PolyA tail element, construction body and application of polyA tail element

Abstract

The invention discloses a polyA tail element, a construction body and application of the polyA tail element and the construction body. The polyA tail element is inserted into a sequence capable of mutually complementarily pairing basic groups behind A at any one of 27th to 33rd and 85th to 91st in the direction from 5'to 3 '; or the basic group at the multiple site of the 12th, 13th, 14th or 15th of the polyA tail element is C. The polyA tail element disclosed by the invention is high in correct cloning rate and good in passage stability, and can be used for fermentation production of mRNA template plasmids; a construct or a recombinant expression vector containing the gene has strong stability and high translation efficiency, and can efficiently express a target protein; and a new template plasmid can be quickly transformed and constructed for transcriptional production of new mRNA, so that the production time and cost are saved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polyA tail element, its construct, and its application. Background Technology

[0002] Messenger RNA (mRNA) is a key molecule in the central dogma of heredity. It binds to ribosomes in the cytoplasm, translates to produce target proteins, and performs specific biological functions, making it an essential biomolecule for maintaining life. In recent years, artificially synthesized mRNA drugs have been developed as new and powerful therapeutic tools. As a reservoir of genetic information in post-transcriptional regulation, multiple regions of mRNA play a regulatory role in protein translation. Generally, the structural elements of mRNA are, in order: the 5' cap structure, the 5' untranslated region (5'UTR), the coding sequence (CDS), the 3' UTR, and the 3' poly(A) tail. Figure 1 As shown, reporter gene luciferase (Luc) or enhanced green fluorescent protein (eGFP) are used as CDS examples to illustrate the above mRNA structures. Specifically, the 5' Cap resists degradation by 5'-exonucleases, promotes mRNA stability and translation efficiency; the UTR regulates mRNA transport, translation efficiency, and subcellular localization; and the 3' poly(A) tail prevents 3'-exonuclease degradation of mRNA, enhances its stability, and simultaneously promotes mRNA nuclear export efficiency and target protein translation efficiency.

[0003] The integrity and uniformity of the 3' ploy(A) tail are key indicators for evaluating the efficacy of mRNA drugs. In the in vitro transcription (IVT) synthesis of mRNA drugs, there are two methods for adding the 3' ploy(A) tail: one is enzymatic synthesis, where mRNA is transcribed from linear double-stranded DNA or PCR product templates in vitro, and then the ploy(A) tail is added to the 3' end of the mRNA using ploy(A) polymerase; the other is co-transcription, where the ploy(A) tail already present on the template plasmid DNA or PCR product is directly transcribed. Because co-transcription simplifies the IVT synthesis process and significantly shortens production time, it has become the mainstream method for mRNA production. Correspondingly, the fermentation and replication of template plasmids by *E. coli* has become one of the key upstream steps in mRNA drug production. Stable, replicable, and passaged template plasmids are crucial for the yield and purity of mRNA synthesized in the IVT reaction and for ensuring the in vivo translation rate of mRNA. The ploy(A) tail constructed on conventional template plasmids typically consists of 100-120 consecutive A bases. However, this continuous A tail leads to sequence instability during plasmid DNA replication and passage, frequently resulting in A base loss in the ploy(A) tail of progeny plasmids obtained through fermentation replication. This causes tail shortening and poor integrity, affecting the uniformity of mRNA produced through subsequent transcription and protein translation efficiency, ultimately compromising the druggability of mRNA drugs. Furthermore, designing, screening, and constructing mRNA template plasmids from scratch is time-consuming and costly. Providing a ready-made mRNA template plasmid that can be quickly used to change different CDS and transcribe different mRNAs can significantly save production time and costs. Summary of the Invention

[0004] To address the following issues in existing technologies: 1. The 3' poly(A) tail of mRNA template plasmids is prone to base loss during fermentation production, resulting in poor passage stability; 2. The 3' poly(A) tail of mRNA produced by existing technologies affects the uniformity and translation efficiency of mRNA; 3. Existing technologies involve long construction times and high costs for de novo construction of mRNA template plasmids, this invention provides a polyA tail element, its construct, and its application.

