Method for producing modified mRNA construct using non-enzymatic extension reaction and pharmaceutical composition comprising same

Abstract

The present invention relates to a method for producing a modified mRNA construct in which a nucleotide derivative is coupled to the 3' terminus of mRNA by non-enzymatic extension using a nucleotide activated with imidazole or 2-aminoimidazole or a derivative thereof, and to enhancement of antigen protein translation efficiency resulting therefrom. When the modified mRNA construct of the present invention is applied to expression of an anticancer gene, an apoptosis-inducing effect in cancer cells can increase, and an anticancer effect in which EZH2 expression is reduced can be exhibited.

Description

Method for preparing a modified mRNA structure using a non-enzymatic extension reaction and a pharmaceutical composition comprising the same

[0001] The present invention relates to a method for preparing a modified mRNA structure using a non-enzymatic extension reaction and a pharmaceutical composition containing the same. More specifically, the invention relates to a method for preparing a modified mRNA structure with improved stability and protein translation efficiency by non-enzymatically extending the 3′ end of the mRNA using an imidazole or a nucleotide derivative activated with 2-aminoimidazole, and a pharmaceutical composition containing the same.

[0002]

[0003] Recently, messenger RNA (mRNA) has garnered attention as a promising therapeutic platform in various biopharmaceutical fields, including vaccines, gene therapies, and anticancer treatments. Because mRNA can directly express target proteins within cells, it offers the advantages of simpler manufacturing processes compared to conventional protein drugs and the ability to induce therapeutic effects without the risks associated with gene editing. Due to these characteristics, mRNA-based therapeutics are being actively researched in diverse areas, such as vaccines for infectious diseases, tumor immunotherapy, and the treatment of rare genetic diseases.

[0004] However, mRNA has technical limitations, such as low intracellular stability and a short half-life, as it is easily degraded by ribonucleases (RNases) in vivo. Accordingly, various modification technologies have been developed to improve mRNA stability and increase protein translation efficiency. Representative examples include modification of the 5′ cap structure, control of poly-A tail length, chemical modification of nucleotide bases or sugars, and delivery technologies using lipid nanoparticles (LNPs).

[0005] In particular, since the 3′ terminal poly-A tail of mRNA plays a crucial role in translation initiation and stability, techniques to improve mRNA stability by controlling the length of the poly-A tail or modifying the terminal structure have been proposed. Conventionally, methods using enzymatic poly-A polymerase to extend the poly-A tail or enzymatic capping systems to form the terminal structure have been primarily used. However, these enzyme-based processes have problems such as high manufacturing costs, complex process control, and potential quality variations depending on the enzymatic reaction conditions.

[0006] Meanwhile, although recent studies have reported chemically extending RNA ends using non-enzymatic nucleotide binding reactions, stable and highly reproducible non-enzymatic extension techniques suitable for direct application in the manufacture of mRNA therapeutics are still limited. In particular, there are few technologies that demonstrate actual intracellular protein expression and therapeutic efficacy while simultaneously improving mRNA stability and translation efficiency through non-enzymatic extension.

[0007] Therefore, there is a need for the development of a new non-enzymatic mRNA end extension technology that can effectively modify the 3′ end of mRNA without relying on enzymatic processes to improve intracellular stability and protein translation efficiency.

[0008]

[0009] The technical problem that the present invention aims to solve is to provide a method for manufacturing a novel modified mRNA structure capable of improving mRNA stability and protein translation efficiency within a cell.

[0010] Furthermore, the technical problem to be solved by the present invention is to provide a pharmaceutical composition that exhibits the effects of inducing apoptosis and inhibiting EZH2 expression in cancer cells when the modified mRNA structure is applied to anticancer gene expression.

[0011]

[0012] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0013]

[0014] To achieve the above technical problem, one embodiment of the present invention provides a method for preparing a modified mRNA structure comprising the step of non-enzymatically reacting a purine nucleotide, a pyrimidine nucleotide, or a derivative thereof containing an imidazole or 2-aminoimidazole active group at the 3' end of the mRNA.

[0015] In an embodiment of the present invention, the nucleotide derivative may have one or more functional groups introduced into at least one of a base, a sugar, and a phosphate backbone.

[0016] In an embodiment of the present invention, the functional group may be one or more selected from the group comprising alkyl groups, alkoxy groups, halogens, aryl groups, heteroaryl groups, alkylamino groups, hydroxyl groups, carbonyl groups, thio groups, and fluoro groups.

[0017] In an embodiment of the present invention, the derivative of the nucleotide may be one in which an imidazole or 2-aminoimidazole active group is attached to at least one of L-guanosine monophosphate (L-GMP), inosine monophosphate (IMP), 2′-O-methylguanosine monophosphate (2′-O-Me-GMP), 2′-O-methoxyethylguanosine monophosphate (2′-O-MOE-GMP), and 2′-fluoroguanosine monophosphate (2′-F-GMP).

[0018] In an embodiment of the present invention, 1 to 10 purine nucleotides, pyrimidine nucleotides, or derivatives thereof may be sequentially bound to the 3' end of the mRNA by the reaction.

[0019] In an embodiment of the present invention, the nucleotide or its derivative may be bound to the poly(A) tail end of the mRNA.

[0020] In an embodiment of the present invention, the reaction is Mg 2+ It may be performed in a HEPES buffer solution with a pH of 7 to 9 in the presence of.

[0021] In an embodiment of the present invention, the reaction may be carried out at 37°C for 1 to 3 hours.

[0022] To achieve the above technical problem, another embodiment of the present invention provides a modified mRNA structure.

[0023] In an embodiment of the present invention, the mRNA structure may comprise one or more selected from the group consisting of L-guanosine monophosphate (L-GMP), inosine monophosphate (IMP), 2′-O-methylguanosine monophosphate (2′-O-Me-GMP), 2′-O-methoxyethylguanosine monophosphate (2′-O-MOE-GMP), and 2′-fluoroguanosine monophosphate (2′-F-GMP) at the 3' end.

[0024] In an embodiment of the present invention, the modified mRNA structure may have increased stability or protein translation expression compared to the unmodified mRNA.

[0025] To achieve the above technical objectives, another embodiment of the present invention provides an anticancer pharmaceutical composition comprising a modified mRNA structure.

[0026]

[0027] The present invention relates to a method for preparing a modified mRNA structure comprising a nucleotide derivative, wherein the modified mRNA structure prepared by non-enzymatic extension has improved intracellular stability and increased protein translation efficiency, which can significantly improve the expression level of a target protein.

[0028] In addition, when the modified mRNA structure of the present invention is applied to anticancer gene expression, it can provide an anticancer effect in which apoptosis is effectively induced in cancer cells and the expression of EZH2, which is involved in tumor proliferation and survival, is reduced.

[0029]

[0030] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0031]

[0032] Figures 1 to 16 are schematic diagrams illustrating the synthesis of each compound.

[0033] Figure 17 is a diagram showing the results of confirming non-enzymatic extension through gel electrophoresis and sequencing, wherein (A) is the 20% denaturation PAGE result to confirm non-enzymatic extension for short RNA primers, (B) is the 0.8% agarose gel electrophoresis result to confirm non-enzymatic extension of GFP mRNA, (C) is the 0.8% agarose gel electrophoresis result to confirm non-enzymatic extension of luciferase mRNA, (D) is the 0.8% agarose gel electrophoresis result to confirm non-enzymatic extension of apotin mRNA, (E) is the Sanger sequencing result of control GFP, (F) is the Sanger sequencing result of GFP non-enzymatically extended to 2′O-Me-GMP-2-amino-IM, and (G) is non-enzymatically extended to N7-(2-MePy)-GMP-IM This is the Sanger sequencing result of GFP, and (H) represents the Sanger sequencing result of GFP non-enzymatically extended with GMP-2-amino-IM, respectively.

[0034] Figure 18 is a diagram showing the results of verifying the expression of non-enzymatic extension mRNA through cell imaging, fluorescence, and Western blot, where (A) is the cell imaging result of GFP expression by non-enzymatic extension GFP mRNA, (B) is a bar graph of luciferase expression by non-enzymatic extension luciferase mRNA, (C) is the Western blot analysis result of apotin expression by non-enzymatic extension apotin mRNA, and (D) is the quantitative analysis (bar graph) result of apotin expression corresponding to Figure 18C.

[0035] Figure 19 is a figure showing the results of time-dependent translation and Exo-T treatment experiments of luciferase mRNA to evaluate the stability of non-enzymatically extended mRNA, (A) showing the results of time-dependent expression (12, 24, 36, and 48 hours) of non-enzymatically extended luciferase mRNA using different imidazole compounds, and (B) showing the results of the stability evaluation of non-enzymatically extended luciferase mRNA treated with Exo-T with 2′O-Me-GMP-2-amino-IM compared with control luciferase mRNA.

[0036] Figure 20 is a diagram showing the IC50 measurement results according to MTT analysis, where (A) is the IC50 measurement result of apotin mRNA non-enzymatically extended with 2′O-Me-GMP-2-amino-IM, (B) is the IC50 measurement result of apotin mRNA non-enzymatically extended with GMP-2-amino-IM, and (C) is the IC50 measurement result of apotin mRNA non-enzymatically extended with N7-(2-MePy)-GMP-IM.

[0037] Figure 21 is a diagram showing the results of cell viability and EZH2 expression analysis following non-enzymatically extended mRNA treatment, (A) is a bar graph of time-dependent cell viability of the non-enzymatically extended mRNA treatment group (including the control group), and (B) is the result of RT-qPCR analysis of EZH2 expression to evaluate the apoptosis induced in each treatment group.

[0038] FIG. 22 shows the method and results of confirming the non-enzymatic extension of a guanosine monophosphate imidazolide derivative (L-guanosine monophosphate imidazolide) to the 3′ end of short RNA (14 nt) using guide RNA, (a) degree of non-enzymatic binding according to imidazole concentration under the same time conditions. Lane 1: Guide RNA (11 nt), Lane 2: RNA without introduction of GMP imidazolide derivative, Lane 3: non-enzymatic binding using 10 mM L-GMP imidazolide, Lane 4: non-enzymatic binding using 15 mM L-GMP imidazolide, Lane 5: non-enzymatic binding using 20 mM L-GMP imidazolide, Lane 6: non-enzymatic binding using 25 mM L-GMP imidazolide, Lane 7: non-enzymatic binding using 30 mM L-GMP imidazolide. (b) Degree of non-enzymatic binding over time under conditions of the same L-GMP imidazolide concentration. Lane 1 represents reaction time 0.5 hours, Lane 2 represents reaction time 1 hour, Lane 3 represents reaction time 1.5 hours, Lane 4 represents reaction time 2 hours, Lane 5 represents reaction time 3 hours, and Lane 6 represents reaction time 4 hours.

[0039] Figure 23 shows the results of confirming the non-enzymatic extension of guanosine monophosphate imidazolide derivatives to the 3′ end of short RNA (14 nt) using guide RNA with five synthesized compounds, Lane 1: 25 / 100 bp Ladder, Lane 2: Guide RNA (11 nt), Lane 3: RNA without GMP imidazolide derivatives, Lane 4: Non-enzymatic extension result using L-guanosine monophosphate imidazolide, Lane 5: Non-enzymatic extension result using Inosine monophosphate imidazolide, Lane 6: Non-enzymatic extension result using 2′-O-Methylguanosine monophosphate imidazolide, Lane 7: Non-enzymatic extension result using 2′-O-(2-Methoxyethyl)guanosine monophosphate imidazolide, Lane 8: Non-enzymatic extension using 2′-Fluoroguanosine monophosphate imidazolide Shows the extension result.

[0040] Figure 24 shows an experiment in which only the luciferase gene sequence is replicated from the luciferase-pcDNA3 plasmid and the base sequences necessary to make mRNA are introduced.

[0041] Figure 25 shows the 1% Agarose gel results and analysis data (NanoDrop) after the PCR amplification reaction for DNA replication confirmation. (a) shows the targeted PCR amplification, and (b) the second PCR amplification.

[0042] Figure 26 shows the sequencing analysis process and results for confirming non-enzymatic binding of the 3′ end of luciferase mRNA.

[0043] Figure 27 is sequencing data obtained by converting luciferase mRNA into cDNA to which L-Guanosine monophosphate imidazolide was introduced.

