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CN116375781B - TNA modified cap analogue and preparation method and application thereof - Google Patents

TNA modified cap analogue and preparation method and application thereof Download PDF

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CN116375781B
CN116375781B CN202310300602.8A CN202310300602A CN116375781B CN 116375781 B CN116375781 B CN 116375781B CN 202310300602 A CN202310300602 A CN 202310300602A CN 116375781 B CN116375781 B CN 116375781B
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tna
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cap analogue
modified cap
mrna
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CN116375781A (en
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黄磊
赵万年
沈奇
肖潇
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Shenji Biotechnology (Suzhou) Co.,Ltd.
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Abstract

The invention discloses a TNA modified cap analogue, a preparation method and application thereof, wherein one or two sugar rings in three sugar rings of the cap analogue are connected to a triphosphates chain segment through a 3' position; the triphosphates are arranged in the middle of the triphosphates chain segment, and the two ends of the triphosphates chain segment have no alkyl end groups and/or have alkyl end groups with the C number not more than 3; the cap analogue has the following advantages: (1) high IVT yield and high capping efficiency; (2) high stability after treatment with a uncapping enzyme DCP 2; (3) the translation efficiency of the target mRNA is high.

Description

TNA modified cap analogue and preparation method and application thereof
Technical Field
The invention relates to the technical field of chemistry and bioengineering, in particular to TNA modified cap analogues for In Vitro Transcription (IVT) synthesis of mRNA, a preparation method thereof and application thereof in mRNA synthesis.
Background
In eukaryotic cells, mature mRNA possesses some key structural elements to perform the relevant function, whereas in vitro transcribed mRNA functions by mimicking the structure of endogenous mRNA. The structure of mature mRNA has five main parts, including from 5 'to 3': a 5'cap structure (5' cap), a5 'untranslated region (5' utr), an open reading frame encoding a protein, a3 'untranslated region (3' utr), and a PolyA tail. mRNA "capping" techniques, i.e., techniques that modify the 5' end cap structure of an mRNA.
The cap structure is an essential structure for mRNA translation initiation, providing a signal for ribosomes to recognize mRNA, assisting ribosome binding to mRNA, and allowing translation to begin from AUG. Meanwhile, the cap structure can increase the stability of mRNA and protect the mRNA from being attacked by 5 '. Fwdarw.3' exonuclease.
The naturally-structured cap analogue, as it can be recognized and hydrolyzed by uncapping enzyme (DCP 2), reduces the stability of mRNA in vivo and ultimately reduces the translation efficiency of target mRNA. Accordingly, there is a need in the art to develop a cap analogue of a modified novel structure.
TNA is a non-natural chemical substance similar to DNA and RNA, and its backbone is composed of repeated arrangement of threose through 2 'and 3' phosphodiester linkages. Because TNA has a base pairing ability similar to that of a natural nucleic acid, and the sugar ring of TNA is formed of a simple four-carbon sugar. TNA is capable of folding like RNA to form a functional higher order structure as the genetic material of the original life form. Therefore, the cap analogue of the TNA structure is not easy to hydrolyze by uncapping enzyme (DCP 2 enzyme), has high stability, can avoid the damage of the mRNA cap structure in organisms, and finally improves the translation effect of target mRNA.
CN201910369662.9 by modern tex corporation in the united states, in its specification paragraphs [ 0061 ] and [ 0062 ], mentions the use of Threose Nucleic Acid (TNA) as a modified nucleoside or nucleotide. However, TNA modification as a capping analogue is not mentioned. Nucleosides or nucleotides are two different substances than capping analogues and also play a completely different role in the synthesis of nucleic acids. Nucleosides or nucleotides are the starting materials for the synthesis of nucleic acids, and capping analogs are the necessary structure for mRNA translation initiation, providing a signal for ribosomes to recognize mRNA, aiding ribosome binding to mRNA, and allowing translation to begin from AUG.
The prior art uses TNA structures for more applications as nucleic acid drugs, such as PCT/US2018/035687 and its patents, mentions methods of treating diseases with modified oligonucleotide compositions (e.g. in RNA interference and/or RNAse H mediated knockdown). The addition of TNA modified oligonucleotide analogs is characterized as being capable of indirectly or directly increasing or decreasing the activity of a protein or inhibiting or promoting the expression of a protein, see paragraph 00533. In some embodiments, the provided oligonucleotide is characterized in that it can be used to control cell proliferation, viral replication, and/or any other cell signaling process.