[0005] The objectives of this invention are as follows: 1. To design and provide novel mRNA sequences with high stability and translation efficiency, enabling them to efficiently express target proteins and ensuring the drug-likeness of mRNA; 2. To design and provide novel mRNA template plasmids that can be stably fermented and passaged, preventing base loss of the 3' poly(A) tail during plasmid replication, improving the correct cloning rate of the poly(A) tail, and effectively ensuring the passage stability of the mRNA template plasmid during fermentation; 3. To provide a model mRNA template plasmid as a ready-made modification template, which can be used to obtain new mRNA template plasmids and new mRNAs transcribed from it by replacing the target protein coding sequence in the CDS region, saving production time and costs.

[0006] To solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a polyA tail element, wherein the polyA tail element contains the sequence "GCUCUUCAC" or "GUGAAGAGC" after any of the 27th to 33rd positions (e.g., positions 27, 28, 29, 30, 31, 32, or 33) of consecutive A's from 5' to 3', and contains a sequence after any of the 85th to 91st positions (e.g., positions 85, 86, 87, 88, 89, 90, or 91) that is complementary to the sequence after any of the 27th to 33rd positions (e.g., positions 27, 28, 29, 30, 31, 32, or 33); the polyA tail element contains a ... The polyA tail element contains the sequence “GCUCUUCAC” or “GUGAAGAGC” after any of the 85th to 91st positions (e.g., positions 85, 86, 87, 88, 89, 90, or 91) of consecutive A's in the 5' to 3' direction, and after any of the 27th to 33rd positions (e.g., positions 27, 28, 29, 30, 31, 32, or 33), it contains a sequence that can complement the sequence after any of the 85th to 91st positions (e.g., positions 85, 86, 87, 88, 89, 90, or 91); or, the polyA tail element has a C base at multiples of the 12th, 13th, 14th, or 15th positions in the 5' to 3' direction.

[0007] In this invention, the term "polyA tail element" also refers to the "3' poly(A) tail," "3' poly(A) tail," or "poly(A) tail" as commonly understood in the art. The 3' poly(A) tail is a structural feature of the 3' end of eukaryotic mRNA molecules, typically composed of several hundred (e.g., 100–120) consecutive A (adenine) residues. This polyadenylation tail forms during mRNA maturation and plays a crucial regulatory role in mRNA stability, nucleocytoplasmic transport, translation efficiency, and degradation. Therefore, in this invention, except for the aforementioned definition of a specific sequence ("GCUCUUCAC" or "GUGAAGAGC") or its complementary pair following an A, or a C (cytosine) base, it should be understood that all other bases in the polyA tail element of this invention are A.

[0008] In this invention, the two sequences following the A at any position 27-33 and any position 85-91 can form a hairpin structure through complementary base pairing. Therefore, those skilled in the art will understand that as long as the A at any position 27-33 contains "GCUCUUCAC" or "GUGAAGAGC" (although only one of these sequences is exemplified in the embodiments of this invention, these two sequences can be interchanged), and the sequence following the A at any position 85-91 can form a hairpin structure through complementary base pairing (as is known in the art, the formation of a hairpin structure usually requires at least 4 consecutive complementary bases, so satisfying 4 or more consecutive complementary bases is sufficient), the technical effects of this invention can be expected to be achieved.

[0009] In this invention, the site regions selected at positions 27-33 and 85-91 are advantageous site regions that the inventors have determined through long-term experimentation to be able to form a large hairpin structure while ensuring stability without affecting translation efficiency. Although the examples given in the embodiments of this invention are located at positions 30 and 88 respectively, if the sequence of the polyA tail element is longer or shorter, the insertion site can be moved forward or backward accordingly.

[0010] In this invention, "any one of the 85th to 91st positions" refers to the site region calculated before the insertion sequence at positions 27 to 33 (i.e., which A position corresponds to the initial consecutive A base sequence). Taking mA30hairpin as an example in this embodiment, the 88th position refers to the 88th A base in the initial sequence of 100 consecutive A bases, which corresponds to the 97th (i.e., 88+9) position after the insertion sequence at position 30.

[0011] In this invention, "the base at the 12th, 13th, 14th, or 15th multiple position is C" can be understood as follows: from the 5' to the 3' direction, every 12, 13, 14, or 15 A's are divided into a unit, and then the last A' is replaced with C, with the polyA tails spaced out. Taking mA15pureC in the embodiment of this invention as an example, the base at any multiple of 15 (i.e., the 15th, 30th, 45th, 60th, 75th, and 90th positions) is C.