[0044] Figure 28 is sequencing data obtained by converting luciferase mRNA into cDNA into which inosine monophosphate imidazolide was introduced.

[0045] Figure 29 is sequencing data obtained by converting luciferase mRNA into cDNA into which 2′-O-Methylguanosine monophosphate imidazolide was introduced.

[0046] Figure 30 is sequencing data obtained by converting luciferase mRNA into cDNA into which 2′-O-(2-Methoxyethyl)guanosine monophosphate imidazolide was introduced.

[0047] Figure 31 shows sequencing data obtained by converting luciferase mRNA into cDNA with 2′-Fluoroguanosine monophosphate imidazolide introduced.

[0048] Figure 32 shows the process and result of luciferase gene expression in luciferase mRNA into which a guanosine monophosphate imidazolide derivative was introduced.

[0049]

[0050] The present invention will be described in detail below.

[0051]

[0052] The present invention relates to a method for preparing a modified mRNA structure comprising the step of non-enzymatically reacting a purine nucleotide, a pyrimidine nucleotide, or a derivative thereof containing an imidazole or 2-aminoimidazole active group at the 3' end of the mRNA.

[0053] The above imidazole or 2-aminoimidazole active group can bind to the phosphate group of a nucleotide to form an activated nucleotide in the form of phosphoroimidazolide, and this activation can increase the reactivity of the phosphate group, thereby enabling a non-enzymatic elongation reaction that forms a phosphodiester bond with the 3′ terminal hydroxyl group of mRNA without the intervention of an enzyme.

[0054] In particular, the 2-aminoimidazole active group has enhanced electron-donating properties compared to the imidazole active group, which can form more stable reactive intermediates, thereby improving the efficiency or reproducibility of non-enzymatic extension reactions. Due to these characteristics, nucleotide derivatives containing the 2-aminoimidazole active group can induce effective mRNA terminal extension even under relatively low concentration conditions or short reaction times.

[0055] In addition, non-enzymatic activation methods using imidazole or 2-aminoimidazole active groups offer the advantage of being able to precisely control the 3′ end of mRNA without using expensive enzymes, and may be advantageous in terms of simplifying the manufacturing process, reducing costs, and suitability for mass production.

[0056] The above purine nucleotides, pyrimidine nucleotides, or derivatives thereof may be used as components to form a modified mRNA structure by non-enzymatically binding to the 3′ end of mRNA. The above nucleotides or derivatives thereof have a basic nucleotide structure including a base, a sugar, and a phosphate backbone, and may function as monomers for chemically controlling the terminal structure of mRNA.

[0057] The above purine or pyrimidine nucleotides may be D-type nucleotides found in nature, but are not limited thereto; they may also be L-type nucleotides or nucleotides with modified stereostructures. Such modification of stereostructure can increase resistance to nucleases and improve the stability of mRNA.

[0058] The above nucleotide derivative may have one or more functional groups introduced into at least one of the base, sugar, and phosphate backbone. When the functional group is introduced into the base portion, it can regulate base pairing characteristics, stacking interactions, or interactions during the protein translation process. Base modification may have the effect of further stabilizing the terminal structure of mRNA or inhibiting non-specific binding. When the functional group is introduced into the sugar portion, it may be introduced specifically at the 2′ or 3′ position of the ribose sugar. Such sugar modification may increase resistance to nucleases and contribute to extending the intracellular half-life of mRNA. For example, functional groups such as 2′-O-methyl groups, 2′-O-methoxyethyl groups, or 2′-fluoro groups can improve the chemical stability of mRNA. Additionally, the functional group may be introduced into the phosphate backbone and may alter the charge distribution or stereochemical structure of the phosphate bond. Such modification of the phosphate backbone can contribute to inhibiting degradation by exonucleases or improving the selectivity and efficiency of non-enzymatic binding reactions.

[0059] The above functional group may be one or more selected from the group comprising alkyl groups, alkoxy groups, halogens, aryl groups, heteroaryl groups, alkylamino groups, hydroxyl groups, carbonyl groups, thio groups, and fluoro groups. The above functional group may be introduced alone or in combination of two or more, and may contribute to comprehensively improving the chemical stability of nucleotide derivatives, nuclease resistance, and the intracellular half-life of mRNA constructs.

[0060] The above alkyl or alkoxy groups can contribute to improving the physical stability of the mRNA terminal structure or inhibiting degradation in the intracellular environment by regulating the hydrophobicity of the nucleotides. The above halogen or fluoro groups are highly electronegative functional groups; when introduced into the sugar or base portion, they can increase resistance to nucleases and improve the chemical stability of the mRNA. The above aryl or heteroaryl groups can regulate the stability of the mRNA terminal structure or the characteristics of interactions with proteins by altering the electronic properties or stacking interactions of the base structure. The above alkylamino or hydroxyl groups provide the ability to form hydrogen bonds, which can regulate interactions between nucleotides or the surrounding hydration environment and can subsequently be used for peptide bonding. The above carbonyl or thio groups can be introduced into the phosphate backbone or base portion to alter electron distribution and binding characteristics, which can contribute to regulating the reactivity or selectivity of non-enzymatic binding reactions.

[0061] The above functional group is not limited thereto and may further include functional groups functionally or structurally equivalent thereto.

[0062] In addition, the derivative of the above nucleotide may be in a form to which a biological moiety is attached. The above biological moiety refers to a biologically active substance or functional molecule introduced to confer a specific function to an mRNA structure. By introducing a nucleotide to which such a biological moiety is attached via an imidazole active group to the mRNA terminus, there is technical significance in that additional functions such as targeting or visualization can be directly conferred to the mRNA beyond simple sequence extension.

[0063] The above biological moiety may be, for example, a peptide, a sugar derivative, a lipid, a nucleic acid fragment, or a label molecule. Specifically, the peptide may be a cell-permeable peptide (CPP) or a ligand that binds to a target cell receptor, and the lipid may include cholesterol, etc., to increase cell membrane permeability. The sugar derivative may include GalNAc, etc., which aids in delivery to specific organs, and the nucleic acid fragment may include siRNA or aptamers, etc. Additionally, the label molecule may be a fluorescent molecule, biotin, or a radioisotope-labeled compound. By controlling the type of such biological moiety, it becomes possible to maximize the delivery efficiency of mRNA or track pharmacokinetics within the body.

[0064] The derivative of the above nucleotide may be one in which an imidazole or 2-aminoimidazole active group is attached to at least one of L-guanosine monophosphate (L-GMP), inosine monophosphate (IMP), 2′-O-methylguanosine monophosphate (2′-O-Me-GMP), 2′-O-methoxyethylguanosine monophosphate (2′-O-MOE-GMP), and 2′-fluoroguanosine monophosphate (2′-F-GMP).

[0065] The above L-GMP is an L-type nucleotide having a stereochemical structure different from natural D-type guanosine, and can improve mRNA stability by increasing resistance to nucleases. The above IMP is an inosine-based nucleotide having a structure in which the guanine base is deaminated, and can regulate the structural characteristics of the 3′ end of mRNA through changes in base pairing characteristics. The above 2′-O-Me-GMP is a sugar-modified nucleotide with a methyl group introduced at the 2′ position of ribose, and can improve protein translation efficiency by increasing resistance to ribonucleases and reducing immune stimulation responses. The above 2′-O-MOE-GMP is a structure with a methoxyethyl group introduced at the 2′ position of ribose, and can increase the half-life of mRNA by improving intracellular stability and binding affinity. The above 2′-F-GMP is a nucleotide with a fluoro group introduced at the 2′ position of ribose, and can improve structural stability and confer resistance to nuclease degradation.

[0066] In the above reaction, one to ten purine nucleotides, pyrimidine nucleotides, or derivatives thereof may be sequentially bound to the 3' end of the mRNA. The binding may be achieved through phosphodiester bonds between adjacent nucleotides, thereby allowing the 3' end structure of the mRNA to be extended stepwise.

[0067] The above purine nucleotides, pyrimidine nucleotides, or derivatives thereof may preferably be in the form of 2 to 8, more preferably 4, nucleotides or derivatives thereof linked in succession. If the number of links is less than the above range, the stabilization effect of the 3′-terminal structure is insufficient, which may limit the effect of increasing resistance to intracellular degradation or translation efficiency. On the other hand, if the number of links exceeds the above range, the excessive formation of the extension may cause changes in the 3′-terminal secondary structure of the mRNA or increase the possibility of non-specific binding and the generation of reaction byproducts, which may instead have an adverse effect on translation efficiency or formulation stability. Preferably, it may be in the form of 4 nucleotides or derivatives thereof linked in succession, which is a representative number of links that can be repeatedly secured under conditions where the non-enzymatic extension reaction proceeds relatively efficiently, and can contribute to significantly improving the stability and translation efficiency of the resulting modified mRNA structure.

[0068] The above nucleotide or derivative thereof may be bound to the poly(A) tail end of mRNA. The above nucleotide or derivative thereof may be covalently bonded to the 3′ terminal hydroxyl group of the adenosine nucleotide constituting the poly(A) tail through a phosphodiester bond, thereby forming a structure in which the length of the poly(A) tail is extended. Generally, mRNA contains a poly(A) tail in which a number of adenine nucleotides are repeated at the 3' end, and by binding the above nucleotide or derivative thereof to the end of the poly(A) tail, the 3′ terminal structure of the mRNA can be stabilized and degradation by exonuclease can be inhibited, and as a result, the intracellular half-life of the mRNA can be increased.

[0069] In an embodiment of the present invention, the reaction is Mg 2+It may be carried out in HEPES buffer at pH 7 to 9 in the presence of. The non-enzymatic elongation reaction between the nucleotide or its derivative and the 3′ end of the mRNA is performed using Mg 2+ It is performed in the presence of ions, and the above Mg 2+ Ions can improve the efficiency of the extension reaction by partially neutralizing the negative charge of the phosphate group, thereby reducing the repulsion between nucleotides and stabilizing the formation of the phosphoromidazolide intermediate.

[0070] The above reaction can be carried out in a HEPES buffer having a pH range of 7 to 9, preferably at pH 8. The above pH range can be set to a range that ensures the chemical stability of mRNA while maintaining the reactivity of nucleotides or derivatives activated by imidazole or 2-aminoimidazole. If the range is below the above range, the reaction rate may decrease because the deprotonation of the imidazole activator is not sufficiently carried out, and if the range is above the above range, the hydrolysis or non-specific degradation reaction of mRNA may increase. Therefore, the above range may be a range that considers the balance between the efficiency of the non-enzymatic extension reaction and mRNA stability.

[0071] The above reaction may be carried out for 1 to 3 hours at a temperature range of 25°C to 40°C, and preferably at 37°C for 2 hours. The temperature and time range may be set so that the thermal stability and structural integrity of the mRNA can be maintained while the non-enzymatic binding reaction of nucleotides or derivatives activated by imidazole or 2-aminoimidazole proceeds sufficiently.

[0072] If the reaction temperature is below 25°C, the reactivity of the activated nucleotide or its derivative is reduced, which may decrease the efficiency of extension to the mRNA 3′ terminus or increase the time required to complete the reaction excessively. On the other hand, if the reaction temperature exceeds 40°C, secondary structural changes or chemical degradation reactions of the mRNA may be accelerated, raising concerns about product quality degradation or an increase in non-specific reactions. Therefore, the above temperature range may be set considering the balance between reaction efficiency and mRNA stability.

[0073] If the above reaction time is less than 1 hour, the non-enzymatic extension reaction may not proceed sufficiently, and the number of nucleotides or their derivatives bound may not reach the target level; if the reaction time exceeds 3 hours, further increase in reaction efficiency may be limited, or conversely, the possibility of non-specific degradation of mRNA and generation of byproducts may increase. Therefore, the time range of 1 to 3 hours may be a range for suppressing unnecessary degradation while securing the target extension efficiency.

[0074] The present invention relates to an mRNA structure comprising a nucleotide derivative non-enzymatically bound to the 3′ end.

[0075] Specifically, it may be a modified mRNA structure manufactured according to the above manufacturing method.