The nucleic acid medicine and the capping analogue for capping and modifying mRNA are completely different fields, and TNA structure in the nucleic acid medicine cannot be directly applied to the capping analogue for capping and modifying mRNA, so that the problems of unknown liver toxicity risk, unstable protein translation and the like are brought.
Disclosure of Invention
In view of the above problems, the present invention provides a TNA-modified cap analogue having the following advantages: (1) high IVT yield and high capping efficiency; (2) high stability after treatment with a uncapping enzyme DCP 2; (3) the translation efficiency of the target mRNA is high.
The TNA modified cap analogue has the following structural general formula:
r in the above structural formula a Is a first sugar ring; r is R b Is a second sugar ring;
y is a triphosphate chain segment, and the structure of the triphosphate chain segment accords with the following structural formula:
m and n are each independently 0, 1,2 or 3;
the R is a R is R b There must be at least one sugar ring attached to the triphosphate segment through the 3' position;
R 1 is that
B 3 Are natural, modified, or unnatural nucleobases.
Preferably, R a And R is b Is linked to the triphosphate chain segment through the 3' position.
Preferably, the R a R is R b The structures of (2) are independent; the R is a R is R b Having substituents at the 2',3' and/or 4' positions; the substituent is hydroxy, halogen, alkyl, substituted or unsubstituted O-alkyl, NH-alkyl, S-alkyl, O-aralkyl, S-aralkyl, alkynyl, cycloalkyl, cycloalkenyl, carboxyl, heterocyclyl, cyano, amino, or nitro; and the number of the substituent C is not more than 7.
Preferably, e.g. R a Or R is b Is linked to the triphosphate segment through the 3' position; then the 2',3' and/or 4' positions of the other sugar ringThe alkoxy has a C number of not more than 5.
Further, the TNA modified cap analogues conform to the following structural general formula:
wherein R is a Is thatR b Is->
When R is a Is thatWhen Rb is not->When R is b Is->When R is a Not be->
R 1 Is that
m and n are each independently 0, 1,2 or 3;
R 2 、R 3 、R 4 and R is 5 Each independently is hydrogen, hydroxy, halogen, alkyl, substituted or unsubstituted O-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, alkynyl, cycloalkyl, cycloalkenyl, or carboxyl;
R 6 is H,OH、NH 2 Alkyl, O-alkyl, N-alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or halogen;
R 7 、R 8 each independently is hydrogen, hydroxy, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cyano, substituted or unsubstituted amino, substituted or unsubstituted carboxy, substituted or unsubstituted nitro.
B 1 And B 2 Each independently a natural, modified, or unnatural nucleobase.
Preferably, in the structure of the TNA modified cap analogue, R 2 、R 3 、R 4 And R is 5 Are each independently hydrogen, C 1-5 Alkyl, hydroxy, O-alkyl.
Preferably, in the structure of the TNA modified cap analogue, R 6 Is hydrogen, C 1-5 Alkyl, hydroxy, O-alkyl.
Preferably, in the structure of the TNA modified cap analogue, R 7 、R 8 Are each independently hydrogen, C 1-5 Alkyl or hydroxy.
Further, the TNA modified cap analogue structure is selected from any one of the following:
the use of the TNA modified cap analogues: for in vitro transcription reactions of linear mRNA, RNA was capped.
A method for capping RNA in an in vitro transcription reaction using the TNA modified cap analogue comprising the steps of:
step (1): preparing a DNA template;
step (2): in vitro transcription reactions were carried out, which contained RNA polymerase, nucleoside triphosphates and TNA modified capping analogues according to the invention.
The TNA modified cap analogues have the following advantages: can promote in vitro transcription and improve the translation effect of target mRNA. The TNA modified cap analogue is not easy to hydrolyze by uncapping enzyme, the stability of the cap analogue is increased, the initiation of in vitro transcription can be promoted, and the yield of mRNA is further improved. The nucleic acid of the TNA modified cap analogue can form a stable double-helix structure with a complementary strand through a base complementary pairing principle, so that the degradation of nuclease can be effectively resisted, the mRNA stability is further improved, the expression of protein is increased, and the translation efficiency of mRNA is improved.