[0012] In a specific embodiment of the present invention, the polyA tail element satisfies one or more of the following conditions:

[0013] (1) The polyA tail element comprises 90 to 120 A's;

[0014] (2) The sequence “GCUCUUCAC” is contained after any A in the 27th to 33rd positions;

[0015] (3) The sequence “GUGAAGAGC” is contained after any A in positions 85 to 91;

[0016] (4) Any one of the 27th to 33rd positions is the 30th position;

[0017] (5) Any one of the 85th to 91st positions is the 88th position; and,

[0018] (6) The 3' end of the polyA tail element contains 12 to 15 consecutive A's.

[0019] To solve the above-mentioned technical problems, the second technical solution provided by the present invention is: a polyA tail element, wherein the polyA tail element comprises a nucleotide sequence as shown in SEQ ID NO:5 or SEQ ID NO:4.

[0020] In a specific embodiment of the present invention, the nucleotide sequence of the polyA tail element is shown in SEQ ID NO:5 or SEQ ID NO:4.

[0021] To solve the above-mentioned technical problems, the third technical solution provided by the present invention is: a nucleic acid isolated from the polyA tail element as described in the first or second technical solution.

[0022] In this invention, "transcribed nucleic acid" refers to DNA, which is transcribed from one strand of the DNA as a template according to the base pairing principle to generate the corresponding mRNA.

[0023] In a specific embodiment of the present invention, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO:10 or SEQ ID NO:9.

[0024] In a specific embodiment of the present invention, the sequence of the nucleic acid is shown as SEQ ID NO:10 or SEQ ID NO:9.

[0025] To solve the above-mentioned technical problems, the fourth technical solution provided by the present invention is: a construct of an mRNA transcription template, wherein the construct comprises, from 5' to 3', nucleic acid for transcribing the 5'UTR region, nucleic acid for transcribing the 3'UTR region, and nucleic acid as described in the third technical solution.

[0026] In this invention, the sequences of the 5'UTR and 3'UTR regions can be selected according to conventional practices in the art. The nucleic acids in the 5'UTR and 3'UTR regions of the mRNA transcription template construct of this invention can be directly ligated (in subsequent applications, the target DNA fragment (e.g., a CDS sequence (Coding Sequence, a region in a DNA sequence that encodes a protein), restriction enzyme site, or multiple cloning site (MCS)) can be directly inserted between the nucleic acids in the 5'UTR and 3'UTR regions using genetic engineering techniques such as seamless cloning and homologous recombination).

[0027] Optionally, the nucleotides transcribed in the 5'UTR region and the nucleotides transcribed in the 3'UTR region may also contain a multiple cloning site and / or a CDS sequence.

[0028] In this invention, when inserting a target DNA fragment using enzyme digestion, a multiple cloning site can be inserted between the 5'UTR and 3'UTR nucleic acids. This multiple cloning site includes, but is not limited to, the sequence mentioned in the embodiments of this invention, and may also be composed of other multiple enzyme digestion sites.

[0029] In a specific embodiment of the present invention, the 5'UTR region and / or the 3'UTR region are derived from the UTR sequence of the human endogenous β-globin gene (e.g., Sequence ID: AH001475.2); and / or, the multiple cloning site comprises one or more restriction enzyme sites selected from SacⅠ, NcoⅠ, NotⅠ, and XhoⅠ.

[0030] In a specific embodiment of the present invention, the sequence of the nucleic acid transcribed in the 5'UTR region is as shown in SEQ ID NO:6 and / or the sequence of the nucleic acid transcribed in the 3'UTR region is as shown in SEQ ID NO:7; and / or, the nucleotide sequence of the multiple cloning site is as shown in SEQ ID NO:13.

[0031] In a specific embodiment of the present invention, the CDS sequence is a Luciferase gene sequence or an eGFP gene sequence. The Luciferase gene sequence is preferably as shown in SEQ ID NO:11, and the eGFP gene sequence is preferably as shown in SEQ ID NO:12.

[0032] In this invention, the Luciferase gene sequence or the eGFP gene sequence is used as an example of the CDS sequence, but those skilled in the art will understand that the CDS sequence suitable for this invention can be any DNA sequence, as long as it contains the genetic information required for the synthesis of the target protein.

[0033] To solve the above-mentioned technical problems, the fifth technical solution provided by the present invention is: an isolated nucleic acid, which is mRNA, wherein the nucleic acid comprises, from 5' to 3', a 5'UTR region, a 3'UTR region and a polyA tail element as described in one or two technical solutions; optionally, the 5'UTR region and the 3'UTR region further comprise mRNA complementary to the multiple cloning site and / or CDS sequence.

