A pace structure modified cap analog and uses thereof

Abstract

The application discloses a PACE structure modified cap analog, which is based on the existing cap analog structure, has ribose bases or modified ribose bases at both ends, three ribose structures in the middle and a triphosphonate segment or a phosphonate segment connecting the ribose structures, and has one or more PACE structures or PACE derivative structures on the triphosphonate segment or the phosphonate segment, namely, (alkoxy)alkyl-COOH or a salt thereof with or without substitution on the triphosphonate or the phosphonate, so that the IVT yield and capping efficiency are improved, and the translation efficiency of the target mRNA is improved.

Description

Technical Field

[0001] This invention relates to the fields of chemical and biological engineering technology, specifically to a cap analogue with PACE structure modification and its application. Background Technology

[0002] In eukaryotic cells, mRNA needs to possess several key structures to perform its functions. These structures mainly consist of five parts: the 5' cap, the 5' untranslated region (5'UTR), the open reading frame (ORF) encoding the antigen, the 3' untranslated region (3'UTR), and a poly(A) tail. Among these, the cap plays a crucial role, enhancing mRNA structural stability, improving translation efficiency, and reducing immunogenicity.

[0003] Autologous immune proteins such as RIGI and IFIT can recognize abnormally capped mRNAs, reducing the activity and half-life of exogenous mRNAs within cells, making them less effective in vivo. Therefore, optimizing the mRNA cap structure (capping) is crucial for improving mRNA biological activity. Summary of the Invention

[0004] To address the issue of insufficient immunogenicity in existing cap analogs, this invention provides a PACE-modified cap analog in which one or more phosphonates are replaced with phosphonoacetic acid (salt) structures at the phosphorus position of the triphosphonate or phosphonate segment between the three ribose structures of the cap analog. The PACE-modified cap analog of this invention, due to the presence of substituted or unsubstituted (alkoxy)alkyl-COOH or its salt on the triphosphonate or phosphonate, not only improves IVT yield and capping efficiency but also enhances the translation efficiency of the target mRNA.

[0005] The PACE-modified cap analogue of this invention is based on the existing cap analogue structure, which has ribose bases or modified ribose bases at both ends, three ribose structures in the middle, and a triphosphonate segment or phosphonate segment connecting the ribose structures. This invention constructs a PACE structure or PACE-derived structure having one or more PACE structures or PACE-derived structures in the triphosphonate segment or phosphonate segment, wherein the PACE structure or PACE-derived structure conforms to the following general structural formula:

[0006]

[0007] X in the PACE structure is -CH2COOH or a salt thereof;

[0008] X of the PACE-derived structure is -COOH, a substituted or unsubstituted alkyl-COOH or a salt thereof, a substituted or unsubstituted O-alkyl-COOH or a salt thereof, a substituted or unsubstituted alkyl-O-COOH or a salt thereof, a substituted or unsubstituted alkyl-O-alkyl-COOH or a salt thereof; the alkyl group has no more than 7 carbon atoms; the substituents refer to alkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl groups with no more than 7 carbon atoms.

[0009] The salt refers to a sodium salt, an ammonium salt, or a salt formed with tris(hydroxymethyl)aminomethane. The salt formed by the tris(hydroxymethyl)aminomethane and the cap analogue modified with the PACE structure described in this invention is hereinafter referred to as a tris salt.

[0010] Preferably, X is an alkyl-COOH or a salt thereof, wherein the alkyl group has 1, 2, 3 or 4 C atoms.

[0011] Preferably, X is O-alkyl-COOH, and the alkyl group has 1, 2, 3 or 4 C atoms.

[0012] As a preferred embodiment, the cap analogue modified with the PACE structure conforms to the following general structural formula:

[0013]

[0014] Wherein, R1, R2 and R3 are independently H, OH, alkyl or O-alkyl;

[0015] Any one, two or more of X1, X2, X3 and X4 is X as described in claim 1, and the others are each independently H, OH, NH2, alkyl, O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or halogen;

[0016] B1 and B2 are, independently, natural, modified, or non-natural nucleoside bases.