Interpretation of the terms
"sugar ring" in the present invention includes, but is not limited to R 2 、R 3 、R 4 、R 5 、R 6 And R is 8 As defined in the present invention.
Detailed Description
Chemical synthesis part
Example 1: the method for synthesizing the initial capping oligonucleotide primer containing alpha-L-threose structure by using the intermediates A and B as raw materials, namely the TNA modified cap analogue, comprises the following steps:
intermediate A (2.0 mol) was suspended in a solution containing ZnCl 2 (20.0 mol) in DMF, then intermediate B (1.8 mol) was added to the reaction solution. After stirring the reaction at room temperature for 24 hours, 10L of 0.25M EDTA-Na was used 2 The solution terminated the reaction. The mixture was loaded onto a DEAE Sephadex column. The product is eluted by 0-1.0M sodium chloride aqueous solution in linear gradient, the salt is removed by nanofiltration, the target product is obtained by concentration, the reaction route flow is as follows
Wherein, the compound A is obtained by the following steps:
(1) Weigh 20.0g N 2 After isobutyryl- (alpha-L-threose) guanosine was dissolved in acetonitrile (10V), bis (2-cyanoethyl) -N, N-diisopropylphosphoramidite (1.8 eq.) and tetrazole (1.8 eq.) were added at room temperature, and stirred at room temperature under nitrogen atmosphere for 2 hours. Point plate detection N 2 -isobutyryl- (alpha-L-threose) guanosine has no residues. 70% aqueous t-butyl hydroperoxide (3.0 eq.) was added dropwise to the reaction solution and reacted at room temperature for 1 hour. After the spot plate detection reaction is finished, adding 5% sodium sulfite aqueous solution (10V), extracting by ethyl acetate (3 V.times.3), and spin-drying to obtain an intermediate crude product A1.
(2) The crude intermediate A1 was dissolved in methanol and concentrated ammonia (10V, 1:1), reacted at room temperature for 14 hours, and dried by spin after the completion of the reaction. The crude product is purified by reverse phase preparative chromatography and concentrated to give intermediate A2.
(3) Intermediate A2, triphenylphosphine (2.0 eq.), 2' -dithiodipyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen atmosphere for 15 hours. After the reaction is finished, slowly adding the reaction solution into 4M sodium perchlorate acetone solution, precipitating solid, and fully washing a filter cake with acetone after suction filtration to obtain an intermediate A3.
(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. After the reaction, 10 times of 0.25M EDTA-Na was used 2 The solution terminated the reaction. The reaction solution was purified by ion chromatography to give intermediate A4.
(5) Intermediate A4 was dissolved in 20 volumes of purified water, the reaction solution was cooled to 4 ℃, dimethyl sulfate (6.0 eq.) was slowly added dropwise, the pH was adjusted to no more than 5 with 2M sodium hydroxide during the reaction, the reaction was monitored by hplc, after completion of the reaction, the reaction was washed with dichloromethane and the aqueous phase was purified by ion chromatography to give intermediate A5.
(6) Intermediate A5, triphenylphosphine (2.0 eq.), 2' -dithiodipyridine (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 nitrogen atmosphere, after the reaction was completed, the reaction solution was slowly added to 4M sodium perchlorate acetone solution to precipitate a solid, which was filtered off with suction, and the filter cake was thoroughly washed with acetone to obtain intermediate a. Scheme compound a scheme as follows:
wherein, the compound B is obtained by the following steps:
(1) 200.0g of 2' OMe-rA phosphoramidite monomer and N were weighed out 2 Isobutyryl-2 ',3' -acetylguanosine (1.0 eq.) was dissolved in a single vial with 2.0L of dichloromethane. Tetrazole (2.1 eq.) was added under nitrogen and reacted at 25℃for 3 hours. After completion of the monitoring reaction, 70% t-butyl hydroperoxide aqueous solution was added dropwise to the reaction solution, and the reaction was carried out at 25℃for 1 hour. After completion of the monitoring reaction, a dichloromethane solution of trichloroacetic acid (4.0 eq.) was added dropwise to the reaction mixture, and the mixture was reacted at room temperature for 1 hour. After the completion of the monitoring reaction, the reaction mixture was washed with 10% aqueous sodium sulfite, 10% aqueous sodium bicarbonate and saturated brine, and the organic phase was concentrated and purified by column chromatography to give intermediate B1.