[0034] In a specific embodiment of the present invention, the 5'UTR region and / or the 3'UTR region are derived from the UTR sequence of human endogenous β-globin; and / or, the multiple cloning site includes one or more restriction enzyme sites selected from SacⅠ, NcoⅠ, NotⅠ, and XhoⅠ.

[0035] In a specific embodiment of the present invention, the nucleotide sequence of the 5'UTR region is as shown in SEQ ID NO:1 and / or the nucleotide sequence of the 3'UTR region is as shown in SEQ ID NO:2; and / or, the nucleotide sequence of the multiple cloning site is as shown in SEQ ID NO:13.

[0036] In a specific embodiment of the present invention, the CDS sequence is a Luciferase gene sequence or an eGFP gene sequence. The Luciferase gene sequence is preferably as shown in SEQ ID NO:11, and the eGFP gene sequence is preferably as shown in SEQ ID NO:12.

[0037] In a specific embodiment of the present invention, the mRNA further comprises a 5' Cap, such as m7(2'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, or other cap analogs. The 5' Cap can be added using enzymatic or co-transcriptional methods.

[0038] To solve the above-mentioned technical problems, the sixth technical solution provided by the present invention is: a recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid as described in the third technical solution or the construct as described in the fourth technical solution.

[0039] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is pMV (its map is shown below). Figure 10 As shown, its full-length sequence is, for example, shown in SEQ ID NO:16; and / or, the polyA tail element further includes an enzyme cleavage site, such as BsaⅠ.

[0040] In this invention, the backbone of the recombinant expression vector includes, but is not limited to, the pMV plasmid in the embodiments of this invention, or other plasmids, granules, bacteriophages or viral vectors with RNA polymerase promoters.

[0041] In this invention, a BsaⅠ restriction site is inserted after the ploy(A) tail to prepare an IVT linearized DNA template. This restriction site includes, but is not limited to, BsaⅠ, and may also be other restriction sites or multiple restriction sites.

[0042] To solve the above-mentioned technical problems, the seventh technical solution provided by the present invention is: a transformant, the transformant comprising the polyA tail element as described in technical solution one or technical solution two, the nucleic acid as described in technical solution three, the construct as described in technical solution four, the nucleic acid as described in technical solution five, or the recombinant expression vector as described in technical solution six; the host cell of the transformant is a eukaryote or a prokaryote.

[0043] In this invention, the host cells used include, but are not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, or eukaryotic cells such as yeast, plants, insects and animals.

[0044] In a specific embodiment of the present invention, the prokaryote is Escherichia coli, such as Stbl2.

[0045] To solve the above-mentioned technical problems, the eighth technical solution provided by the present invention is: a kit comprising the nucleic acid as described in the third technical solution, the construct as described in the fourth technical solution, the recombinant expression vector as described in the sixth technical solution, or the transformant as described in the seventh technical solution.

[0046] The kit of this invention can be widely used to guide the design, development and production of RNA transcription templates for prokaryotes or eukaryotes.

[0047] To solve the above-mentioned technical problems, the ninth technical solution provided by the present invention is: a pharmaceutical composition comprising nucleic acid as described in the fifth technical solution, and a pharmaceutically acceptable carrier.

[0048] In a specific embodiment of the present invention, the CDS sequence encodes a drug protein or polypeptide.

[0049] In this invention, the term "drug protein or polypeptide" refers to a biological macromolecular protein or polypeptide (e.g., an antigen or antibody associated with a specific disease) that has the effect of preventing, diagnosing, and / or treating diseases.

[0050] In this invention, the pharmaceutical composition comprises a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing the mRNA of the invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0051] To solve the above-mentioned technical problems, the tenth technical solution provided by the present invention is: the use of the polyA tail element as described in technical solution one or technical solution two, the nucleic acid as described in technical solution three, the construct as described in technical solution four, the nucleic acid as described in technical solution five, the recombinant expression vector as described in technical solution six, the transformant as described in technical solution seven, the kit as described in technical solution eight, or the pharmaceutical composition as described in technical solution nine in the preparation of mRNA or products containing the thereof.

[0052] In a specific embodiment of the present invention, the product is an mRNA vaccine or an mRNA therapeutic drug.

[0053] To solve the above-mentioned technical problems, the eleventh technical solution provided by the present invention is: the application of nucleic acid as described in technical solution three, construct as described in technical solution four, recombinant expression vector as described in technical solution six, transformant as described in technical solution seven, or kit as described in technical solution eight in the preparation of mRNA.