[0076] The mRNA structure may comprise one or more selected from the group consisting of L-guanosine monophosphate (L-GMP), inosine monophosphate (IMP), 2′-O-methylguanosine monophosphate (2′-O-Me-GMP), 2′-O-methoxyethylguanosine monophosphate (2′-O-MOE-GMP), and 2′-fluoroguanosine monophosphate (2′-F-GMP) at the 3' end.

[0077] Detailed explanations regarding matters that overlap with the aforementioned have been omitted.

[0078] The modified mRNA structure may have increased stability or protein translation expression compared to the unmodified mRNA. By introducing a nucleotide or a derivative thereof into the poly-A tail at the 3′ end of the modified mRNA structure, the terminal stability of the mRNA is improved and the rate of intracellular degradation is reduced, thereby extending the time available for translation. The modified mRNA structure may have increased binding efficiency to ribosomes or enhanced interaction with translation initiation factors, and consequently, a higher expression level of the target protein may be observed even when the same amount of mRNA is introduced into the cell. Therefore, the mRNA modified according to the present invention may exhibit a statistically significant increase in expression compared to the unmodified mRNA.

[0079] The present invention relates to a pharmaceutical composition for anticancer purposes comprising the modified mRNA structure.

[0080] The above modified mRNA construct may be an mRNA encoding a protein that is expressed in cancer cells and induces apoptosis, and said protein may induce inhibition of cancer cell proliferation, cell cycle arrest, or activation of apoptosis pathways.

[0081] In addition, the modified mRNA structure may have an increased intracellular half-life and improved translation efficiency by having a nucleotide or a derivative thereof attached to the 3′ end, thereby inducing a higher level of anticancer protein expression compared to the same dosage. Accordingly, the anticancer pharmaceutical composition may exhibit a tumor growth inhibitory effect by reducing EZH2 expression in cancer cells or increasing the expression of apoptosis-related genes.

[0082] The above-described pharmaceutical composition for anticancer purposes may further comprise a pharmaceutically acceptable carrier, excipient, or diluent, wherein the carrier may be a buffer solution, physiological saline, lipid nanoparticles, liposomes, polymer delivery systems, or a combination thereof. Additionally, the composition may be prepared as an injectable, lyophilized formulation, suspension, or formulation encapsulated in lipid nanoparticles, and may be administered via various routes including intravenous, intramuscular, subcutaneous, or intratumoral administration.

[0083]

[0084] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0085]

[0086] Experimental method

[0087] 1. General Information

[0088] DNA and RNA oligonucleotides were purchased from Bioneer (South Korea), and dNTP mixture (dATP, dTTP, dCTP, and dGTP; 10 mM each), rNTPs (rATP, rUTP, rCTP, and rGTP; 100 mM each), RNase inhibitor, 2× TOPsimple™DyeMIX-Tenuto PCR master-mix, and T7 RNA polymerase were provided by Enzynomics (South Korea). Ultrapure water was obtained from Enzynomics (South Korea). 40% acrylamide / bis solution was supplied by BIORAD (USA), and extra pure urea was provided by DAEJUNG Chemicals (South Korea). The luciferase plasmid (Vector ID: VB220810-1198vwd) was purchased from VectorBuilder, and the GFP gene source, pCFE-GFP, was provided by Thermo Fisher's 1-Step Human Coupled IVT Kit. The apotin gene was provided by IDT Technologies (South Korea). The luciferase substrate, “Luciferase Assay System,” was supplied by Promega, and the polystyrene-coated 96-well cell culture plates were provided by Corning. The 60 mL cell culture flasks were supplied by SPL Life Sciences. The 3′-O-Me-m7G(5′)ppp(5′)G RNA Cap Structure Analog (ARCA), Exonuclease T, and DNase I were purchased from New England Biolabs, and the NeoGreen gel pre-staining material was obtained from NeoScience. Bioneer's 'AccuPrep PCR / Gel Purification Kit' was used for mRNA purification, and the RNA Clean & Concentrator was purchased from Zymo Research (South Korea).The Cell / Virus RNA Extraction Kit was obtained from BIOCODE (South Korea).

[0089] Ethanol, fetal bovine serum (FBS), penicillin / streptomycin, and trypsin were purchased from Sigma Aldrich. Lipofectamine™MessengerMAX™Transfection Reagent, SYBR Green PCR Mastermix, Opti-MEM, DMEM, and a Heracell 240i CO₂ incubator were supplied by Thermo Fisher. The Colibri+ LB 916 microvolume spectrophotometer (nanodrop), Ver. 25B05, and Centro LB 960 microplate luminometer were purchased from Berthold Technologies (Germany). An Atto Ez-Capture instrument was used for dot blot and western blot band imaging. The anti-apotin antibody was purchased from Abcam (South Korea), and the β-actin monoclonal antibody was supplied by Thermo Fisher. The anti-rabbit IgG HRP-linked antibody was purchased from Cell Signaling. Human embryonic kidney (HEK)293 cells were provided by Cytion (USA), and the human liver cancer cell line (HEPG2) and mouse macrophage cell line (RAW264.7) were provided by the Laboratory of Lipid Biochemistry at Gachon University.

[0090] The chemicals used to synthesize the three types of imidazole compounds were purchased from Sigma-Aldrich (USA) and used without further purification. The imidazole compounds were synthesized at the Laboratory of Synthetic Biology and Medicinal Chemistry at Jeonbuk National University (JBNU). The PAGE apparatus was purchased from CBS Scientific (USA), and the agarose electrophoresis apparatus (AGE) was purchased from Mupid-2plus (ADVANCE, Japan). 1 H, 13 C and 31 P NMR spectra were measured using a Bruker AVANCE III-HD 400 MHz Fourier transform spectrometer installed at the Future Energy Convergence Core Center (FECC). UV-Vis spectra were acquired at room temperature using a Cary Series UV-Vis spectrophotometer (Agilent Technologies) with a quartz cuvette with a 1 cm optical path length, and changes in absorbance were measured immediately after UV irradiation of the sample solution in the cuvette. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorescence spectrometer.

[0091] A solution was prepared by dissolving 40% acrylamide / bis solution (2.5 mL), 10× TBE buffer (0.5 mL), and urea (2.5 g) in a water bath. To this, 20% ammonium persulfate solution (20 μL) and TEMED (5 μL) were added to prepare a 20% modified polyacrylamide gel solution (total 5 mL). The gel solution was injected into a PAGE apparatus (CBS Scientific Adjustable Height Vertical, USA) and electrophoresed at 180 V for 12 hours. Subsequently, the gel was stained with EtBr solution for 10 minutes and washed with water for 5 minutes. NeoGreen was added during the preparation of the 0.8% agarose gel, and electrophoresis was performed at 135 V for 20 minutes. Gel images were captured using a mobile device under a transilluminator. Graphs, including fluorescence data, were created using Origin 6.0 software, and all chemical structural formulas were created using ChemDraw Professional software.

[0092]

[0093] 2. Non-enzymatic 4G extension

[0094] The addition of four guanosines (GGGG) to the poly(A) tail end of mRNA is related to mRNA stability, translation efficiency, secondary structure formation, and nuclear export. Guanosines can increase mRNA stability to facilitate efficient translation by protecting mRNA from exonuclease degradation. Additionally, the secondary structure formed by the G-quadruplex enhances ribosome recruitment and further stabilizes mRNA by strengthening binding to specific proteins that promote translation. Furthermore, it has been reported in single-strand RNA that RNA template elongation by multiple nucleotides containing up to four base pairs is the most efficient compared to single or double nucleotide elongation.

[0095]

[0096] 3. Synthesis of Imidazole-Activated Guanosine-Based Compounds

[0097] 3.1. Synthesis of N7-(pyridin-2-ylmethyl)guanosine 5′-monophosphate triethylammonium salt

[0098] Triethylammonium guanosine 5′-monophosphate (566 mg, 1 mmol) was dissolved in dimethyl sulfoxide (DMSO, 3 mL), and 2-(bromomethyl)-pyridine (860 mg, 5 mmol) was added and reacted at room temperature for 24 hours. The reaction mixture was added to an acetone (50 mL) solution containing sodium perchlorate (0.5 g), cooled at 4°C for 30 minutes, and centrifuged at 5000 rpm for 8 minutes. The resulting solid was washed with acetone, centrifuged again, and dried under vacuum. The dried solid was dissolved in water and loaded onto DEAE Sepharose fast-flow resin. The aqueous solution of the solid was eluted using 1 M TEAB buffer (pH 7.5) under conditions of a 0–30% gradient and a flow rate of 3 mL / min. The fraction containing the product was recovered, concentrated using a rotary evaporator, and then freeze-dried to obtain a fine white powder product in the form of a triethylamine salt.

[0099]

[0100] 3.2. Synthesis of 2′-O-methylguanosine 5′-monophosphate triethylammonium salt

[0101] 2′-O-methylguanosine (446 mg, 1.5 mmol) was dissolved in trimethyl phosphate (3 mL) and stirred at 0°C under a nitrogen atmosphere, after which POCl₃ (186 μL, 2 mmol) was dropwise. The reaction was stirred at 0°C for 4 hours, after which 1 M TEAB buffer was added to stop the reaction. This was followed by additional stirring at 0°C for 10 minutes, and then stirring at room temperature for 20 minutes. The reaction mixture was washed with ethyl acetate, diluted to a total volume of 20 mL with distilled water, and loaded onto a DEAE Sepharose column. Subsequently, the target product was eluted using a 0–1 M TEAB buffer (pH 7.5) gradient. The fraction containing the product was collected, concentrated, and freeze-dried to obtain the 2′-O-methylguanosine 5′-monophosphate TEA salt.

[0102]

[0103] 3.3. General Synthesis Method of Nucleoside-5′-phosphoroimidazolide

[0104] The triethylammonium salt of nucleoside-5′-monophosphate (1 mmol) was dissolved in anhydrous DMSO (4 mL), and then triethylamine (0.85 mL, 6 mmol), imidazole or 2-aminoimidazole (3 equivalents), and 2,2′-dithiodipyridine (DPDS) (0.65 g, 3 mmol) were added and stirred. After 5 minutes, triphenylphosphine (0.78 g, 3 mmol) dissolved in anhydrous DMSO (4 mL) was added and stirred at room temperature for 5 hours. The reaction mixture was added to an acetone (50 mL) solution containing sodium perchlorate (0.5 g), cooled at 4°C for 30 minutes, and centrifuged at 5000 rpm for 8 minutes. The resulting solid was washed with acetone to remove residual imidazole and triphenylphosphine, cooled and centrifuged again, and then vacuum dried at room temperature to obtain the final product.

[0105]

[0106] 4. Non-enzymatic extension reaction conditions

[0107] The total reaction proceeded in two steps: first, mRNA was prepared, followed by non-enzymatic extension. The in vitro transcription reaction mixture contained the gene of interest (GFP / luciferase / apotin; approximately 250 ng of dsDNA containing a T7 promoter and 30 poly-A tail), 4 μL of rNTPs (1 μL each of 10 mM rATP, rCTP, rUTP, and 2.5 mM rGTP), 10 mM DTT, 25 units of RNase inhibitor, 1× T7 RNA polymerase buffer (40 mM Tris-HCl, 10 mM MgCl₂, 10 mM DTT, and 2 mM spermidine, pH 7.9 at 25°C), 75 units of T7 RNA polymerase, and 1 μL of ARCA cap 10 mM solution (4-fold ratio to rGTP). The final volume was adjusted to 15 μL with sterile water, and the mixture was reacted at 37°C for 90 minutes. The transcribed mRNA was purified using an RNA spin column and eluted with sterile water.

[0108] For non-enzymatic extension, purified mRNA (12 μg) was mixed with gRNA (approx. 12 μg), 50 mM MgCl₂, 200 mM HEPES, and 20 mM imidazole-activated guanosine derivative monomers, and reacted at 37°C for 90 minutes. The reaction results were confirmed by 0.8% agarose gel electrophoresis using NeoGreen staining, and residual substances were removed by agarose gel-cut spin-column purification. Concentration and purity were measured using Nano-Drop after each purification step.

[0109]

[0110] 5. Cell Culture and Real-Time qPCR

[0111] HEK-293, HEPG2, and RAW 264.7 cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium) in a humidified cell culture incubator maintained at 37°C and 5% CO₂ until 80–90% confluence was reached, followed by subculture with trypsin treatment. The culture medium was prepared to contain 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Cells were administered mRNA treated differently according to the experimental purpose and cultured for the required incubation time.