[0017] Preferably, B1 and B2 are adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T), respectively.

[0018] Preferably, B1 and B2 are adenine (A) and guanine (G), respectively.

[0019] Furthermore, the PACE structure-modified cap analogue can be any of the following structures:

[0020]

[0021] Preferably, the PACE-modified cap analogue is a salt of any of the following structures, wherein the salt is a sodium salt, ammonium salt, or salt formed from tris(hydroxymethyl)aminomethane:

[0022]

[0023]

[0024] The application of the PACE structure-modified cap analogue described in this invention involves capping RNA in an in vitro transcription reaction for the in vitro transcription preparation of linear mRNA; the steps are as follows:

[0025] Step (1): Prepare DNA template;

[0026] Step (2): Perform an in vitro transcription reaction, wherein the reaction system contains RNA polymerase, nucleoside triphosphonic acid and the cap analog of the PACE structure modification.

[0027] The PACE-modified cap analogs described in this invention have a more stable conformation and are less susceptible to recognition and hydrolysis by nucleases. Therefore, PACE-structured cap analogs have the following advantages: (1) high IVT yield and high capping efficiency; (2) high translation efficiency of the target mRNA.

[0028] Terminology Explanation

[0029] In this invention, the terms "base", "natural base" and "nucleoside base" can be used interchangeably, including but not limited to adenine (A), guanine (G), cytosine (C), uracil (U), or thymine (T).

[0030] "Modified base" and "modified nucleoside base" can be used interchangeably. They refer to substances obtained by replacing one or more hydrogen atoms of a natural base, such as, but not limited to, 7-methylguanine.

[0031] The "ribose structure" in this invention includes, but is not limited to, and its isomers, derivatives, etc. Structures in which any functional group is replaced, and their isomers, derivatives, etc.

[0032] The "triphosphonate segment" in this invention includes, but is not limited to, And its isomers, derivatives, salts, etc., wherein one or more of X1, X2, X3 are -COOH, -CH2COOH, substituted or unsubstituted alkyl-COOH, substituted or unsubstituted O-COOH, substituted or unsubstituted O-alkyl-COOH, substituted or unsubstituted alkyl-O-alkyl-COOH, etc.

[0033] The "phosphonate segment" in this invention includes, but is not limited to, And its isomers, derivatives, salts, etc., wherein X4 is -COOH, -CH2COOH, substituted or unsubstituted alkyl-COOH, substituted or unsubstituted O-COOH, substituted or unsubstituted O-alkyl-COOH, substituted or unsubstituted alkyl-O-alkyl-COOH, etc. Detailed implementation method:

[0034] Example

[0035] Synthesis section

[0036] Example 1: Synthesis method of PACE structure-modified cap analogues using intermediates A and B as raw materials

[0037] Intermediate A (2.0 mol) was suspended in a DMF solution containing ZnCl2 (20.0 mol), and intermediate B (1.8 mol) was added to the reaction solution under ice bath conditions. After stirring at room temperature for 24 hours, the reaction was terminated with 10 L of 0.25 M EDTA-Na2 solution. The mixture was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M sodium chloride aqueous solution, desalted by nanofiltration, and concentrated to obtain the target product. The reaction pathway is shown in the following equation:

[0038]

[0039] Intermediate A is obtained through the following steps:

[0040] (1) Weigh 50.0 g of guanosine and dissolve it in 500.0 mL of trimethyl phosphate. Cool the reaction solution to 0 °C and slowly add phosphorus oxychloride (1.8 eq.) dropwise under a nitrogen atmosphere. After stirring at 0 °C for 4 hours, quench the reaction with water. Wash twice with dichloromethane to remove most of the trimethyl phosphate. After removing the residual organic solvent by aqueous phase concentration under reduced pressure, purify by reversed-phase preparative liquid chromatography and concentrate to obtain intermediate A1.