[0054] The nucleic acid, mRNA transcription template, or recombinant expression vector of the present invention that transcribes the polyA tail element can be used as a template to transcribe and produce mRNA. The method of transcription to produce mRNA includes, but is not limited to, in vitro transcription. The transcription raw material nucleotide NTPs include, but are not limited to, natural or artificially synthesized, modified or unmodified NTPs.

[0055] To solve the above-mentioned technical problems, the twelfth technical solution provided by the present invention is: the application of the polyA tail element as described in technical solution one or technical solution two, the nucleic acid as described in technical solution three, the construct as described in technical solution four, the nucleic acid as described in technical solution five, the recombinant expression vector as described in technical solution six, the transformant as described in technical solution seven, the kit as described in technical solution eight, or the pharmaceutical composition as described in technical solution nine in the preparation of products containing mRNA.

[0056] In a specific embodiment of the present invention, the product is an mRNA vaccine or an mRNA therapeutic drug.

[0057] mRNA prepared by transcription using the polyA tail element of the present invention—a construct or recombinant expression vector of nucleic acid or mRNA transcription template—has high translation efficiency and can be widely used in the production of mRNA vaccines, mRNA therapeutics, and other formulations. Examples of delivery systems used include, but are not limited to, polymers, exosomes, liposomes, and emulsions.

[0058] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0059] The reagents and raw materials used in this invention are all commercially available.

[0060] The positive and progressive effects of this invention are as follows:

[0061] This invention can rapidly and effectively facilitate the design of mRNAs for the production of their encoded target proteins or for gene regulation. This invention designs and optimizes the 3'ploy(A) tail sequence, successfully obtaining mRNA sequences with high translation efficiency, which can efficiently guide mRNA production. Both the A15pureC and A30hairpin ploy(A) tail sequences significantly promote mRNA translation efficiency, with A30hairpin showing a better promoting effect. Furthermore, the novel mRNA template plasmids designed and optimized in this invention exhibit high passage stability during fermentation; their ploy(A) tails showed no significant base loss after 15 generations of continuous plate culture. The correct cloning rate of the ploy(A) tail of the A15pureC sequence reached 91.2%-92.5%, while the correct cloning rate of the ploy(A) tail of the A30hairpin sequence reached 100%. In addition, this invention provides a ready-made modified template for the construction of mRNA template plasmids, which can replace the CDS region sequence to quickly obtain new mRNA template plasmids for subsequent mRNA transcription production, greatly saving production time and cost. Attached Figure Description

[0062] Figure 1 This is a schematic diagram of the mRNA structure.

[0063] Figure 2 This is an electrophoresis image of an mRNA template plasmid.

[0064] Figure 3 The results are statistical results of passage sequencing of the non-ploy(A) tail region of the mRNA template plasmid.

[0065] Figure 4 The statistical results of base loss rate (a) and stability (b) of the tail region of the mRNA template plasmid ploy (A) are presented.

[0066] Figure 5 This is a congealed electrophoresis image of a plasmid template that can transcribe Luciferase mRNA and eGFP mRNA.

[0067] Figure 6 Electrophoresis diagram of the purified products of a single enzyme digestion of a plasmid template plasmid that can transcribe Luciferase mRNA and eGFP mRNA.

[0068] Figure 7 Purified mRNA products that can translate Luciferase and eGFP were synthesized for IVT.

[0069] Figure 8 The fluorescein MFI values ​​were detected for each Luciferase mRNA transfection group.

[0070] Figure 9 Fluorescence microscopy images (a) and MFI values ​​(b) of each eGFP mRNA transfection group.

[0071] Figure 10 This is a pMV plasmid map. Detailed Implementation

[0072] This invention mainly involves the following experimental steps:

[0073] Step 1: mRNA sequence design

[0074] The mRNA sequence components designed in this invention include 5'Cap, 5'UTR, CDS, 3'UTR, and 3'ploy(A) tail, as shown in Table 1. The 5'Cap is capped using enzymatic or co-transcription methods and does not need to be reflected in the sequence design process.

[0075] Based on the different 3'ploy(A) sequences, three RNA sequences can be obtained: mA100-mRNA, mA15pureC-mRNA, and mA30hairpin-mRNA.

[0076] Step 2: Design of mRNA template sequence and construction of template plasmid

[0077] The mRNA template sequence designed in this invention is a DNA sequence obtained by replacing "U" with "T" in the 5'UTR, CDS, 3'UTR and poly(A) tail regions of the mRNA. The specific sequence is shown in Table 2.