[0112] Total RNA was isolated from treated cells using the spin-column extraction method of the Cell / Virus RNA Extraction Kit. cDNA synthesis was performed on the extracted RNA using EZH2-specific reverse primers, and other reaction conditions followed the manufacturer's instructions. Subsequently, RT-qPCR was performed using EZH2-specific primers and the SYBR Green PCR mastermix. Real-time qPCR data were analyzed using StepOne software version 2.3, and the results were expressed as relative mRNA expression levels compared to the negative control, normalized to the internal control. The negative control refers to RT-qPCR data obtained under conditions without target cDNA. Each sample was analyzed in at least three replicates.

[0113]

[0114] 6. Western blot and dot-blot analysis

[0115] After extracting proteins from cells treated for Western blot analysis, they were separated on a 15% SDS-PAGE gel and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA) for immunoblotting. To evaluate dsRNA removal after non-enzymatic extension, purified mRNA was directly transferred to nitrocellulose paper for dot-blot analysis. Subsequently, the membrane was reacted with primary antibodies anti-apotin (1:1000), anti-bax (1:1000), anti-dsRNA (1:1000), and anti-β-actin (1:1000), followed by treatment with an HRP-linked secondary antibody. Signal detection on the membrane was performed using chemiluminescence.

[0116]

[0117] 7. MTT Analysis

[0118] For the MTT assay, HEPG2 and RAW 264.7 cell lines were co-cultured at a 1:10 ratio to evaluate the effects of non-enzymatically extended apotin mRNA expression levels. All other conditions were maintained as previously described. Cells were seeded into 96-well cell culture plates at a density of approximately 1–2 × 10⁴ cells per well and treated with mRNA at concentrations ranging from 3.5 μg / mL to 60 μg / mL, followed by 24 hours of incubation. Subsequently, time-dependent apoptosis assays were performed at 12, 24, 36, and 48 hours to calculate the IC50 values ​​for each treatment group. After each incubation period, 15 μL of MTT reagent (5 mg / mL) was added, and the mixture was incubated at 37°C for 4 hours. The generated formazan product was dissolved in 100 μL of DMSO and mixed by gently shaking at room temperature for 10 minutes, after which the absorbance (OD) was measured at 490 nm using a spectrophotometer (MRX A2000 Microplate reader). As a control, only HEPG2 and RAW 264.7 cells co-cultured with the medium were used.

[0119]

[0120] 8. Non-enzymatic prolongation mechanism

[0121] The non-enzymatic extension mechanism facilitated by imidazole compounds involves a series of steps that activate nucleotides, promote monomer addition, form reactive intermediates, increase nucleophilicity, and enhance reaction rates. The combination of these factors enables the successful replication of RNA templates without an enzymatic apparatus. A key step in this process is the formation of covalent intermediates; specifically, the 5′-5′ imidazolium bridged dinucleotide formed by the nucleophilic attack of the phosphate group of the protonated monomer by imidazole N3 of the aprotonated monomer. This covalent intermediate subsequently reacts with a primer to promote its extension. The involvement of imidazole is an essential element enabling the non-enzymatic replication of RNA templates.

[0122] The key features of this mechanism were identified through kinetic, structural, and chemical structure-activity relationship (SAR) analyses. In particular, the stability of intermediates bridged with 2-aminoimidazole was confirmed to have a significant effect on their efficacy as nucleotide activators. This stability enables the maintenance of high steady-state concentrations, which facilitates template binding and primer extension. Furthermore, these intermediates enhance reactivity by increasing the nucleophilicity of specific functional groups within the nucleotides and promote efficient and rapid primer extension.

[0123]

[0124] 9. Preparation of Buffer Solution

[0125] A 1.0 M Triethylammonium Bicarbonate (TEAB) solution was prepared as follows (based on 2 L, performed in a fume hood). 1.7 L of cold deionized water was placed in a 2 L beaker, and triethylamine (279 mL, 202 g) was slowly added while stirring. Finely crushed dry ice was added over 6 hours at 0 ℃ while measuring the pH of the solution until it reached the pH range of 7.0–8.0. After confirming that all dry ice had dissolved, the solution was returned to room temperature and transferred to a 2 L volumetric flask. The final volume was adjusted to 2 L using deionized water, and the pH was accurately measured for use in the experiment.

[0126]

[0127] 10. Chemical Synthesis of Guanosine-Based Modified Nucleotides

[0128] 10.1. Synthesis of L-Guanosine monophosphate imidazolide

[0129] The L-Guanosine monophosphate imidazolide (3) compound was synthesized by optimization (Fig. 1). The reagents used in the experiment were purchased commercially and used without further purification. The solvents used for the reaction and purification were all anhydrous, which had been treated to remove moisture, and the trimethyl phosphate used in the monophosphate reaction was further dried using molecular Sieves (4 Å). All reactions were carried out in a nitrogen atmosphere (N2 atmosphere). 1 H-NMR, 31 The P-NMR spectrum was measured using a 400 MHz Bruker Avance III HD Fourier transform NMR spectrometer with D2O as the solvent and tetramethylsilane as the internal standard.

[0130]

[0131] 10.1.1. L-Guanosine monophosphate triethylammonium salt (2)

[0132] 700 mg of L-Guanosine nucleoside (1) dried in a freeze-dryer was dissolved in anhydrous trimethyl phosphate (7 mL), and the reaction solution was heated at 40 °C for 15 minutes. After confirming that the solute was well dissolved in the solvent, the reaction solution was cooled in an ice bath for 15 minutes. Phosphoryl chloride (0.3 mL, 3.21 mmol, 1.3 eq.) was slowly added at 0 °C, and the reaction mixture was stirred for 4 hours. 1.0 M triethylammonium bicarbonate buffer (5 mL) was slowly added to neutralize and stop the reaction. L-guanosine monophosphate, from which the solvent was removed by freeze-drying the reaction mixture, was purified by connecting a cation exchange chromatography column (DEAE Sepharose Fast Flow anion exchange) to a Combi Flash Rf 200 Ion Exchange Chromatography system under conditions of 1.0 M TEAB (triethylammonium bicarbonate, pH 7.5), H2O, and a flow rate of 3 mL / min. As a result of purification, L-guanosine monophosphate triethylammonium salt (266 mg) in the form of a pale yellow foam was obtained (Fig. 2).

[0133] 1 H-NMR (400 MHz, DO) δ8.16 (s, 1H, 8-H), 5.88 (d, J = 6.0 Hz, 1H, 1′-H), 4.74 (t, J = 5.6 Hz, 1H, 2′-H), 4.46 (t, J = 4.3 Hz, 1H, 3′) -H), 4.29 (m, J = 3.3 Hz, 1H, 4′-H), 3.97 (m, J = 4.3 Hz, 2H, 5′-H)

[0134] 31 P-NMR (162 MHz, D2O) δ3.73.

[0135]

[0136] 10.1.2. L-Guanosine monophosphate imidazolide sodium salt (3)

[0137] Compound (2) (250 mg) dried in a freeze dryer was mixed with imidazole (300 mg, 4.41 mmol, 10 eq.) and 2,2′-dithiodipyridine (387 mg, 1.76 mmol, 4 eq.) and dissolved in dimethyl sulfoxide (3 mL). After dissolving by stirring at room temperature for 20 minutes, triethylamine (0.3 mL, 2.20 mmol, 5 eq.) was added and stirred for an additional 10 minutes at room temperature. Triphenyl phosphine (461 mg, 1.76 mmol, 4 eq.) dissolved in 3 mL of dimethyl sulfoxide was slowly added, and the reaction mixture was stirred for 14 hours. A solution of 1% NaClO4 dissolved in acetone was cooled and used as the acetone washing solution. The reaction mixture was dropped dropwise into the prepared acetone washing solution and centrifuged at 8,000 rpm for 4 minutes to remove the supernatant. This process was repeated 5 times and dried under reduced pressure to obtain a white solid L-guanosine monophosphate imidazolide sodium salt (3) (220 mg). Quantitative analysis was performed in aqueous solution using a UV-vis spectrophotometer (Agilent Technologies Cary 100) (Fig. 3).

[0138] 1H-NMR (400 MHz, DO) δ7.87 (s, 1H, Im(2-H)), 7.81 (s, 1H, 8-H), 7.13 (s, 1H, Im(5-H)), 6.92 (s, 1H, Im(4-H)), 5.82 (d, J = 5.16 Hz, 1H, 1′-H), 4.75 (t, J = 5.32 Hz, 1H, 2′-H), 4.40 (t, J = 4.60 Hz, 1H, 3′-H), 4.24 (m, 1H, 4′-H), 4.08 (m, J = 4.64 Hz, 2H, 5′-H).

[0139] 31 P-NMR (162 MHz, D2O) δ-7.96

[0140]

[0141] 10.2. Synthesis of Inosine Monophosphate Imidazolide

[0142] The inosine monophosphate imidazolide (6) compound was synthesized by optimization (Fig. 4). The reagents used in the experiment were purchased commercially and used without further purification. The solvents used for the reaction and purification were all anhydrous, which had been treated to remove moisture, and the trimethyl phosphate used in the monophosphate reaction was further dried using molecular Sieves (4 Å). All reactions were carried out in a nitrogen atmosphere (N2 atmosphere). 1 H-NMR, 31 The P-NMR spectrum was measured using a 400 MHz Bruker Avance III HD Fourier transform NMR spectrometer with D2O as the solvent and tetramethylsilane as the internal standard.

[0143]

[0144] 10.2.1. Inosine monophosphate triethylammonium salt (5)

[0145] 700 mg of inosine nucleoside (4) dried in a freeze-dryer was dissolved in anhydrous trimethyl phosphate (7 mL). The reaction solution was heated at 40 °C for 15 minutes. After confirming that the solute was well dissolved in the solvent, the reaction solution was cooled in an ice bath for 15 minutes. Phosphoryl chloride (0.34 mL, 3.61 mmol, 1.3 eq.) was slowly added at 0 °C, and the reaction mixture was stirred for 4 hours. 1.0 M triethylammonium bicarbonate buffer (5 mL) was slowly added to neutralize and stop the reaction. The inosine monophosphate, from which the solvent was removed by freeze-drying the reaction mixture, was purified by connecting a cation exchange chromatography column (DEAE Sepharose Fast Flow anion exchange) to a Combi Flash Rf 200 Ion Exchange Chromatography at 1.0 M TEAB (triethylammonium bicarbonate, pH 7.5), H2O, and a flow rate of 3 mL / min. As a result of purification, a pale yellow foam-like inosine monophosphate triethylammonium salt (5) (338 mg) was obtained (Fig. 5).

[0146] 1 H-NMR (400 MHz, DO) δ8.53 (s, 1H, 2-H), 8.21 (s, 1H, 8-H), 6.13 (d, J = 5.78 Hz, 1H, 1′-H), 4.77 (s, 1H, 2′-H), 4.51 (t, J = 4.32 Hz, 1H, 3′-H), 4.37 (t, J = 1.59 Hz, 1H, 4′-H), 4.05 (q, J = 3.27 Hz, 2H, 5′-H).

[0147] 31 P-NMR (162 MHz, D2O) δ2.40.

[0148]

[0149] 10.2.2. Inosine monophosphate imidazolide sodium salt (6)

[0150] Compound (5) (330 mg) dried in a freeze-dryer was mixed with imidazole (410 mg, 6.02 mmol, 10 eq.) and 2,2′-dithiodipyridine (530 mg, 2.41 mmol, 4 eq.) and dissolved in dimethyl sulfoxide (3 mL). After dissolving by stirring at room temperature for 20 minutes, triethylamine (0.42 mL, 3.01 mmol, 5 eq.) was added and stirred for an additional 10 minutes at room temperature. Triphenyl phosphine (632 mg, 2.41 mmol, 4 eq.) dissolved in 3 mL of dimethyl sulfoxide was slowly added, and the reaction mixture was stirred for 14 hours. A solution of 1% NaClO4 dissolved in acetone was cooled and used as the acetone washing solution. The reaction mixture was dropped dropwise into the prepared acetone washing solution and centrifuged at 8,000 rpm for 4 minutes to remove the supernatant. This process was repeated 5 times and dried under reduced pressure to obtain a white solid inosine monophosphate imidazolide sodium salt (6) (250 mg). Quantitative analysis was performed in aqueous solution using a UV-vis spectrophotometer (Agilent Technologies Cary 100) (Fig. 6).

