney epithelial cell line HEK293T (LipoMessengerMAX transfection reagent). Figure 1 As shown, when pH = 3.4, the expression efficiency is the best.

[0106] 2) Optimization of reaction time

[0107] In this experiment, circular RNA expressing mCherry fluorescent protein was modified and purified RNA was collected at different time points after modification. 0.5 μg of modified purified RNA was transfected into 2×10 5 Human embryonic kidney epithelial cell line HEK293T (LipoMessengerMAX transfection reagent). Figure 2 As shown, when the time is , the expression efficiency is best.

[0108] 3) Optimization of reaction reagents

[0109] This example further tests the effects of different imidazole derivatives and adducts on RNA modification. 20mM Histamine: histidine, AL-IM: 5-ethynyl imidazole (cas 57121-48-9), IM: imidazole, reaction conditions 0.1g / L RNA, 100mM CDI, pH=3.4, and the expression efficiency is tested according to the above experimental method.

[0110] The results are as follows Fig.13 As shown, imidazole was significantly superior to histidine and AK-IM.

[0111] Similarly, 100 mM TCDI (N,N-thiocarboxydiimidazole, cas 160-65-2) and CDI: N,N-carboxydiimidazole were used, and the reaction conditions were 0.1 g / L RNA, 20 mM imidazole, pH = 3.4, and the expression efficiency was tested according to the above experimental method.

[0112] The results are as follows Fig.14 As shown, CDI has a more obvious effect.

[0113] 4) Optimization of imidazole concentration

[0114] After the reaction of 0.1 g / L mCherry expression RNA with different concentrations of imidazole and CDI at pH = 3.4, 0.5 μg of modified and purified RNA was transfected into 2×105 human embryonic kidney epithelial cell line HEK293T and its fluorescence intensity was measured 72 hours later. The color of the heat map represents the comparison of the fluorescence intensity of the experimental group and the fluorescence intensity of the unmodified RNA.

[0115] The results are as follows Fig.12 As shown, 100 mM CDI and 20 mM imidazole have significantly excellent effects.

[0116] Example 2 Stability test of modified RNA

[0117] In this experiment, agarose gel electrophoresis was used to detect the integrity of RNA. Fetal bovine serum (FBS) contains a large amount of RNA nuclease, which can degrade RNA molecules the moment they come into contact with them. The degraded RNA molecules form oligonucleotides, which have a smaller molecular weight than the undegraded RNA. Therefore, agarose gel electrophoresis can determine the integrity of RNA molecules based on their size.

[0118] 1. Experimental methods

[0119] The experiment was divided into four groups with two factors (modification or not, addition of FBS or not). FBS (2 v / v%) was added to the modified RNA (0.5 μg / μL, 10 μg in total) and unmodified RNA (0.5 μg / μL, 10 μg in total) prepared in Example 1. The control group was not treated and reacted at room temperature for 1 hour. The samples were subjected to 1.5% agarose gel electrophoresis (15 V / cm, 15 minutes).

[0120] 2. Experimental results

[0121] like Figure 3As shown, after adding 2 v / v% FBS and incubating at room temperature for 60 minutes, the unmodified RNA was degraded and its band position shifted; oligonucleotides with smaller molecular weight were formed; while the position of the modified RNA did not change, indicating that it was not sensitive to enzyme degradation. The modified RNA has an inhibitory effect on the degradation of RNA nucleases, making the RNA molecule more stable. Example 3 Translation efficiency test of modified RNA

[0122] 1. Experimental methods

[0123] Referring to Example 1, this experiment used circular RNA expressing different proteins, including mCherry fluorescent protein (GenBank: MZ027319.1), chicken ovalbumin (OVA, Gene ID: 396058), the new crown spike protein binding domain (receptor binding domain, RBD, Gene ID: 43740568) and firefly luciferase protein (fireflyluciferase, F-Luc, Gene ID: 116160065) for verification.

[0124] 1) Fluorescence detection experiment

[0125] The mCherry fluorescent protein and firefly luciferase protein transfection operations were performed in the human embryonic kidney epithelial cell line HEK293T as follows:

[0126] 0.5 μg of modified purified RNA was transfected into 2×10 5 Human embryonic kidney epithelial cell line HEK293T (LipoMessengerMAX transfection reagent)

[0127] The fluorescence signal was detected using a laser confocal microscope and an enzyme-labeled instrument 72 hours after transfection.

[0128] 2) Expression experiments

[0129] The transfection operation of chicken ovalbumin OVA and the new coronavirus spike protein binding domain RBD was carried out in the human embryonic kidney epithelial cell line HEK293T cells as follows:

[0130] 0.5 μg of modified purified RNA was transfected into 2×10 5 Human embryonic kidney epithelial cell line HEK293T (LipoMessengerMAX transfection reagent).

[0131] 72 hours after transfection, immunoblotting was performed to detect the expression levels of OVA and RBD: RBD is a secreted protein, so the RBD protein content in the cell culture supernatant and cell lysate was detected simultaneously; OVA is a cell membrane surface protein, so the OVA protein content in the cell lysate was detected; actin is a protein constantly expressed in each cell and is used as a protein internal reference in the experiment to ensure that the total amount of protein in each sample is consistent.