[0078] A BsaⅠ restriction site was inserted after all ploy(A) tails to prepare IVT linearized DNA templates for single digestion of the plasmid. Simultaneously, a multiple cloning site was inserted between the 5'UTR and 3'UTR of the template plasmid. This site allows for rapid insertion or replacement of the CDS sequence via restriction enzyme digestion and ligation, resulting in three mRNA template DNA sequences: dA100-mRNA, dA15pureC-mRNA, and dA30hairpin-mRNA.

[0079] The designed mRNA template DNA sequence was obtained using whole-genome synthesis technology and constructed into the T7 promoter of the cloning vector plasmid pMV to obtain the corresponding recombinant plasmid. The plasmid was then subjected to E. coli transformation culture, single-clone colony PCR, and sequencing of positive clones to screen for template plasmids with correct sequences.

[0080] Step 3: Passage stability test of mRNA template plasmid

[0081] The correctly sequenced template plasmids dA100-mRNA / pMV, dA15pureC-mRNA / pMV, and dA30hairpin-mRNA / pMV were transformed into *E. coli* and cultured overnight on plates to obtain generation P0. Ten uniformly sized single colonies were randomly selected, numbered, and passaged continuously to generation P15 using plate culture. Ten single colonies from generations P1, P5, P10, and P15 were sequenced, and the sequencing results of the mRNA template region were analyzed to determine the fermentation and passage stability of the mRNA template plasmid.

[0082] Step 4: Replacement of CDS region sequence in mRNA template plasmid

[0083] The Luciferase and eGFP gene sequences were obtained using whole-genome synthesis technology, and then constructed into the CDS region of the mRNA template plasmid through enzyme digestion and ligation. The recombinant plasmid was transformed into Enterobacter spp. Stbl2 cells, and single-clone colony PCR and positive clones were sequenced to screen out the template plasmids Luc-dA100-mRNA / pMV, Luc-dA15pureC-mRNA / pMV, Luc-dA30hairpin-mRNA / pMV, and eGFP-dA100-mRNA / pMV, eGFP-dA15pureC-mRNA / pMV, and eGFP-dA30hairpin-mRNA / pMV with correct sequences that can transcribe Luciferase mRNA and eGFP mRNA.

[0084] Step 5: In vitro synthesis of mRNA via co-transcription method

[0085] The above recombinant template plasmids were extracted, digested with BsaI, and purified to obtain purified linearized template DNA, which was used for IVT transcription to produce the corresponding Luciferase mRNA and eGFP mRNA.

[0086] Step 6: Validation of mRNA expression in vitro

[0087] The purified Luc-mA100-mRNA, Luc-mA15pureC-mRNA, Luc-mA30hairpin-mRNA, and eGFP-mA100-mRNA, eGFP-mA15pureC-mRNA, and eGFP-mA30hairpin-mRNA were transfected into Hek293T cells, respectively. Twenty-four hours after transfection, the expression levels of Luciferase or eGFP were measured to determine the translation efficiency of these mRNAs.

[0088] The technical solution of the present invention will be described in detail below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments, in order to illustrate the present invention. Therefore, the embodiments provided below can serve as a guide for those skilled in the art to make further improvements and optimizations, but do not constitute a limitation on the present invention in any way.

[0089] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to common techniques or conditions described in the literature in this field, or according to the product instructions. Also, unless otherwise specified, the reagents, materials, instruments, and equipment used in the examples are commercially available. The experiments in the final examples used three replicates, and the results are shown as mean ± standard deviation. Two-way ANOVA with multiple comparisons was used to analyze statistical differences in the data. "*" means p < 0.05, indicating a statistically significant difference; "**" means p < 0.01, indicating a highly statistically significant difference; and "***" means p < 0.001, indicating an extremely statistically significant difference.