[0151] 1H-NMR (400 MHz, DO) δ8.21 (s, 1H, Im(2-H)), 8.16 (s, 1H, 2-H), 7.81 (s, 1H, 8-H), 7.12 (d, J = 1.17 Hz, 1H, Im(5-H)), 6.92 (d, J = 0.79 Hz, 1H, Im(4-H)), 6.04 (d, J = 5.24 Hz, 1H, 1′-H), 4.76 (d, J = 5.22 Hz, 1H, 2′-H), 4.42 (t, J = 4.73 Hz, 1H, 3′-H), 4.29 (m, 1H, 4′-H), 4.09 (q, J = 4.17 Hz, 2H, 5′-H).

[0152] 31 P-NMR (162 MHz, D2O) δ-8.00.

[0153]

[0154] 10.3. Synthesis of 2′-O-Methylguanosine monophosphate imidazolide

[0155] The compound 2′-O-Methylguanosine monophosphate imidazolide (9) was synthesized by further optimizing the reaction based on the published paper and patent. The reagents used in the experiment were purchased commercially and used without further purification. All solvents used for the reaction and purification were anhydrous, which had been treated to remove moisture, and the trimethyl phosphate used in the monophosphate reaction was further dried using molecular Sieves (4 Å). All reactions were carried out in a nitrogen atmosphere (N2 atmosphere). 1 H-NMR, 31 The P-NMR spectrum was measured using a 400 MHz Bruker Avance III HD Fourier transform NMR spectrometer with D2O as the solvent and tetramethylsilane as the internal standard (Fig. 7).

[0156]

[0157] 10.3.1. 2′-O-Methylguanosine monophosphate triethylammonium salt (8)

[0158] 2′-O-methylguanosine nucleoside (7) (110 mg) dried in a freeze-dryer was dissolved in anhydrous trimethyl phosphate (2 mL). The reaction solution was heated at 40 °C for 15 minutes. After confirming that the solute was well dissolved in the solvent, the reaction solution was cooled in an ice bath for 15 minutes. Phosphoryl chloride (0.05 mL, 0.48 mmol, 1.3 eq.) was slowly added at 0 °C, and the reaction mixture was stirred for 4 hours. 1.0 M triethylammonium bicarbonate buffer (1.5 mL) was slowly added to neutralize and stop the reaction. The 2′-O-methylguanosine monophosphate, from which the solvent was removed by freeze-drying the reaction mixture, was purified by connecting a cation exchange chromatography column (DEAE Sepharose Fast Flow anion exchange) to a CombiFlash Rf 200 Ion Exchange Chromatography system with 1.0 M TEAB (triethylammonium bicarbonate, pH 7.5) and H2O at a flow rate of 3 mL / min. As a result of purification, a pale yellow foamy 2′-O-methylguanosine monophosphate triethylammonium salt (8) (110 mg) was obtained (Fig. 8).

[0159] 1H-NMR (400 MHz, D2O) δ8.17 (s, 1H, 8-H), 5.99 (d, J = 6.08 Hz, 1H, 1′-H), 4.65 (t, J = 2.38 Hz, 1H, 2′-H), 4.52 (q, J = 3.71 Hz, 1H, 3′-H), 4.33 (m, 1H, 4′-H), 4.05 (m, 2H, 5′-H), 3.46 (s, 3H, OCH3).

[0160] 31 P-NMR (162 MHz, D2O) δ2.02.

[0161]

[0162] 10.3.2. 2′-O-Methylguanosine monophosphate imidazolide sodium salt (9)

[0163] Compound (8) (110 mg) dried in a freeze-dryer was mixed with imidazole (200 mg, 2.93 mmol, 10 eq.) and 2,2′-dithiodipyridine (258 mg, 1.17 mmol, 4 eq.) and dissolved in dimethyl sulfoxide (3 mL). After dissolving by stirring at room temperature for 20 minutes, triethylamine (0.20 mL, 1.47 mmol, 5 eq.) was added and stirred for an additional 10 minutes at room temperature. Triphenyl phosphine (308 mg, 1.17 mmol, 4 eq.) dissolved in 3 mL of dimethyl sulfoxide was slowly added, and the reaction mixture was stirred for 14 hours. A solution of 1% NaClO4 dissolved in acetone was cooled and used as the acetone washing solution. The reaction mixture was dropped dropwise into the prepared acetone washing solution and centrifuged at 8,000 rpm for 4 minutes to remove the supernatant. This process was repeated 5 times and dried under reduced pressure to obtain a white solid 2′-O-methylguanosine monophosphate imidazolide sodium salt (9) (95 mg). Quantitative analysis was performed in aqueous solution using a UV-vis spectrophotometer (Agilent Technologies Cary 100) (Fig. 9).

[0164] 1 H-NMR (400 MHz, DO) δ7.92 (s, 1H, Im(2-H)), 7.82 (s, 1H, 8-H), 7.14 (d, J = 1.38 Hz, 1H, Im(5-H)), 6.93 (s, 1H, Im(4-H)), 5.91 (t, J =2.37 Hz, 1H, 1′-H), 4.53 (d, J = 3.83 Hz, 2H, 2′-H, 4′-H), 4.25 (m, 1H, 3′-H), 4.10 (q, J = 3.00 Hz, 2H, 5′-H), 3.45 (s, 3H, OCH3).

[0165] 31 P-NMR (162 MHz, D2O) δ-7.96.

[0166]

[0167] 10.4. Synthesis of 2′-O-(2-Methoxyethyl)guanosine monophosphate imidazolide

[0168] The compound 2′-O-(Methoxyethyl)guanosine monophosphate imidazolide (12) was synthesized by further optimizing the reaction based on the published paper and patent. The reagents used in the experiment were purchased commercially and used without further purification. All solvents used for the reaction and purification were anhydrous, which had been treated to remove moisture, and the trimethyl phosphate used in the monophosphate reaction was further dried using molecular Sieves (4 Å). All reactions were carried out in a nitrogen atmosphere (N2 atmosphere). 1 H-NMR, 31 The P-NMR spectrum was measured using a 400 MHz Bruker Avance III HD Fourier transform NMR spectrometer with D2O as the solvent and tetramethylsilane as the internal standard (Fig. 10).

[0169]

[0170] 10.4.1. 2′-O-(2-Methoxyethyl)guanosine monophosphate triethylammonium salt (11)

[0171] 2′-O-(methoxyethyl)guanosine nucleoside (10) (110 mg) dried in a freeze-dryer was dissolved in anhydrous trimethyl phosphate (2 mL). The reaction solution was heated at 40 °C for 15 minutes. After confirming that the solute was well dissolved in the solvent, the reaction solution was cooled in an ice bath for 15 minutes. Phosphoryl chloride (0.04 mL, 0.419 mmol, 1.3 eq.) was slowly added at 0 °C, and the reaction mixture was stirred for 4 hours. 1.0 M triethylammonium bicarbonate buffer (2 mL) was slowly added to neutralize and stop the reaction. The 2′-O-(methoxyethyl)guanosine monophosphate, from which the solvent was removed by freeze-drying the reaction mixture, was purified by connecting a cation exchange chromatography column (DEAE Sepharose Fast Flow anion exchange) to a Combi Flash Rf 200 Ion Exchange Chromatography at 1.0 M TEAB (triethylammonium bicarbonate, pH 7.5) and H2O at a flow rate of 3 mL / min. As a result of purification, a pale yellow foam-like 2′-O-(2-methoxyethyl)guanosine monophosphate triethylammonium salt (11) (60 mg) was obtained (Fig. 11).

[0172] 1H-NMR (400 MHz, D2O) δ8.18 (s, 1H, 8-H), 5.99 (d, J = 6.44 Hz, 1H, 1′-H), 4.68 (q, J = 3.85 Hz, 1H, 2′-H), 4.61 (q, J = 2.66 Hz, 1H 3′-H), 4.34 (q, J = 1.41 Hz, 1H, 4′-H), 4.04 (m, J = 2.49 Hz, 2H, 5′-H), 3.78 (m, J = 4.37 Hz, 2H, Ethyl(1-H)), 3.53 (m, J = 2.50 Hz, 2H, Ethyl(2-H)), 3.22 (s, 3H, OCH3).

[0173] 31 P-NMR (162 MHz, D2O) δ2.36.

[0174]

[0175] 10.4.2. 2′-O-(2-Methoxyethyl)guanosine monophosphate imidazolide sodium salt (12)

[0176] Compound (11) (45 mg) dried in a freeze dryer was mixed with imidazole (73 mg, 1.07 mmol, 10 eq.) and 2,2′-dithiodipyridine (95 mg, 0.43 mmol, 4 eq.) and dissolved in dimethyl sulfoxide (2 mL). After dissolving by stirring at room temperature for 20 minutes, triethylamine (0.07 mL, 0.54 mmol, 5 eq.) was added and stirred for an additional 10 minutes at room temperature. Triphenyl phosphine (113 mg, 0.43 mmol, 4 eq.) dissolved in 2 mL of dimethyl sulfoxide was slowly added, and the reaction mixture was stirred for 14 hours. A solution of 1% NaClO4 dissolved in acetone was cooled and used as the acetone washing solution. The reaction mixture was dropped dropwise into the prepared acetone washing solution and centrifuged at 8,000 rpm for 4 minutes to remove the supernatant. This process was repeated 5 times and dried under reduced pressure to obtain a white solid 2′-O-(2-Methoxyethyl)guanosine monophosphate imidazolide sodium salt (12) (40 mg). Quantitative analysis was performed in aqueous solution using a UV-vis spectrophotometer (Agilent Technologies Cary 100) (Fig. 12).

[0177] 1H-NMR (400 MHz, DO) δ7.92 (s, 1H, Im(2-H)), 7.85 (s, 1H, 8-H), 7.16 (d, J = 1.39 Hz, 1H, Im(5-H)), 6.95 (s, 1H, Im(4-H)), 5.91 (d, J =5.97 Hz, 1H, 1′-H), 4.72 (t, J = 5.57 Hz, 1H, 2′-H), 4.48 (q, J = 2.85 Hz, 1H, 3′-H), 4.28 (m, 1H, 4′-H), 4.12 (q, J = 3.27 Hz, 2H, 5′-H), 3.76 (m, J = 4.44 Hz, 2H, Ethyl(1-H)), 3.53 (quin, J = 2.96 Hz, 2H, Ethyl(2-H)), 3.23 (s, 3H, OCH3).

[0178] 31 P-NMR (162 MHz, D2O) δ-7.97.

[0179]

[0180] 10.5. Synthesis of 2′-Fluoroguanosine monophosphate imidazolide

[0181] The compound 2′-Fluoroguanosine monophosphate imidazolide (15) was synthesized by further optimizing the reaction based on the published paper and patent. The reagents used in the experiment were purchased commercially and used without further purification. All solvents used for the reaction and purification were anhydrous, which had been treated to remove moisture, and the trimethyl phosphate used in the monophosphate reaction was further dried using molecular Sieves (4 Å). All reactions were carried out in a nitrogen atmosphere (N2 atmosphere). 1 H-NMR, 31 P-NMR, 19The F-NMR spectrum was measured using a 400 MHz Bruker Avance III HD Fourier transform NMR spectrometer with DMSO-d6 as the solvent and tetramethylsilane as the internal standard (Fig. 13).

[0182]

[0183] 10.5.1. 2′-Fluoroguanosine monophosphate triethylammonium salt (14)

[0184] 2′-fluoroguanosine nucleoside (13) (100 mg) dried in a freeze-dryer was dissolved in anhydrous trimethyl phosphate (2 mL). The reaction solution was heated at 40 °C for 15 minutes. After confirming that the solute was well dissolved in the solvent, the reaction solution was cooled in an ice bath for 15 minutes. Phosphoryl chloride (0.04 mL, 0.456 mmol, 1.3 eq.) was slowly added at 0 °C, and the reaction mixture was stirred for 4 hours. 1.0 M triethylammonium bicarbonate buffer (2 mL) was slowly added to neutralize and stop the reaction. The 2′-fluoroguanosine monophosphate, from which the solvent was removed by freeze-drying the reaction mixture, was purified by connecting a cation exchange chromatography column (DEAE Sepharose Fast Flow anion exchange) to a Combi Flash Rf 200 Ion Exchange Chromatography at 1.0 M TEAB (triethylammonium bicarbonate, pH 7.5) and H2O at a flow rate of 3 mL / min. As a result of purification, a pale yellow foamy 2′-fluoroguanosine monophosphate triethylammonium salt (14) (55 mg) was obtained (Fig. 14).