(2) Intermediate B1 was dissolved in acetonitrile (10V), and 1.8eq. Of bis (2-cyanoethyl) -N, N-diisopropylphosphoramidite, 1.8eq. Of tetrazole were added and stirred at room temperature for 2 hours under nitrogen atmosphere. After the completion of the reaction, a 70% aqueous t-butyl hydroperoxide solution (1.2 eq.) was added dropwise to the reaction mixture, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed, the mixture was monitored and dried by spin drying, methanol and concentrated ammonia water (10V, 1:1) were added to the spin flask, and the mixture was reacted at room temperature for 14 hours, and after the reaction was completed, the mixture was dried by spin drying. The crude product is purified by ion chromatography and then concentrated to obtain an intermediate B, and the reaction route flow is as follows:
example 2: the initial capping oligonucleotide primer containing alpha-L-threose structure and taking intermediates C and D as raw materials, namely the TNA modified cap analogue, is synthesized by the following steps:
the starting capped oligonucleotide primers of example 2 were obtained by reference to the synthesis of the target product of example 1, starting from intermediates C and D. The reaction route flow is as follows:
wherein, the compound C is obtained by the following steps:
(1) 5.0g of guanosine was weighed and dissolved in 50.0mL of trimethyl phosphate, and the reaction solution was cooled to 0℃and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under nitrogen atmosphere. After stirring at 0℃for 4 hours, the reaction was quenched by water. Most of the trimethyl phosphate was removed by washing twice with methylene chloride. Concentrating under reduced pressure to remove residual organic solvent, purifying with reversed phase preparative liquid chromatography, and concentrating to obtain intermediate C1.
(2) Intermediate C1, triphenylphosphine (2.0 eq.), 2' -dithiodipyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen for 15 hours. After the reaction is finished, slowly adding the reaction solution into 4M sodium perchlorate acetone solution, precipitating solid, and fully washing a filter cake with acetone after suction filtration to obtain an intermediate C2.
(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 C2 was added to the reaction solution in portions and stirred at room temperature for 5 hours. After the reaction, 10 times of 0.25M EDTA-Na was used 2 The solution terminated the reaction. The reaction solution was purified by ion chromatography to obtain intermediate C3.
(4) Intermediate C3 was dissolved in 20 volumes of purified water, the reaction solution was cooled to 4℃and dimethyl sulfate (6.0 eq.) was slowly added dropwise, the pH was adjusted to no more than 5 with 2M sodium hydroxide during the reaction, the reaction was monitored by HPLC, after completion of the reaction, the reaction was washed with dichloromethane, and the aqueous phase was purified by ion chromatography to give intermediate C4.
(5) Intermediate C4, triphenylphosphine (2.0 eq.), 2' -dithiodipyridine (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 nitrogen atmosphere, after the reaction was completed, the reaction solution was slowly added to 4M sodium perchlorate acetone solution to precipitate a solid, which was suction filtered, and the filter cake was thoroughly washed with acetone to obtain intermediate C. The reaction scheme is as follows
Compound C scheme, equation ():
wherein, the compound D is obtained by the following steps:
(1) 200.0g of N was weighed out 6 -benzoyl- (alpha-L-threose) adenosine phosphoramidite monomer and N 2 Isobutyryl-2 ',3' -acetylguanosine (1.0 eq.) was dissolved in a single vial with 2.0L of dichloromethane. Tetrazole (2.1 eq.) was added under nitrogen and reacted at 25℃for 3 hours. After completion of the monitoring reaction, 70% t-butyl hydroperoxide aqueous solution was added dropwise to the reaction solution, and the reaction was carried out at 25℃for 1 hour. After completion of the monitoring reaction, a dichloromethane solution of trichloroacetic acid (4.0 eq.) was added dropwise to the reaction mixture, and the mixture was reacted at room temperature for 1 hour. After the completion of the monitoring reaction, the reaction mixture was washed with 10% aqueous sodium sulfite, 10% aqueous sodium bicarbonate and saturated brine, and the organic phase was concentrated and purified by column chromatography to give intermediate D1.