[0090] Example 1: Design of mRNA sequences

[0091] The typical mRNA components include 5'Cap, 5'UTR, CDS, 3'UTR, and 3'ploy(A). The 5'Cap is added using enzymatic or co-transcriptional methods and does not need to be included in the mRNA sequence design. The specific sequences of each component in the designed mRNA sequence are shown in Table 1. The 5'UTR is named m5'UTR, and its sequence is shown in SEQ ID NO:1; the CDS can be any RNA sequence; the 3'UTR is named m3'UTR, and its sequence is shown in SEQ ID NO:2; the control ploy(A) tail sequence mA100 is an unoptimized sequence of 100 consecutive A bases, and its sequence is shown in SEQ ID NO:3; the preferred ploy(A) tail sequence 1 is called mA15pureC, which is formed by replacing the A bases at positions 15, 30, 45, 60, 75, and 90 of the A100 sequence with C bases, and its sequence is shown in SEQ ID NO:3. As shown in NO:4, the substituted C bases are indicated in bold and italics; the preferred ploy(A) tail sequence 2, called mA30hairpin, is formed by inserting a 9bp sequence “GCUCUUCAC” after the 30th A base of the A100 sequence, inserting a 9bp hairpin sequence “GUGAAGAGC” after the 88th A base, and adding 3 more A bases at the end. Its sequence is shown in SEQ ID NO:5, with the inserted sequences indicated in bold and underlined. Depending on the 3' ploy(A) sequence, three RNA sequences can be obtained: mA100-mRNA, mA15pureC-mRNA, and mA30hairpin-mRNA.

[0092] Table 1. Design of mRNA sequence components

[0093]

[0094] Example 2: Design of mRNA template sequence and construction of template plasmid

[0095] Step 1: mRNA template DNA sequence design

[0096] Replacing the "U" in each component sequence of the mRNA with the "T" in the DNA sequence yields the corresponding mRNA template DNA sequence. The specific sequences of each component in the obtained mRNA template sequence are shown in Table 2. The 5'UTR is referred to as d5'UTR, as shown in SEQ ID NO:6; the 3'UTR is referred to as d3'UTR, as shown in SEQ ID NO:7; the control ploy(A) tail sequence dA100 is shown in SEQ ID NO:8; and the candidate ploy(A) tail sequences dA15pureC and dA30hairpin are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. A BsaⅠ restriction site (5'GGTCTCN3') is inserted after all ploy(A) tails for single-enzyme digestion of the plasmid to prepare an IVT linearized DNA template for mRNA production. Simultaneously, a 40 bp multiple cloning site (MCS) sequence 5'GAGCTCCTCAGCT is inserted between the 5'UTR and 3'UTR of the template plasmid. CCATGG TCAT GCA (SEQ ID NO:13), introducing restriction enzyme sites SacⅠ (GAGCTC) and NcoⅠ ( CCA TGG), NotⅠ and XhoⅠ Rapid insertion can be achieved through enzyme digestion and ligation. Alternatively, the CDS sequence can be replaced to construct a new mRNA template. Based on the different 3'ploy(A) sequences, three mRNA template DNA sequences can be obtained: dA100-mRNA, dA15pureC-mRNA, and dA30hairpin-mRNA.

[0097] Step 2: Construction and screening of mRNA template plasmids

[0098] The designed mRNA template DNA sequence was modified by adding restriction enzyme sites Xba I (TCTAGA) and Hind III (AAGCTT) to its head and tail, respectively. The entire genome was synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and then inserted into the self-constructed cloning vector plasmid pMV (its map is shown below). Figure 10As shown in SEQ ID NO:16, the MCS region following the T7 RNA polymerase promoter yielded three different recombinant plasmids corresponding to different ploy(A) tail sequences: dA100-mRNA / pMV, dA15pureC-mRNA / pMV, and dA30hairpin-mRNA / pMV. The three recombinant plasmids were transformed into *E. coli* competent cells Stbl2 and plated on Amp... + After overnight incubation at 37°C on LB plates, single clones were picked from the plates for colony PCR. Positive clones were sent for sequencing, and template plasmids with the correct sequences were selected.

[0099] The correct template plasmids dA100-mRNA / pMV, dA15pureC-mRNA / pMV, and dA30hairpin-mRNA / pMV contain the corresponding 5'UTR and 3'UTR sequences as shown in SEQ ID NO:6 and SEQ ID NO:7, respectively, while their 3'ploy(A) tail sequences are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. Figure 2 The results of 1.2% agarose gel electrophoresis of mRNA template plasmids dA100-mRNA / pMV (2709bp), dA15pureC-mRNA / pMV (2709bp), and dA30hairpin-mRNA / pMV (2730bp) are shown.