[0185] 1 H-NMR (400 MHz, DMSO-d6) δ10.73 (s, 1H, 1-H), 7.91 (s, 1H, 8-H), 6.73 (s, 2H, NH2), 6.00 (dd, J = 6.71 Hz, 1H, 1′-H), 5.32 (m, J= 12.10 Hz, 1H, 3′-OH), 4.58 (m, J = 5.88 Hz, 1H, 2′-H), 4.02 (m, J= 4.16 Hz, 1H, 3′-H, 4′-H), 3.88 (m, J = 4.86 Hz, 2H, 5′-H).

[0186] 31 P-NMR (162 MHz, DMSO-d6) δ0.03.

[0187] 19 F-NMR (377 MHz, DMSO-d6) δ-204.23.

[0188]

[0189] 10.5.2. 2′-Fluoroguanosine monophosphate imidazolide sodium salt (15)

[0190] Compound (14) (45 mg) dried in a freeze dryer was mixed with imidazole (84 mg, 1.24 mmol, 10 eq.) and 2,2′-dithiodipyridine (109 mg, 0.50 mmol, 4 eq.) and dissolved in dimethyl sulfoxide (2 mL). After dissolving by stirring at room temperature for 20 minutes, triethylamine (0.08 mL, 0.62 mmol, 5 eq.) was added and stirred for an additional 10 minutes at room temperature. Triphenyl phosphine (130 mg, 0.50 mmol, 4 eq.) dissolved in 2 mL of dimethyl sulfoxide was slowly added, and the reaction mixture was stirred for 14 hours. A solution of 1% NaClO4 dissolved in acetone was cooled and used as the acetone washing solution. The reaction mixture was dropped dropwise into the prepared acetone washing solution and centrifuged at 8,000 rpm for 4 minutes to remove the supernatant. This process was repeated 5 times and dried under reduced pressure to obtain a white solid 2′-fluoroguanosine monophosphate imidazolide sodium salt (15) (38 mg). Quantitative analysis was performed in aqueous solution using a UV-vis spectrophotometer (Agilent Technologies Cary 100) (Fig. 15).

[0191] 1H-NMR (400 MHz, DMSO-d6) δ10.67 (s, 1H, 1-H), 7.89 (s, 1H, Im(1-H)), 7.61 (s, 1H, 8-H), 7.08 (d, J = 1.13 Hz, 1H, Im(5-H)), 6.84 (s, 1H, Im(4-H)), 6.64 (s, 2H, NH2), 5.98 (dd, J = 6.70 Hz, 1H, 1′-H), 5.80 (d, J = 5.02 Hz, 1H, 3′-OH), 5.34 (m, J = 12.22 Hz, 1H, 2′-H), 4.34 (m, J = 5.01 Hz, 1H, 3′-H), 3.94 (d, J = 3.72 Hz, 1H, 4′-H), 3.81 (m, J = 5.57 Hz, 2H, 5′-H).

[0192] 31 P-NMR (162 MHz, DMSO-d6) δ-10.12.

[0193] 19 F-NMR (377 MHz, DMSO-d6) δ-205.86.

[0194]

[0195] 10.6. Synthesis of guanosine monophosphorothioate triethylammonium salt (17)

[0196] The guanosine monophosphorothioate (17) compound was synthesized by optimization (Fig. 16). The reagents used in the experiment were purchased commercially and used without further purification. All solvents used for the reaction and purification were anhydrous, which had been treated to remove moisture, and the trimethyl phosphate used in the monophosphate reaction was further dried using molecular Sieves (4 Å). All reactions were carried out in a nitrogen atmosphere (N2 atmosphere). 1 H-NMR, 31The P-NMR spectrum was measured using a 400 MHz Bruker Avance III HD Fourier transform NMR spectrometer with D2O as the solvent and tetramethylsilane as the internal standard.

[0197] 300 mg of guanosine nucleoside (16), dried in a freeze-dryer, was dissolved in anhydrous trimethyl phosphate (4.5 mL). The reaction solution was heated at 40 °C for 15 minutes. After confirming that the solute was well dissolved in the solvent, the reaction solution was cooled in an ice bath for 15 minutes. 2,6-Lutidine (0.37 mL, 3.18 mmol, 3.0 eq.) was dissolved in the reaction solution, and thiophosphoryl chloride (0.16 mL, 1.59 mmol, 1.5 eq.) was slowly added at 0 °C, and the reaction mixture was stirred for 4 hours. 1.0 M triethylammonium bicarbonate buffer (5 mL) was slowly added to neutralize and stop the reaction. The guanosine monophosphorothioate, from which the solvent was removed by freeze-drying the reaction mixture, was purified by connecting a cation exchange chromatography column (DEAE Sepharose Fast Flow anion exchange) to a Combi Flash Rf 200 Ion Exchange Chromatography at 1.0 M TEAB (triethylammonium bicarbonate, pH 7.5) and H2O at a flow rate of 3 mL / min. As a result of purification, a pale yellow foam of guanosine monophosphorothioate triethylammonium salt (17) (30 mg) was obtained.

[0198] 1H-NMR (400 MHz, DO) δ8.25 (d, J = 1.42 Hz, 1H, 8-H), 5.93 (d, J = 6.50 Hz, 1H, 1′-H), 4.82 (d, J = 5.40 Hz, 1H, 2′-H), 4.52 (q, J = 2.42 Hz, 1H, 3′-H), 4.35 (s, 1H, 4′-H), 4.06 (d, J = 3.80 Hz, 2H, 5′-H).

[0199] 31 P-NMR (162 MHz, D2O) δ43.21.

[0200]

[0201] 11. Confirmation of Non-enzymatic Elongation of Short RNA Using Guide RNA

[0202] 11.1. Oligonucleotides Used in the Experiment

[0203] Oligonucleotide sequence information used in the experiment Name Sequence (5′ → 3′) Template 1 [FAM] GGCGAUGUGAAAAA (Short RNA) Primer 1 CUUUUUCACAU (Guide RNA 1)

[0204] Five synthesized guanosine monophosphate imidazolide derivatives were measured using a Cary Series UV-Vis Spectrophotometer and prepared as 100 mM concentration samples. To confirm the potential for non-enzymatic elongation at the mRNA Poly(A) tail, guide RNA was used in short RNA to verify that the guanosine monophosphate imidazolide derivatives non-enzymatically elongated the 3′ end. First, concentration-dependent and time-dependent studies were conducted using L-guanosine monophosphate imidazolide, and it was confirmed that the five derivatives synthesized under optimized conditions non-enzymatically elongated the 3′ end of short RNA. The oligonucleotides used in the experiment were ordered from Bionia. Additionally, to confirm the experimental results after electrophoresis, the oligonucleotides with elongated base sequences were used as oligonucleotides with 6-carboxyfluorescein (FAM) fluorescent dye added to the 5′ end.

[0205]

[0206] 11.2. Optimization of Non-Enzymatic Extension Conditions

[0207] 11.2.1. Concentration-dependent studies

[0208] FAM-labeled RNA T1 (3 μL, 20 mM), P1 (3.5 μL, 23.3 mM), HEPES (3 μL, 200 mM, pH 8), and MgCl2 (1.5 μL, 20 mM) were mixed in reaction tubes. Subsequently, 10 mM, 15 mM, 20 mM, 25 mM, and 30 mM L-guanosine monophosphate imidazolide and nuclease-free water were added to each reaction tube, and a final reaction solution volume of 15 μL was reacted at 37 °C for 2 hours. Afterward, the differences in each reaction were observed via 20% Urea PAGE (30 x 12 cm) gel electrophoresis.

[0209]

[0210] 11.2.2. Time-dependent research

[0211] FAM-labeled RNA T1 (3 μL, 20 mM), P1 (3.5 μL, 23.3 mM), HEPES (200 mM, pH 8), and MgCl2 (20 mM) were mixed in reaction tubes. Subsequently, L-guanosine monophosphate imidazolide (3 μL, 20 mM) and nuclease-free water (1 μL) were added to each reaction tube and mixed. 15 μL of the reaction solution was incubated at 37°C for 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours, respectively. Afterward, the differences in each reaction were observed via 20% Urea PAGE (30 x 12 cm) gel electrophoresis.

[0212]

[0213] 11.3. Non-enzymatic elongation of short RNA

[0214] FAM-labeled RNA T1 (3 μL, 20 mM), P1 (3.5 μL, 23.3 mM), HEPES (200 mM, pH 8), and MgCl2 (20 mM) were mixed in a reaction tube. Then, five guanosine monophosphate imidazolide derivatives (6 μL each, 20 mM) were added to each reaction tube, and the mixture was reacted for 2 hours in 30 μL of reaction solution at 37°C. Subsequently, the differences in each reaction were observed via 20% Urea PAGE (30 x 12 cm) gel electrophoresis.

[0215]

[0216] 12. Confirmation of Non-Enzymatic Elongation of Luciferase mRNA 3′ Poly(A)Tail ​​and Biological Evaluation

[0217] 12.1. Oligonucleotides used in the experiment

[0218]

[0219]

[0220] 12.2. DNA Replication

[0221] 12.2.1. Primary PCR Amplification of Luciferase Gene Sequence

[0222] PCR was performed using FP1 and RP1 to replicate only the luciferase gene sequence from the luciferase-pcDNA3 plasmid. Luciferase-pcDNA3 template (100 ng, 1.0 μL), FP1 (1.5 μL, 10 mM), RP1 (1.5 μL, 10 mM), Nuclease-free water (6.0 μL), and 2X Topsimple™ DyeMix Tenuto DNA Polymerase (10.0 μL) were mixed in a 0.2 mL PCR tube, and PCR was performed under conditions of 95 ℃-50 ℃-72 ℃ for 35 cycles. After confirming the reaction results by 1% Agarose gel electrophoresis, the PCR product band corresponding to the luciferase gene was cut and extracted using the AccuPrep®PCR / Gel Purification kit (Bioneer).

[0223]

[0224] 12.2.2. Secondary PCR Amplification of Luciferase Gene Sequence

[0225] PCR was performed using FP2 and RP1 to insert a T7 promoter, Kozak sequence, into the 5′ end of the purified luciferase gene template and a 30 nt poly(T) sequence into the 3′ end. Luciferase gene template (100 ng, 1.0 μL), FP2 (1.5 μL, 10 mM), RP1 (1.5 μL, 10 mM), Nuclease-free water (6.0 μL), and 2x Topsimple™DyeMix Tenuto DNA Polymerase (10.0 μL) were mixed in a 0.2 mL PCR tube, and PCR was performed under conditions of 95 ℃-50 ℃-72 ℃ for 35 cycles. After confirming the reaction results through 1% Agarose gel electrophoresis, the PCR product was extracted by cutting the band corresponding to the luciferase gene using the AccuPrep®PCR / Gel Purification kit (Bioneer).

[0226]

[0227] 12.3. mRNA preparation

[0228] 15 μL of reaction solution containing 10X T7 RNA Polymerase Buffer (1.5 μL), dithiothreitol (DTT, 1.5 μL, 10 mM), rATP (1 μL, 10 mM), rUTP (1 μL, 10 mM), rGTP (1 μL, 10 mM), rCTP (1 μL, 10 mM), Clean cap (1 μL, 10 mM), RNase inhibitor (0.5 μL), Nuclease-free water (3.5 μL), and T7 RNA Polymerase (1 μL, 50 U / μL) was reacted for 2 hours at 37°C. Subsequently, mRNA was purified using the Zymo Research Oligo Clean & Concentrator kit.

[0229]

[0230] 12.4. Non-enzymatic Prolongation

[0231] Purified mRNA (10 μg), guide RNA 2 (3 μL, 30 μM), HEPES (6 μL, 200 mM), MgCl2 (3 μL, 20 mM), and five guanosine monophosphate imidazolide derivatives (6 μL each, 20 mM) were placed in separate reaction tubes and reacted at 37°C for 2 hours. Subsequently, non-enzymatically extended mRNA was purified using the Zymo Research Oligo Clean & Concentrator kit.