[0041] (2) Intermediate A1, triphenylphosphine (2.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature for 15 hours under a nitrogen atmosphere. After the reaction was completed, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution, and a solid precipitated. The filter cake was thoroughly washed with acetone after filtration to obtain intermediate A2.

[0042] (3) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate A2 was added to the reaction solution in portions and stirred at room temperature for 5 hours. After the reaction was completed, the reaction was terminated with 10 times the volume of 0.25 MEDTA-Na2 solution. The reaction solution was purified by ion chromatography to obtain intermediate A3.

[0043] (4) Dissolve intermediate A3 in 20 times the volume of purified water, cool the reaction solution to 4°C, slowly add dimethyl sulfate (6.0 eq.), and adjust the pH to not exceed 5 with 2M sodium hydroxide during the process. Detect the reaction by HPLC. After the reaction is completed, wash with dichloromethane and purify the aqueous phase by ion chromatography to obtain intermediate A4.

[0044] (5) Intermediate A4, triphenylphosphine (2.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature for 10 hours under a nitrogen atmosphere. After the reaction was completed, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid. The solid was filtered, and the filter cake was thoroughly washed with acetone to obtain intermediate A. The reaction route is shown in the following equation.

[0045]

[0046] Compound B is obtained through the following steps:

[0047] (1) Weigh 200.0g of 2'OMe-rA phosphoridamide monomer containing the PACE structure and N 2 Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetraazole (2.1 eq.) was added under nitrogen purging, and the reaction was carried out at 25 °C for 3 hours. After the reaction was completed, 70% aqueous tert-butyl hydroperoxide solution was added dropwise to the reaction solution, and the reaction was carried out at 25 °C for 1 hour. After the reaction was completed, trichloroacetic acid (4.0 eq.) in dichloromethane solution was added dropwise to the reaction solution, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was washed with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate B1.

[0048] (2) Intermediate B1 was dissolved in acetonitrile (10V), and 1.8 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.8 eq. of tetrazolium were added. The mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was complete, 70% aqueous solution of tert-butyl hydrogen peroxide (1.2 eq.) was added dropwise to the reaction solution, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed, the solution was evaporated to dryness. Methanol (5V) and DBU (2.5 eq.) were added to the rotary flask, and the mixture was reacted at room temperature for 4 hours. Then, concentrated ammonia (5V) was added, and the mixture was reacted at room temperature for 14 hours. The reaction was then monitored, and the solution was evaporated to dryness after the reaction was completed. The crude product was purified by ion chromatography and concentrated to obtain intermediate B. The reaction route is shown in the following equation:

[0049]

[0050] Example 2: Synthesis method of PACE structure-modified cap analogues using intermediates C and D as raw materials

[0051] Using intermediates C and D as raw materials, the cap analogue of Example 2 was obtained by referring to the synthesis method of the target product in Example 1. The reaction route is shown in the following equation:

[0052]

[0053] Compound C is obtained through the following steps:

[0054] (1) Take 50g N 2 -Isobutyryl-2',3'-acetylguanosine was dissolved in dichloromethane (10V), and 1.8 eq. of intermediate C1 and 1.8 eq. of tetrazolium were added. The mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. N was detected by TLC. 2 After the disappearance of -isobutyryl-2',3'-acetylguanosine, 70% tert-butylhydrogen peroxide aqueous solution (1.2 eq.) was added dropwise to the reaction solution, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was washed with 10% sodium sulfite aqueous solution and saturated brine, respectively, and the organic phase was concentrated to obtain intermediate C2.

[0055] (2) Intermediate C2 was dissolved in methanol (5V), DBU (2.5 eq.) was added, and the mixture was reacted at room temperature for 4 hours. Then, concentrated ammonia (5V) was added, and the mixture was reacted at room temperature for 14 hours. The mixture was then evaporated to dryness after the reaction was completed. The crude product was purified by ion chromatography and concentrated to obtain intermediate C3.