[0132] 2. Experimental results

[0133] 1) Fluorescence detection results

[0134] Figure 4 Fluorescence microscopy images of 2×10 5 The human embryonic kidney epithelial cell line HEK293T was transfected with equal masses (0.5 μg) of mCherry-modified or unmodified circular RNA using LipoMessengerMAX transfection reagent, and images were collected 72 hours after transfection: From the intuitive fluorescence intensity, the modified RNA has stronger fluorescence. After HEK293T cells were lysed with cell lysis buffer at different time points after transfection, the supernatant was taken to detect the fluorescence intensity. It can be seen that the expression level of the modified RNA has been significantly enhanced, which is more than 3 times that of the unmodified one. The same experimental steps were used to detect the expression level of F-Luc using luciferase substrate. The results of the circular RNA expressing F-Luc 72 hours after transfection were consistent with the above (such as Figure 5 shown).

[0135] 2) Western blot results

[0136] The results are as follows Figure 6 As shown, the expression levels of OVA and RBD in the modified RNA were higher than those in the unmodified RNA.

[0137] Example 4 Effect of modified RNA on antigen presentation

[0138] Dendritic cells (DC) are one of the important antigen-presenting cells (APC) in the immune system. They have the function of recognizing and presenting foreign antigens and are an essential component for inducing specific immunity. One of the mechanisms of action of existing RNA vaccines is to transfect dendritic cells to express exogenous proteins and directly induce antigen presentation.

[0139] 1. Experimental methods

[0140] 0.5 μg of modified purified RNA was transfected into 2×10 5Mouse bone marrow-derived dendritic cells (BMDC) (LipoMessengerMAX transfection reagent)

[0141] 2. Experimental results

[0142] After mouse bone marrow derived dendritic cells (BMDC) were transfected with equal mass OVA-modified or unmodified circular RNA using LipoMessengerMAX transfection reagent, flow cytometry was used to detect surface antigen presentation epitopes 24 hours later to evaluate the degree of antigen presentation. It can be seen that compared with unmodified RNA, the number of antigen presenting cells induced by modified RNA is greater, that is, the degree of antigen presentation is stronger, indicating that modified RNA can be used as a vaccine medium to improve antigen presentation and induce efficient immune response ( Figure 7 , 8 ).

[0143] Example 5 RNA modification result verification

[0144] 1. Experimental methods

[0145] This experiment used two verification methods; for large RNA molecules (circular RNA expressed by mCherry, with a molecular weight of more than 10kDa), the spectral absorption ratio of 207nm and 260nm in Nanodrop was used to confirm whether the modification was successful (the solvent was water). The 207:260 ratio in unmodified RNA was between 1.2-1.3, while the ratio of modified RNA was greater than 2.5. In order to more accurately confirm the efficiency of chemical modification and its chemical structure, an 18-nucleotide model RNA (model-RNA, AUCCUGCCGACUACGCCA, SEQ ID NO: 1) was used after modification (0.1mg / ml RNA, 0.1MN, N'-carbonyldiimidazole and 0.02M imidazole, pH 3.4, reaction 6h), and matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOFMS) was used for analysis.

[0146] 2. Experimental results

[0147] The modified and purified model RNA was used to verify its modification status and modified chemical groups using MADEL-TOF MS. Fig. 9 The mass spectrometry mass-to-charge ratio peak diagram is shown, the RNA mass is, the RNA-sodium ion mass is. 5702.4, and the subsequent small peak chemical modification structure is as follows Fig. 9 As shown: the amount of modified RNA accounts for 30% of the total RNA.

[0148] The modification results of large RNA molecules are as follows Fig.11 As shown, the 207:260 ratio of the modified RNA was significantly increased, proving its successful modification.

Claims

1. Any one of the methods a1) to a2), characterized in that: a1) Methods for improving RNA stability; a2) Methods for improving RNA translation efficiency; The method comprises the following steps: reacting imidazole or its derivative, adduct, and RNA; The adduct includes N,N'-carbonyldiimidazole.

2. The method according to claim 1, characterized in that: The RNA includes at least one of circular RNA, linear RNA, double-stranded RNA, and stem-loop RNA.

3. The method according to claim 1, characterized in that: The imidazole or its derivatives include at least one of imidazole, ethynyl imidazole and histidine.

4. The method according to claim 1, characterized in that: The ratio of the imidazole or its derivative, adduct and RNA is (175-225 mM): (800-1200 mM): 1 g / L.

5. The method according to claim 4, characterized in that: The reaction is carried out in an acidic buffer; Preferably, the pH of the acidic buffer is 3.0 to 5.0; Preferably, the acidic buffer comprises at least one of glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-citric acid buffer, and acetic acid-sodium acetate buffer.

6. An RNA molecule, characterized in that: The RNA molecule has the following modification:

7. The method for preparing the RNA molecule according to claim 6, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

8. Use of the method according to any one of claims 1 to 5 or the RNA molecule according to claim 6 in preparing a product; The product includes at least one of a drug, a vaccine, a reagent, and a kit; Preferably, the medicament comprises pharmaceutically acceptable excipients.

9. A vaccine comprising the RNA molecule of claim 6; Preferably, the vaccine comprises an adjuvant.

10. A kit comprising the vaccine according to claim 9.

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