[0232]

[0233] 12.5. Reverse transcription

[0234] 20 μL of reaction solution containing cDNA RP (1 μL, 5 mM), 10X TOPscript™ RT Buffer (2.0 μL), dNTP Mixture (2 μL, 2.5 mM each), RNase inhibitor (0.5 μL), Nuclease-free water (12 μL), and TOPscript™ Reverse Transcriptase (1 μL) was reacted with extracted mRNA (100 ng) for 1 hour at 55 ℃.

[0235]

[0236] 12.6. cDNA PCR Amplification

[0237] A reaction solution containing cDNA (6 μL), cDNA FP, RP (2 μL each, 10 μM), and 2X Topsimple™ DyeMix Tenuto DNA Polymerase (10.0 μL) were mixed in a 0.2 mL PCR tube, and PCR was performed under conditions of 95 ℃-50 ℃-72 ℃ for 35 cycles. After confirming the reaction results by 1% Agarose gel electrophoresis, the PCR product band corresponding to the luciferase gene was cut and extracted using the AccuPrep® PCR / Gel Purification kit (Bioneer).

[0238]

[0239] 12.7. Transfection

[0240] 12.7.1. HEK 293 Cell Culture

[0241] To remove DMSO contained in the frozen HEK 293 cell line, it was mixed with 10% DMEM media in a 1:1 ratio and centrifuged at 3000 rpm for 3 minutes to remove the supernatant. It was mixed with fresh DMEM media, evenly sprayed into a 7 mL SPL Cell Culture Flask, and cultured for 24 hours at 37 ℃ under 5% CO2 level conditions.

[0242]

[0243] 12.7.2. mRNA Transduction into HEK 293 Cells

[0244] Twelve hours before transfection, culture HEK 293 cells in each well of a 96-well plate to establish a homogeneous environment. (1 × 10⁶ 4After dissolving purified mRNA (1 μg) in Opti-MEM (5 μL) (cells / well, 100 μL), the mixture was combined with Lipofectamine™MassengerMAX™ Transfection Reagent (1.5 μL) dissolved in an equal volume of Opti-MEM and left at room temperature for 10 minutes. Subsequently, the mixed solution was slowly added to each well of a 96-well plate in which HEK 293 cells were uniformly cultured. After confirming that the cells were evenly distributed using an EVOS®FL Cell Imaging System fluorescence microscope, the results were measured after incubation for 24 hours at 37°C and 5% CO2 level.

[0245]

[0246] 12.8. Luciferase Gene Expression

[0247] After incubating in an incubator for 24 hours, check the cell status using an EVOS®FL Cell Imaging System fluorescence microscope. Then, remove the media, add 30 μL of 1X Cell Culture Lysis Reagent, and incubate for 5 minutes at 37°C under 5% humidity. Afterward, carefully remove the cells and slowly pipette them into a 96-well plate for analysis. Add 150 μL of luciferase substrate and mix carefully, then measure the luminescence of each well using a Centro XS LB 960 Microplate Luminometer.

[0248]

[0249] result

[0250] 1. Selection of Modified Nucleotides (XNA)

[0251] In this study, two types of guanosine derivative XNAs were selected alongside natural nucleotides based on their unique chemical properties that can enhance mRNA stability and provide potential biological benefits. N7-(2-MePy)-GMP was selected as the base-modified nucleotide; the introduction of a pyridine ring into the nucleobase is believed to improve mRNA interactions with ribosomes, potentially leading to increased translation efficiency. 2′O-Me-GMP was selected as the sugar-modified nucleotide; the methyl group located at the 2′ position of the ribose sugar is considered to protect mRNA from enzymatic degradation and reduce inflammatory responses, thereby enabling efficient translation and sustained protein expression in therapeutic settings. Collectively, the XNAs selected in this study are expected to improve mRNA stability and translation efficiency, making them useful tools for biological and therapeutic applications. Meanwhile, although N7-(2-MePy)-GMP-IM was obtained as a pure product, the conversion rates of GMP-2-amino-IM and 2′O-Me-GMP-2-amino-IM were only 70% and 43%, respectively, leaving a mixture containing 30% and 57% of unreacted starting materials, GMP and 2′O-Me-GMP, respectively. However, under biological reaction conditions, the starting materials do not participate in the non-enzymatic extension process, and non-enzymatic extension occurs only when the guanosine monophosphate derivative is activated by imidazole. Therefore, the presence of unreacted GMP derivatives does not interfere with this reaction.

[0252]

[0253] 2. Verification of the Hypothesis

[0254] In this study, the genes of interest (GFP, luciferase, or apotin) were prepared via PCR to include a T7 promoter for T7 RNA polymerase-mediated transcription at the 5′ end and a 30-poly-A tail for non-enzymatic extension at the 3′ end. The corresponding mRNA was synthesized via in vitro transcription and then purified using a spin column. The purified transcription mRNA was modified by adding a guanosine-based natural or modified nucleotide unit activated with imidazole or 2-aminoimidazole to gRNA (which is complementary to the 30-poly-A tail and contains four “C” bases added to its 5′ end), and then non-enzymatically extending the mRNA after the 3′-terminal poly-A tail using the complementary strand of the gRNA as a template.

[0255] To demonstrate non-enzymatic extension, the same reaction procedure targeting 4-base extension was first performed using commercially available short RNA primers and shorter gRNAs. Previous studies have reported non-enzymatic extensions ranging from 1 minute to 120 minutes, indicating that efficiency improves with increasing reaction time, with most primer non-enzymatic extensions occurring particularly after 60 minutes. Additionally, other researchers have confirmed that 2-aminoimidazole induces more efficient extension than imidazole. Based on this, the reaction time in this study was set to 90 minutes to secure maximum efficiency for non-enzymatic extension. This reaction was confirmed by 20% denatured polyacrylamide gel electrophoresis (PAGE) using EtBr staining, and non-enzymatic extension was demonstrated by the observation of mRNA bands shifted upward relative to the short RNA primers (Fig. 17A). As a result of the reaction, it was confirmed that non-enzymatic extension proceeded efficiently for each compound when using either imidazole or 2-aminoimidazole (Figs. 17A, 17B, 17C, and 17D). After confirmation via PAGE analysis, this study proceeded with experiments on genes of interest. Since large mRNAs have minimal base differences, there are limitations in evaluating them solely by gel electrophoresis; therefore, short RNA primers were initially used to clearly demonstrate non-enzymatic reactions. To prepare functional and stable mRNA, an ARCA cap was introduced at the 5′ end, and a 30-poly-A tail was retained at the 3′ end to protect it from degradation. Primer extension was performed by reacting the freshly purified transcribed mRNA for a certain period of time in a mixture of designed gRNA, 200 mM HEPES, 50 mM MgCl₂, and 20 mM imidazole monomer.After the reaction was completed, agarose gel electrophoresis was performed, and mRNA bands located directly below each corresponding gene template band were observed (Figs. 17B, 17C, and 17D), and further purification was performed.

[0256] For large mRNAs, since 4-base extensions are very small relative to the mRNA size (~400 / 780 / 1700 bp) and are difficult to clearly distinguish using gel electrophoresis alone, the extensions were verified through Sanger sequencing (Figs. 17E, 17F, 17G, and 17H). Sequencing samples were prepared by performing reverse transcription using specific hexamer reverse primers during the initial cDNA synthesis step, followed by amplification via extension PCR. Extension PCR was necessary due to the limitations of Sanger sequencing to mitigate noise signals that could occur at primer binding sites caused by modifications concentrated at the 3′ end of the mRNA. Sequencing of each sample treated non-enzymatically with three 2-aminoimidazole / imidazole compounds confirmed the addition of an extended G at the end of the 30 poly-A tail (Figs. 17E, 17F, 17G, and 17H). During the sequencing process, DNA polymerase may slip in repetitive base sequences (e.g., regions with multiple repeated A or G bases), which may lead to the insertion of additional bases. Accordingly, in the GFP control group sequencing, an additional A was observed after the 3′-terminal 30 poly-A tail (Fig. 17E), and in the non-enzymatic treatment group, an additional G was observed after the intended 4G extension (Figs. 17F, 17G, and 17H).

[0257]

[0258] 3. Expression Analysis and Discussion

[0259] After the non-enzymatic extension reaction was successfully performed, the intracellular expression of non-enzymatically extended luciferase, GFP, and apotin mRNAs was analyzed. The translation efficiency of the non-enzymatically extended mRNAs was evaluated by comparing the results using the three imidazole compounds with the control mRNA (without extension). Cells were transfected for 24 hours using 1 μg of mRNA in 100 μL of complete medium (DMEM, 10% FBS, and 1% penicillin / streptomycin) in 96-well plates. Prior to transfection, a dot-blot analysis was performed to confirm the presence of dsRNA in the purified non-enzymatically extended mRNA. The purification process using a Cellulose and RNA purification kit was confirmed to effectively remove potential dsRNA and transcription byproducts.

[0260] For the treated GFP mRNA, immediate expression was evaluated through cell imaging and fluorescence measurements (Fig. 18A). Analysis of the tested compounds [GMP-2-amino-IM, 2′O-Me-GMP-2-amino-IM, and N7-(2-MePy)-GMP-IM] revealed that GFP mRNA treated with the glyco-modified 2′O-Me-GMP-2-amino-IM exhibited significantly higher expression levels compared to the control group and other treatment groups. GFP mRNA treated with GMP-2-amino-IM showed only a slight increase in expression compared to the control mRNA (no extension). The expression intensity of N7-(2-MePy)-GMP-IM treated mRNA was lower than that of the control group. Corresponding GFP expression was measured by recording the fluorescence spectrum.

[0261] After incubating luciferase mRNAs treated differently for a certain period, the medium was removed, and cells were lysed using 50 μL of 1× lysis buffer provided with the luciferase substrate. To quantify the luminescence signal, 100 μL of luciferin substrate was dispensed into a 96-well white plate, followed by the addition of 20 μL of cell lysate. Luciferase luminescence was then measured using a luminometer, and the results are shown in the bar graph in Fig. 18B. Luciferase expression also exhibited an expression pattern similar to GFP depending on each treatment condition.

[0262] For the apotin expression analysis, HEPG2 and RAW 264.7 cells were co-cultured at a ratio of 10:1, and other conditions were maintained identically to the previous experiment. The apotin expression patterns for each treated mRNA were evaluated via western blot analysis, and the results for the control and sugar-modified treatment groups were consistent with previously observed expression patterns. In contrast, the expression of the natural and base-modified treatment groups was lower than that of the control group. Western blot band intensities were quantified using Gel Analyzer software, and the relative change in expression for each treatment group was calculated by setting the expression level of the untreated apotin mRNA control to 1.0 (Figs. 18C and 18D).

[0263]

[0264] 4. mRNA stability analysis

[0265] Using this system, the stability of non-enzymatically extended mRNA was compared with control mRNA (when extension was not performed). Generally, the half-life of mRNA is known to be approximately 16.4 hours. First, stability based on the mRNA half-life was evaluated by observing the expression patterns of luciferase mRNA over time. This was measured through time-dependent translation analysis from 12 to 48 hours. Among the tested compounds [GMP-2-amino-IM, 2′O-Me-GMP-2-amino-IM, and N7-(2-MePy)-GMP-IM], mRNA treated with the sugar-modified 2′O-Me-GMP-2-amino-IM exhibited the highest expression levels over time and showed the strongest mRNA half-life among the tested samples (Fig. 19A).

[0266] To verify the results of Fig. 19A, the stability pattern of mRNA modified with 2′O-Me-GMP-2-amino-IM was subsequently determined by another method and compared to control mRNA (no treatment). The RNase activity of the Exo-T enzyme, which degrades single-stranded RNA in the 3′→5′ direction, was evaluated. After performing Exo-T treatment on control mRNA and 2′O-Me-GMP-2-amino-IM-treated mRNA for different durations, their respective expression patterns were analyzed. As a result, the treated mRNA exhibited a longer half-life and enhanced expression compared to the control. This experiment demonstrates that biological mRNA degradation correlates with other factors that can negatively affect translation, emphasizing the importance of mRNA stability (Fig. 19B).