[0056] (3) Dissolve intermediate C3 in 20 times the volume of purified water, cool the reaction solution to 4°C, slowly add dimethyl sulfate (6.0 eq.), and adjust the pH to no more than 5 with 2M sodium hydroxide during the process. Detect the reaction by HPLC. After the reaction is completed, wash with dichloromethane and purify the aqueous phase by ion chromatography to obtain intermediate C.

[0057]

[0058] Compound D is obtained through the following steps:

[0059] (1) Weigh 200.0g of 2'OMe-rA phosphorous amide monomer and N 2 Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetraazole (2.1 eq.) was added under nitrogen purging, and the reaction was carried out at 25 °C for 3 hours. After the reaction was completed, 70% aqueous tert-butyl hydroperoxide solution was added dropwise to the reaction solution, and the reaction was carried out at 25 °C for 1 hour. After the reaction was completed, trichloroacetic acid (4.0 eq.) in dichloromethane solution was added dropwise to the reaction solution, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was washed with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate D1.

[0060] (2) Intermediate D1 was dissolved in acetonitrile (10V), and 1.8 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.8 eq. of tetrazolium were added. The mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was completed, 70% aqueous solution of tert-butyl hydrogen peroxide (1.2 eq.) was added dropwise to the reaction solution, and the mixture was allowed to react at room temperature for 1 hour. After the reaction was completed, the solution was evaporated to dryness. Methanol and concentrated ammonia (10V, 1:1) were added to the rotary flask, and the mixture was allowed to react at room temperature for 14 hours. The reaction was then monitored, and the solution was evaporated to dryness after the reaction was completed. The crude product was purified by ion chromatography and concentrated to obtain intermediate D2.

[0061] (3) Intermediate D2, triphenylphosphine (2.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature for 15 hours under a nitrogen atmosphere. After the reaction was completed, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution, and a solid precipitated. The filter cake was thoroughly washed with acetone after filtration to obtain intermediate D3.

[0062] (4) Triethylamine phosphate (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate A3 was added to the reaction solution in portions and stirred at room temperature for 5 hours. The reaction was terminated with 10 volumes of 0.25 MEDTA-Na2 solution. The reaction solution was purified by ion chromatography to obtain intermediate D4.

[0063] (5) Intermediate D4, triphenylphosphine (2.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature for 10 hours under a nitrogen atmosphere. After the reaction was completed, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid. The solid was filtered, and the filter cake was thoroughly washed with acetone to obtain intermediate D. The reaction route is shown in the following equation.

[0064]

[0065] Example 3: Synthesis method of PACE structure-modified cap analogues using intermediates E and F as raw materials

[0066] Using intermediates E and F as raw materials, the cap analogue of Example 3 was obtained by referring to the synthesis method of the target product in Example 1. The reaction route is shown in the following equation:

[0067]

[0068] Compound E is obtained through the following steps:

[0069] (1) Dissolve intermediate A1 in 20 times the volume of purified water, cool the reaction solution to 4°C, slowly add dimethyl sulfate (6.0 eq.), and adjust the pH to not exceed 5 with 2M sodium hydroxide during the process. Detect the reaction by HPLC. After the reaction is completed, wash with dichloromethane and purify the aqueous phase by ion chromatography to obtain intermediate E1.

[0070] (2) Intermediate E1, triphenylphosphine (2.0 eq.), 2,2'-dithiopyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature for 10 hours under a nitrogen atmosphere. After the reaction was completed, the reaction solution was slowly added to a 4M sodium perchlorate acetone solution to precipitate a solid. The solid was filtered, and the filter cake was thoroughly washed with acetone to obtain intermediate E. The reaction route is shown in the following equation.