(2) Intermediate D1 was dissolved in acetonitrile (10V), and 1.8eq. Of bis (2-cyanoethyl) -N, N-diisopropylphosphoramidite, 1.8eq. Of tetrazole were added and stirred at room temperature under nitrogen atmosphere for 2 hours. After the completion of the reaction, 70% of an aqueous t-butyl hydroperoxide solution was added dropwise to the reaction mixture, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed, the mixture was monitored and dried by spin drying, methanol and concentrated ammonia water (10V, 1:1) were added to the spin flask, and the mixture was reacted at room temperature for 14 hours, and after the reaction was completed, the mixture was dried by spin drying. The crude product is purified by ion chromatography and then concentrated to obtain an intermediate D, and the reaction route flow is as follows:
example 3 initial capping oligonucleotide primers containing an alpha-L-threose Structure starting from intermediates A and D, namely TNA modified cap analogues according to the invention, were synthesized as follows:
the starting capped oligonucleotide primers of example 3 were obtained by reference to the synthesis of the target product of example 1, starting from intermediates a and D. The reaction route flow is as follows:
EXAMPLE 4 Synthesis of threose Structure starting capped oligonucleotide primers Using intermediates E and B as starting materials
The starting capped oligonucleotide primers of example 4 were obtained by reference to the synthesis of the target product of example 1, starting from intermediates E and B. The reaction route flow is as follows:
wherein, the compound E is obtained by the following steps:
(1) 25.0g of (3 '-deoxy-3' -hydroxymethyl-furanose ring) guanosine was weighed and dissolved in 250.0mL of trimethyl phosphate, the reaction solution was cooled to 0℃and phosphorus oxychloride (1.8 eq.) was slowly added dropwise under nitrogen atmosphere. After stirring at 0℃for 4 hours, the reaction was quenched by water. Most of the trimethyl phosphate was removed by washing twice with methylene chloride. Concentrating under reduced pressure to remove residual organic solvent, purifying with reversed phase preparative liquid chromatography, and concentrating to obtain intermediate E1.
(2) Intermediate E1, triphenylphosphine (2.0 eq.), 2' -dithiodipyridine (2.0 eq.), imidazole (8.0 eq.), and triethylamine (1.0 eq.) were dissolved in DMF and stirred at room temperature under nitrogen for 15 hours. After the reaction is finished, slowly adding the reaction solution into 4M sodium perchlorate acetone solution, precipitating solid, and fully washing a filter cake with acetone after suction filtration to obtain an intermediate E2.
(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 E2 was added to the reaction mixture in portions and stirred at room temperature for 5 hours. Use 1 after the reaction0 times the volume of 0.25M EDTA-Na 2 The solution terminated the reaction. The reaction solution was purified by ion chromatography to obtain intermediate E3.
(4) Intermediate E3 was dissolved in 20 volumes of purified water, the reaction solution was cooled to 4℃and dimethyl sulfate (6.0 eq.) was slowly added dropwise, the pH was adjusted to no more than 5 with 2M sodium hydroxide during the reaction, the reaction was monitored by HPLC, after completion of the reaction, the reaction was washed with dichloromethane and the aqueous phase was purified by ion chromatography to give intermediate E4.
(5) Intermediate E4, triphenylphosphine (2.0 eq.), 2' -dithiodipyridine (2.0 eq.), imidazole (8.0
eq.) and triethylamine (1.0 eq.) are dissolved in DMF and stirred at room temperature for 10 hours under nitrogen atmosphere, after the reaction is completed, the reaction solution is slowly added into 4M sodium perchlorate acetone solution to precipitate solid, suction filtration is carried out, and the filter cake is fully washed with acetone to obtain intermediate E. The reaction scheme is as follows
Compound E scheme, equation ():
comparative example 1 Synthesis method of primer for initially capping oligonucleotides Using intermediates C and B as raw materials
The initial capping oligonucleotide primers of comparative example 1 were obtained by the synthesis method of the target product of reference example 1 using intermediates C and B as raw materials. The reaction route flow is as follows:
biological test part
1. Determination of mRNA in vitro transcription yield and capping efficiency
In vitro synthesis of mRNA using the TNA nucleoside cap analogues: linear endonuclease cleaves plasmids; extracting the linearized DNA template; mRNA is synthesized by in vitro transcription. The cap analogues of examples 1-4 and comparative example 1 were used as cap structures, respectively.