[0267]

[0268] 5. Analysis of Apoptosis in Cancer Cells

[0269] Apoptin is known to induce cancer cell death and reduce cell viability, motility, and invasiveness by disrupting mitochondrial pathways and G2 / M checkpoints. To track apoptosis induced by non-enzymatically treated modified apoptin mRNA, a 24-hour MTT assay was first performed to compare the viability of treated cells with that of untreated control apoptin mRNA. Based on the MTT results, the IC50 values ​​for each treatment group were calculated. According to Figure 20, the IC50 value of the control apoptin (no treatment) was 2.6 μg. The IC50 values ​​of the treated mRNA were 1.5 μg for the 2′O-Me-GMP-2-amino-IM treatment group, 2.2 μg for the GMP-2-amino-IM treatment group, and 2.8 μg for the N7-(2-MePy)-GMP-IM treatment group, respectively. These IC50 values ​​correlated with the apotin expression patterns of each treatment group (Fig. 20, Fig. 18C, and Fig. 18D). The highest apoptotic activity (i.e., the lowest IC50 value) was observed in apotin mRNA extended with the glyco-modified 2′O-Me-GMP-2-amino-IM, which means that the high expression of the correspondingly treated apotin mRNA induced stronger apoptosis in cancer cells (Fig. 20A).

[0270]

[0271] 6. Analysis of EZH2 Expression via Apoptosis

[0272] To date, we evaluated the expression of apotin mRNA treated with different imidazole compounds using western blot analysis and monitored cancer cell viability through apoptosis. Subsequently, to investigate the effects of differences in apoptosis rates on cellular epigenetics, we conducted additional experiments targeting the EZH2 gene, which is correlated with apoptosis. In cancer cells, apoptosis is closely linked to epigenetic changes. The expression of genes that play a crucial role in epigenetic regulation, such as EZH2, can have a significant impact on apoptosis. It has been reported that inhibiting EZH2 expression promotes apoptosis and induces cell cycle arrest in cancer cells. Understanding these epigenetic changes and their impact on apoptosis can lead to more effective cancer treatment strategies.

[0273] First, to determine the point at which apoptosis induced by the treated mRNA becomes more pronounced than in the control group (no treatment), time-dependent translation experiments were performed to measure cell viability. As a result, the difference between each treatment group became more distinct after 48 hours, and a decrease in cell viability of approximately 40% to 50% was observed due to apoptosis induced by each treatment (Fig. 21A). Accordingly, the treatment time was extended to more than 48 hours before performing the EZH2 expression analysis. Total RNA was extracted after treatment, and EZH2 expression was measured by performing Real-Time qPCR following reverse transcription (Fig. 21B). EZH2 expression was compared with two control groups. One was normal HEK-293 cells (no transduction), which showed normal EZH2 expression, and the other was cancer HEP-G2 cells (no transduction), which generally showed EZH2 overexpression. As a result, significant epigenetic responses were observed, particularly in the apotin mRNA treatment group in which non-enzymatic extension with 2′O-Me-2-amino-IM was performed. In this case, EZH2 expression levels were significantly reduced or CT values ​​were distinctly increased, which was consistent with the expected expression pattern. EZH2 is well known to promote the survival and proliferation of cancer cells by downregulating tumor suppressor genes and upregulating oncogenes. Therefore, the reduction in EZH2 expression in cancer cells through treatment with apotin mRNA modified with 2′O-Me-2-amino-IM implies that selectively downregulating oncogenes could be a promising approach for cancer treatment. Expression levels consistent with each control group were also observed in the treatment groups using natural and base-modified imidazole compounds. Specifically, in the GMP-2-amino-IM treated group, EZH2 expression was slightly lower or CT values ​​were slightly higher than in the control group, whereas in the N7-(2-MePy)-GMP-IM treated group, EZH2 expression was higher or CT values ​​were lower than in the control group.According to recent studies, EZH2 activity can be regulated by non-enzymatically modified mRNA (one of the post-transcriptional modification methods), and it has been suggested that these modifications can perform important biological functions for PRC2 target genes in the process of cancer cell development, where EZH2 and PRC2 (polycomb repressive complex 2) act as key regulators of genes involved in cell growth and differentiation.

[0274]

[0275] 7. Optimization of Non-Enzymatic Extension Conditions

[0276] Prior to confirming the non-enzymatic binding of synthesized guanosine monophosphate imidazolide derivatives to the poly(A) tail ends of luciferase mRNA, we planned an experiment to verify non-enzymatic binding at the short RNA stage and optimize reaction conditions. In 20% urea pAGE, RNA with FAM fluorescent dye added to the 5′ end was used to facilitate identification of the target RNA. Following electrophoresis, the bands were stained in methylene blue and observed under UV light. As a result, non-enzymatic extension bands began to appear at a concentration of 10 mM for the guanosine monophosphate imidazolide derivatives; however, as the concentration increased, the bands became progressively darker, becoming clearly visible at 20 mM. Beyond this point, although the bands became slightly clearer with increasing concentration, the difference was insignificant, and since increasing the concentration resulted in an excessive amount, 20 mM was deemed the most suitable concentration. In the time-dependent experiment, the extension band was observed even at 30 minutes after the start of the reaction, but a tendency for it to become progressively darker over time was confirmed. Although it implies that non-enzymatic binding increases over time, considering the stability of mRNA, it was concluded that the optimal reaction time is 2 hours (Fig. 22).

[0277]

[0278] 8. Non-enzymatic elongation of short RNA

[0279] It was confirmed that all reactions using guanosine monophosphate imidazolide derivatives on 20% urea pAGE resulted in extension of more than 1 nt. In particular, non-enzymatic extension using inosine monophosphate imidazolide was observed to extend up to 2 nt. This indicates that the extension was greater than that of the guide RNA designed for a 1 nt extension. The role of the guide RNA facilitates the introduction of guanosine monophosphate imidazolide derivatives, allowing for extension of more bases than designed. This implies that during subsequent non-enzymatic extension of luciferase mRNA, more derivatives than designed may be extended. The non-enzymatic binding affinities predicted from the gel data were found to be IMP·Im > 2′-O-(2-Methoxyethyl)GMP·Im > 2′-O-MethylGMP·Im > 2′-FluoroGMP·Im > L-GMP·Im (Fig. 23).

[0280]

[0281] 9. Non-enzymatic elongation of luciferase mRNA

[0282] 9.1. DNA replication

[0283] Forward and reverse primers containing the nucleotide sequences constituting the 5′ and 3′ ends of the luciferase gene from the luciferase-pcDNA3 plasmid containing the luciferase gene sequence were ordered from Cosmogenetech, and a PCR amplification reaction was performed using them. A replicated luciferase gene band approximately 1.7 kb in length was found below the 2 kb ladder on a 1% agarose gel containing Neo green. After purification, a luciferase gene template used in the experiment was obtained.

[0284] Subsequently, experiments were conducted to introduce the nucleotide sequences constituting mRNA into the 5′ and 3′ ends of the luciferase gene template. At the 5′ end, a T7 promoter was introduced to enable recognition by T7 RNA polymerase, and a Kozak sequence was introduced to allow ribosomes to accurately and efficiently recognize the start cordon during transcription. At the 3′ end, the corresponding nucleotide sequence was introduced to enable the introduction of the mRNA's poly(A) tail. Similarly, these forward and reverse primers were ordered from Cosmogenetech and used to perform PCR amplification reactions. The replicated luciferase gene band, approximately 1.7 kb in length, was found below the 2 kb ladder on a 1% agarose gel containing Neo green. After purification, a modified luciferase gene template used in transcription experiments was obtained (Figs. 24 and 25).

[0285]

[0286] 9.2. Sequence Analysis of Non-Enzymatic Extension Samples

[0287] Primers were designed to obtain only nucleotide sequences of an appropriate length for Sanger sequencing analysis. After non-enzymatic introduction, cDNA was obtained by reverse transcribing luciferase mRNA, and PCR was performed using the designed primers. This sample was submitted to Cosmogenetech for DNA sequencing analysis along with complementary primers to obtain nucleotide sequence information for the non-enzymatically extended Guanosine derivative (Fig. 26).

[0288] Sequence analysis revealed that guanine sequences were observed following the 3′poly(A) tail in all samples. Interestingly, similar to the results observed in short RNA, a much larger number of guanosine monophosphate imidazolides were extended than the number of designed induction derivatives in the guide RNA. It was confirmed that at least four, and up to six, additional induction derivatives were introduced compared to the designed four sequences. Non-enzymatic binding affinity was also found to follow the trend observed in short RNA (Figs. 27, 28, 29, 30, and 31).

[0289]

[0290] 9.3. Analysis of Non-Enzymatically Extended Luciferase mRNA Expression in HEK 293 Cells

[0291] Five guanosine monophosphate imidazolide derivatives were non-enzymatically introduced into the prepared luciferase mRNA, and 1 μg was transfected into each well of HEK 293 cells. The cells were incubated for 24 hours in an incubator at 37°C and 5% CO2 levels, and luminescence in each well was measured using a Centro XS LB 960 Microplate Luminescence Analyzer for luciferase gene expression analysis. As a result, it was confirmed that luciferase expression increased for four of the substances, excluding L-guanosine monophosphate imidazolide, compared to luciferase mRNA that had not undergone non-enzymatic elongation. Translation efficiency and mRNA stability influence the determination of luciferase expression levels; since all samples contained the same luciferase gene, their translation efficiencies were similar. Therefore, it was confirmed that guanosine monophosphate imidazolide bound to the 3′ poly(A) tail terminal affected the stability of mRNA, resulting in a difference in luciferase expression levels (Fig. 32).

Claims

1. A method for preparing a modified mRNA structure comprising the step of non-enzymatically reacting a purine nucleotide, a pyrimidine nucleotide, or a derivative thereof containing an imidazole or 2-aminoimidazole active group at the 3' end of the mRNA.

2. In Claim 1, A method for preparing a modified mRNA structure, wherein the nucleotide derivative has at least one functional group introduced into at least one of a base, a sugar, and a phosphate backbone.

3. In Claim 2, A method for preparing a modified mRNA structure, wherein the functional group is one or more selected from the group comprising alkyl groups, alkoxy groups, halogens, aryl groups, heteroaryl groups, alkylamino groups, hydroxyl groups, carbonyl groups, thio groups, and fluoro groups.

4. In Claim 1, A method for preparing a modified mRNA structure, wherein the derivative of the above nucleotide is formed by attaching an imidazole or 2-aminoimidazole active group to at least one of L-guanosine monophosphate (L-GMP), inosine monophosphate (IMP), 2′-O-methylguanosine monophosphate (2′-O-Me-GMP), 2′-O-methoxyethylguanosine monophosphate (2′-O-MOE-GMP), and 2′-fluoroguanosine monophosphate (2′-F-GMP).

5. In Claim 1, A method for preparing a modified mRNA structure in which 1 to 10 purine nucleotides, pyrimidine nucleotides, or derivatives thereof are sequentially bound to the 3' end of the mRNA by the above reaction.

6. In Claim 5, A method for preparing a modified mRNA structure in which the above nucleotide or derivative thereof is attached to the poly(A) tail end of the mRNA.

7. In Claim 1, The above reaction is Mg 2+ A method for preparing a modified mRNA structure, which is performed in a HEPES buffer with a pH of 7 to 9 in the presence of.

8. In Claim 1, A method for preparing a modified mRNA structure, wherein the above reaction is carried out at 37°C for 1 to 3 hours.

9. mRNA structure prepared by the method of Claim 1.

10. In Claim 9, The mRNA structure is a modified mRNA structure comprising one or more selected from the group consisting of L-guanosine monophosphate (L-GMP), inosine monophosphate (IMP), 2′-O-methylguanosine monophosphate (2′-O-Me-GMP), 2′-O-methoxyethylguanosine monophosphate (2′-O-MOE-GMP), and 2′-fluoroguanosine monophosphate (2′-F-GMP) at the 3' end.

11. In Claim 9, The above modified mRNA structure is a modified mRNA structure that has increased stability or protein translation expression compared to non-modified mRNA.

12. An anticancer pharmaceutical composition comprising the modified mRNA structure of Claim 9.

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