[0071]

[0072] Compound F was obtained through the following steps: Phosphorylated acetic acid (3.0 eq.) and zinc chloride (8.0 eq.) were suspended in anhydrous DMF and stirred at room temperature for 5 minutes. Intermediate D3 was added to the reaction solution in portions, and the mixture was stirred at room temperature for 5 hours. The reaction was terminated with 10 times its volume of 0.25 M EDTA-Na2 solution.

[0073] The reaction solution was purified by ion chromatography to obtain intermediate F.

[0074]

[0075] Example 4: Synthesis method of PACE structure-modified cap analogues using intermediates A and H as raw materials

[0076] Using intermediates A and H as raw materials, the cap analogue of Example 4 was obtained by referring to the synthesis method of the target product in Example 1. The reaction route is shown in the following equation:

[0077]

[0078] Compound H is obtained through the following steps:

[0079] (1) Dissolve 50g of intermediate D1 in dichloromethane (10V), add 1.8 eq. of intermediate C1 and 1.8 eq. of tetrazolium, and stir at room temperature for 2 hours under a nitrogen atmosphere. After intermediate D1 is completely consumed, 70% tert-butyl hydrogen peroxide aqueous solution (1.2 eq.) is added dropwise to the reaction solution, and the reaction is carried out at room temperature for 1 hour. After the reaction is completed, the reaction solution is washed with 10% sodium sulfite aqueous solution and saturated brine, respectively, and the organic phase is concentrated to obtain intermediate H1.

[0080] (2) Intermediate H1 was dissolved in methanol (5V), DBU (2.5 eq.) was added, and the mixture was reacted at room temperature for 4 hours. Concentrated ammonia (5V) was added, and the mixture was reacted at room temperature for 14 hours. The reaction was then checked by rotary evaporation. The crude product was purified by ion chromatography and concentrated to obtain intermediate H.

[0081]

[0082] Example 5: Synthesis method of PACE structure-modified cap analogues using intermediates A and I as raw materials

[0083] Using intermediates A and I as raw materials, the cap analogue of Example 5 was obtained by referring to the synthesis method of the target product in Example 1. The reaction route is shown in the following equation:

[0084]

[0085] Compound I is obtained through the following steps:

[0086] (1) Dissolve 30g of intermediate B1 in dichloromethane (10V), add 1.8 eq. of intermediate C1 and 1.8 eq. of tetrazolium, and stir at room temperature for 2 hours under a nitrogen atmosphere. After intermediate B1 is completely consumed, 70% tert-butyl hydrogen peroxide aqueous solution (1.2 eq.) is added dropwise to the reaction solution, and the reaction is carried out at room temperature for 1 hour. After the reaction is completed, the reaction solution is washed with 10% sodium sulfite aqueous solution and saturated brine, respectively, and the organic phase is concentrated to obtain intermediate I1.

[0087] (2) Intermediate I1 was dissolved in methanol (5V), DBU (2.5 eq.) was added, and the mixture was reacted at room temperature for 4 hours. Then concentrated ammonia (5V) was added, and the mixture was reacted at room temperature for 14 hours. The mixture was then evaporated to dryness after the reaction was completed. The crude product was purified by ion chromatography and concentrated to obtain intermediate I.

[0088]

[0089] Example 6: Synthesis method of PACE structure-modified cap analogues using intermediates A and J as raw materials

[0090] Using intermediates A and J as raw materials, the cap analogue of Example 6 was obtained by referring to the synthesis method of the target product in Example 1. The reaction route is shown in the following equation:

[0091]

[0092] Compound J is obtained through the following steps:

[0093] (3) Weigh 150.0g of the 2'OMe-rA phosphoridamide monomer containing the PACE-derived structure and N 2 Isobutyryl-2',3'-acetylguanosine (1.0 eq.) was dissolved in 2.0 L of dichloromethane in a single-necked flask. Tetraazole (2.1 eq.) was added under nitrogen purging, and the reaction was carried out at 25 °C for 3 hours. After the reaction was completed, 70% aqueous tert-butyl hydroperoxide solution was added dropwise to the reaction solution, and the reaction was carried out at 25 °C for 1 hour. After the reaction was completed, trichloroacetic acid (4.0 eq.) in dichloromethane solution was added dropwise to the reaction solution, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was washed with 10% sodium sulfite aqueous solution, 10% sodium bicarbonate aqueous solution, and saturated brine, respectively. The organic phase was concentrated and purified by column chromatography to obtain intermediate J1.