The reaction system is shown in Table 1:
TABLE 1 in vitro transcription reaction System
Remarks: in the experimental process, the volume of materials required by the system is calculated first, and then the sample is added. Firstly, adding sterile and sterile water into a system, then sequentially adding 10 Xbuffer, NTPs and cap analogues, mixing uniformly, lightly centrifuging, then adding nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase and linearized DNA template, fully mixing uniformly, lightly centrifuging, incubating at 37 ℃ for 2 hours, adding DNase I1U, continuously incubating at 37 ℃ for 30 minutes to remove the DNA template, and purifying mRNA by using a magnetic bead purification method. The purified mRNA was dissolved in sterile, enzyme-free water and the mRNA yield was measured using a Nanodrop One instrument.
Liquid chromatography mass spectrometry (LC-MS) was used to detect IVT capping rates of mRNA of different starting cap analogs; firstly, a section of DNA probe with a label matched with the initial base of mRNA of a transcription product needs to be designed, a general label is a biotin label, a streptavidin-labeled magnetic bead is washed and then incubated with the synthesized DNA probe, mRNA and 10X RNase H reaction buffer for 30 minutes at room temperature, the DNA probe, mRNA and 10X RNase H reaction buffer are slowly mixed while being incubated, and then 20ul RNase H (5U/ul) is added to incubate for 3 hours at 37 ℃ and uniformly mixed every half hour. After the incubation, the beads were washed, 100. Mu.L of 75% methanol heated to 80℃was added to the washed beads, the mixture was heated to 80℃on a hot plate, kept for 3 minutes, and the supernatant was then sucked up on a magnetic rack and dried at room temperature for 45 minutes to 10. Mu.L using an evaporation centrifuge. The sample was then resuspended in 50. Mu.l of 100. Mu.M EDTA/1% MeOH and used for LC-MS analysis to determine RNA capping during transcription. Since the capped and uncapped bases are significantly different in molecular weight, the difference in molecular mass can be used to determine the capping rate of mRNA transcription initiated by different cap analogs.
As can be seen from Table 2, the TNA nucleoside cap analogues of examples 1-4 were transcribed into the corresponding target mRNA, and the TNA nucleoside cap analogues of examples 1,2, and 4 were significantly superior in terms of yield and capping rate to comparative example 1.
TABLE 2 in vitro transcription yield and capping Rate of mRNA
Numbering device Yield (μg) Capping Rate (%)
Example 1 110 98.3
Example 2 108 96.5
Example 3 95 89.2
Example 4 118 95.3
Comparative example 1 98 93.5
2. Study of stability of uncapping enzyme on hat Structure
30pmol of RNAs purified by polyacrylamide gel electrophoresis PAGE are mixed with 50U of mRNA uncapping enzyme (New England Biolabs) and 1 XMDE buffer solution, and then the mixture is subjected to enzymatic reaction at 37 ℃ for 45min. The enzymatic reactions were subjected to PAGE electrophoresis, stained with SYBR Green II (Lonza), and the gel images after electrophoresis were visualized on a Typhoon FLA7000 (GE Healthcare) instrument. The ratio of intensity of the bands of capped RNA to uncapped RNA electrophoresis was counted using Image Quant (GE Healthcare) software, and the uncapping efficiency of the TNA structured cap analogues after treatment with uncapping enzyme (DCP 2 enzyme) was calculated. Statistical tests were performed using the Dunnett test of KaleidaGraph (Synergy) software. From the data in Table 3, it can be seen that the TNA structural cap analogues of examples 1,2,3,4 have significantly lower uncapping rates than comparative example 1 after treatment with uncapping enzyme (DCP 2 enzyme).
TABLE 3 detection of uncapping Rate after uncapping enzyme treatment
Numbering device Uncapping rate (%)
Example 1 7.2
Example 2 29.3
Example 3 10.5
Example 4 9.7
Comparative example 1 52.5
3. Detection of translation efficiency of mRNA
The in vitro transcription was performed using the eGFP coding sequence as a DNA template and the cap analogues of examples 1-4 and comparative example 1 as the starting material, and the in vitro transcribed mRNA was transfected into 293T cells.
293T cells were expressed as (0.5-1). Times.10 5 Individual cells were plated (24-well plate) and transfection experiments were recommended using cells within 50 passages. Cells were required to be passaged again 24 hours prior to transfection, and the addition of antibiotics to the medium had no effect on the transfection effect. The cell density at transfection is generally 60-80% and 2. Mu.g mRNA per well is transfected, and the transfection reagent is selected from Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and is manipulated by reference to its method of use. The transfected cells were placed at 37℃and CO 2 In the incubator, after 4-6 hours of transfection, the medium was replaced with fresh complete medium. CO at 37 DEG C 2 After incubation for 24 hours in the incubator, the fluorescence intensity of GFP was observed by a fluorescence microscope.
The results are shown in Table 4. The mRNA synthesized by in vitro transcription of examples 1,2,4 was significantly more efficiently translated into protein in the cell than comparative example 1, while none caused significant cell death. The above experimental data indicate that the TNA-containing structural cap analogs of the present application can be used with high efficacy for in vitro transcription of mRNA and intracellular protein expression.
TABLE 4 translation expression efficiency of mRNA in cells

Claims (8)

1. A TNA modified cap analogue, characterized in that the TNA modified cap analogue corresponds to the following structural general formula:
，
wherein R is a Is thatOr->；R b Is->Or->；
When R is a Is thatWhen Rb is not->The method comprises the steps of carrying out a first treatment on the surface of the When R is b Is->When R is a Not be->；
R 1 Is that；
m and n are each independently 0, 1;
R 2 、R 3 、R 4 and R is 5 Are each independently hydrogen, hydroxy, halogen, C 1-5 Alkyl, O-C 1-5 An alkyl group;
R 6 is H, OH, NH 2 、C 1-5 Alkyl, O-C 1-5 An alkyl group;
R 7 、R 8 are respectively and independentlyHydrogen, hydroxy, halogen, C 1-5 An alkyl group;
the Ra and Rb must have at least one sugar ring attached to the triphosphate segment through the 3' position;
B 1 and B 2 Each independently a natural nucleobase.
2. The TNA modified cap analogue according to claim 1, characterised in that R a Or R is b Is linked to the triphosphate segment through the 3' position; the other sugar ring having hydroxy groups or O-C groups at the 2',3' and/or 4' positions 1-5 An alkyl group.
3. The TNA modified cap analogue according to claim 1, characterised in that in the structure of the TNA modified cap analogue R 2 、R 3 、R 4 And R is 5 Are each independently hydrogen, C 1-5 Alkyl, hydroxy, O-C 1-5 An alkyl group.
4. The TNA modified cap analogue according to claim 1, characterised in that in the structure of the TNA modified cap analogue R 6 Is hydrogen, C 1-5 Alkyl, hydroxy, O-C 1-5 An alkyl group.
5. The TNA modified cap analogue according to claim 1, characterised in that in the structure of the TNA modified cap analogue R 7 、R 8 Are each independently hydrogen, C 1-5 Alkyl or hydroxy.
6. A TNA modified cap analogue, characterised in that the TNA modified cap analogue is selected from any one of the following:
；
；
；
。
7. use of the TNA modified cap analogue according to any of claims 1-6, characterised in that: for in vitro transcription of linear mRNA, RNA was capped.
8. The use according to claim 7, characterized by the specific steps of:
step (1): preparing a DNA template;
step (2): performing an in vitro transcription reaction comprising an RNA polymerase, a nucleoside triphosphate and a TNA modified capping analogue according to any one of claims 1 to 6.
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WO2022051677A1 (en) * 2020-09-04 2022-03-10 Verve Therapeutics, Inc. Compositions and methods for capping rnas
CN115109110A (en) * 2022-06-22 2022-09-27 江苏申基生物科技有限公司 Initial capped oligonucleotide primer containing hexa-membered sugar ring structure and preparation method and application thereof
CN116143854A (en) * 2022-11-08 2023-05-23 江苏申基生物科技有限公司 Ribose ring modified mRNA cap analogue and preparation method and application thereof

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WO2022051677A1 (en) * 2020-09-04 2022-03-10 Verve Therapeutics, Inc. Compositions and methods for capping rnas
CN115109110A (en) * 2022-06-22 2022-09-27 江苏申基生物科技有限公司 Initial capped oligonucleotide primer containing hexa-membered sugar ring structure and preparation method and application thereof
CN116143854A (en) * 2022-11-08 2023-05-23 江苏申基生物科技有限公司 Ribose ring modified mRNA cap analogue and preparation method and application thereof

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