[0094] (4) Intermediate J1 was dissolved in acetonitrile (10V), and 1.8 eq. of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide and 1.8 eq. of tetrazolium were added. The mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was completed, 70% aqueous solution of tert-butyl hydrogen peroxide (1.2 eq.) was added dropwise to the reaction solution, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed, the mixture was evaporated to dryness. Methanol (5V) and DBU (2.5 eq.) were added to the rotary flask, and the mixture was reacted at room temperature for 4 hours. Then, concentrated ammonia (5V) was added, and the mixture was reacted at room temperature for 14 hours. The reaction was then monitored, and the mixture was evaporated to dryness after the reaction was completed. The crude product was purified by ion chromatography and concentrated to obtain intermediate J. The reaction route is shown in the following equation:

[0095]

[0096] Comparative Example 1: Synthesis of Cap Analogs Using Intermediates A and D2 as Raw Materials

[0097] Using intermediates A and D2 as raw materials, the cap analogue of Comparative Example 1 was obtained by referring to the synthesis method of the target product in Example 1. The reaction route is shown in the following equation:

[0098]

[0099] Bioactivity assay

[0100] 1. Determination of mRNA in vitro transcription yield and capping efficiency

[0101] In vitro synthesis of mRNA using cap analogs modified with PACE structure: plasmids were digested with linear endonucleases; linearized DNA templates were extracted; and mRNA was synthesized through in vitro transcription. Cap analogs from Examples 1-6 and Comparative Example 1 were used as cap structures, respectively.

[0102] The reaction system is shown in Table 1:

[0103] Table 1 In vitro transcription reaction system

[0104] system Dosage T7 RNA polymerase 50U 10X buffer 2μl 100mM ATP 1μl 100mM GTP 1μl 100mM CTP 1μl 100mM N1-Me-pUTP 1μl 100mm cap analogue 1μl Inorganic pyrophosphatase 0.05U nuclease inhibitors 20U sterile enzyme-free water Make up to 20 μl template 1μg

[0105] Note: During the experiment, first calculate the required volume of materials for the system, and then add the samples. First, add sterile, enzyme-free water to the system, followed by 10X buffer, NTPs, and cap analogue. Mix well and centrifuge gently. Then add nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template. Mix thoroughly and centrifuge gently. Incubate at 37°C for 2 hours, then add 1U of DNase I and incubate at 37°C for another 30 minutes to remove the DNA template. Purify the mRNA using magnetic beads. Dissolve the purified mRNA in sterile, enzyme-free water and use a Nanodrop One instrument to detect the mRNA yield.

[0106] Liquid chromatography-mass spectrometry (LC-MS) was used to detect the IVT capping rate of mRNAs with different initiation cap analogs. First, a labeled DNA probe matching the initiation base of the transcription product mRNA needs to be designed, typically using biotin. Streptavidin-labeled magnetic beads are washed and then incubated with the synthesized DNA probe, mRNA, and 10×RNase H reaction buffer at room temperature for 30 minutes, mixing slowly while incubating. Then, 20 μL of RNase H (5 U / μL) is added and incubated at 37°C for 3 hours, mixing every half hour. After incubation, the magnetic beads are washed, and 100 μL of 75% methanol heated to 80°C is added. The mixture is heated to 80°C on a hot plate for 3 minutes, then the supernatant is aspirated and dried using an evaporative centrifuge at room temperature for 45 minutes to 10 μL. The sample is then resuspended in 50 μL of 100 μM EDTA / 1% MeOH and is ready for LC-MS analysis to determine the RNA capping status during the transcription reaction. Since capped and uncapped bases differ significantly in molecular weight, the capping rate of mRNA transcription initiated by different capped analogs can be determined by using the difference in molecular weight.

[0107] As shown in Table 2, the cap analogs modified with PACE structure in Examples 1-6 can all be transcribed into the corresponding target mRNAs, and the yield and capping rate of the cap analogs modified with PACE structure in Examples 1-6 are significantly better than those in Comparative Example 1.

[0108] Table 2. In vitro transcription yield and capping rate of mRNA

[0109] serial number Yield (μg) Capping rate (%) Example 1 108 98.3 Example 2 107 97.5 Example 3 110 98.2 Example 4 96 97.8 Example 5 102 95.0 Example 6 104 95.7 Comparative Example 1 98 93.5

[0110] 2. Detection of mRNA translation efficiency

[0111] Using the eGFP coding sequence as a DNA template, in vitro transcription was performed using the cap analogues of Examples 1-6 and Comparative Example 1 as the starting material. The mRNA synthesized by in vitro transcription was transfected into 293T cells.

[0112] 293T cells were used at a rate of (0.5-1)×10 5Cells were seeded in 24-well plates, with cells up to passage 50 recommended for transfection. Cells should be passaged again 24 hours before transfection. Adding antibiotics to the culture medium does not affect transfection efficiency. A cell density of 60-80% is ideal at transfection. Transfect 2 μg mRNA per well using Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and follow its instructions. After transfection, cells were incubated at 37°C in a CO2 incubator for 4-6 hours, then the medium was replaced with fresh complete culture medium. After incubation at 37°C in a CO2 incubator for 24 hours, the fluorescence intensity of GFP was observed using a fluorescence microscope.

[0113] The results are shown in Table 3. The mRNA synthesized via in vitro transcription in Examples 1-6 showed significantly higher efficiency in translating into proteins within cells compared to Comparative Example 1, without inducing significant cell death. These experimental data demonstrate that the PACE-modified cap analogues described in this application can be efficiently applied to in vitro transcription of mRNA and intracellular protein expression.

[0114] Table 3 Translational expression efficiency of intracellular mRNA

[0115]

Claims

1. A cap analogue modified with a PACE structure, characterized in that, PACE-modified cap analogues conform to the following structural formula: ， Wherein, R1 and R2 are OH; R3 is an O-alkyl group; the alkyl group has 1 carbon atom. Any one or two of X1, X2, X3 and X4 are X, and the rest are OH independently; X is -CH2COOH, -COOH, or a salt thereof; B1 and B2 are adenine (A) and guanine (G) respectively.

2. The cap analogue modified with the PACE structure according to claim 1, characterized in that, The salt refers to a sodium salt, an ammonium salt, or a salt formed with tris(hydroxymethyl)aminomethane.

3. The cap analogue modified with the PACE structure according to claim 1 or 2, characterized in that, It can be any of the following structures: ， ， ， ， ， 。 4. The cap analogue modified with the PACE structure according to claim 1 or 2, characterized in that, It is a salt form of any of the following structures, wherein the salt form is a sodium salt, ammonium salt, or salt formed from tris(hydroxymethyl)aminomethane: ， ， ， ， ， 。 5. The application of the cap analogue modified with the PACE structure according to any one of claims 1-4, characterized in that, RNA is capped in the in vitro transcription reaction for the in vitro transcription preparation of linear mRNA.

6. The application according to claim 5, characterized in that, The application steps are as follows: Step (1): Prepare DNA template; Step (2): Perform an in vitro transcription reaction, wherein the reaction system contains RNA polymerase, nucleoside triphosphonic acid and the cap analog of the PACE structure modification.

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