[0100] Table 2. Design of mRNA template DNA sequence components

[0101]

[0102] Example 3: Stability test of mRNA template plasmid after passage

[0103] Step 1: Subculturing of mRNA template plasmids

[0104] The correctly sequenced template plasmids dA100-mRNA / pMV, dA15pureC-mRNA / pMV, and dA30hairpin-mRNA / pMV were transformed into E. coli Stbl2 and plated on Amp. + After overnight incubation on LB plates, P0 generation transformation plates were obtained. Ten uniformly sized single colonies were randomly selected from each plate, numbered, and then inoculated onto the correspondingly numbered Amp plates using a 10 μL sterile pipette tip. + The cells were cultured overnight at 37°C on LB plates to obtain generation P1. The cells were then continuously subcultured using the same method described above until generation P15.

[0105] Step 2: Sample delivery, sequencing, and result analysis of mRNA template plasmids

[0106] Ten single colonies from generations P1, P5, P10, and P15 were all sent for sequencing of the mRNA template region using the universal primer pair M13F / M13R (Table 3). Generally, non-highly repetitive regions of plasmids exhibit good replication stability and are less prone to mutations such as base loss, substitution, and insertion. However, the highly repetitive A sequence in the ploy(A) tail region makes it susceptible to base deletion during replication. Therefore, the sequencing results of the non-ploy(A) tail region and the ploy(A) tail region in the plasmid were statistically analyzed separately. The statistical analysis results of the passage sequencing of the non-ploy(A) tail region of the mRNA template plasmid are shown in [Table 3]. Figure 3 The results showed that, up to the P15 generation, none of the 10 single colonies submitted for testing, containing copies of three different mRNA template plasmids (dA100-mRNA / pMV, dA15pureC-mRNA / pMV, and dA30hairpin-mRNA / pMV) exhibited any base loss, substitution, or insertion mutations in the non-ploy(A) tail regions. The correct cloning rate was 100% for all three mRNA template plasmids, indicating good passage stability of the non-ploy(A) tail regions. The corresponding sequencing statistical analysis results of the ploy(A) tail regions of the mRNA template plasmids are shown below. Figure 4 The results showed that with increasing passage number, the deletion rate of the ploy(A) tail region in the template plasmid dA100-mRNA / pMV gradually increased. Figure 4 (a) The mean correct cloning rate gradually decreased from 18.4% in generation P1 to 39.4% in generation P15. Figure 4 (b) Poor stability; while the deletion rate of the ploy(A) tail region in the template plasmids dA15pureC-mRNA / pMV and dA30hairpin-mRNA / pMV was significantly reduced. Figure 4 a) and stability were significantly improved. Figure 4 (b) Among them, the dA15pureC-mRNA / pMV plasmid experienced a small number of base losses during passage of the poly(A) tail, with a mean correct cloning rate of 91.2%-92.5%; while in the dA30hairpin-mRNA / pMV, no base deletions occurred during passage of the poly(A) tail region, and the correct cloning rate of the poly(A) tail was 100%, demonstrating good uniformity and excellent stability. These results indicate that the preferred poly(A) tail sequences A15pureC and A30hairpin can prevent base loss of the 3' poly(A) tail during plasmid replication, exhibiting excellent passage stability and promoting stable fermentation production of mRNA template plasmids.

[0107] Table 3. Primers for mRNA template sequence sequencing

[0108] Primer name SEQ ID NO: specific sequence M13F 14 TGTAAAACGACGGCCAGT M13R 15 CAGGAAACAGCTATGACC

[0109] Example 4: CDS region sequence replacement in mRNA template plasmid

[0110] The reporter genes Luciferase and eGFP were selected as example CDS for sequence replacement in the CDS region of the mRNA template plasmid. First, Beijing Liuhe Huada Genomics Co., Ltd. was commissioned to synthesize the Luciferase (SEQ ID NO:11) and eGFP (SEQ ID NO:12) gene fragments according to the sequences shown in Table 4 using a whole-genome synthesis method. NotⅠ and XhoⅠ restriction sites were added upstream and downstream of the genes, respectively. Then, the Luciferase and eGFP gene sequences were constructed into the mRNA template plasmid by NotⅠ / XhoⅠ double digestion and T4 ligase ligation, respectively, and transformed into E. coli competent cells Stbl2. After Amp... + After plating on LB agar plates and incubating overnight at 37°C, single clones were picked from the plates for colony PCR. Positive clones were sent for sequencing to screen for corresponding template plasmids with correct sequences. The final results yielded recombinant template plasmids capable of transcribing Luciferase mRNA: Luc-dA100-mRNA / pMV (4359bp), Luc-dA15pureC-mRNA / pMV (4359bp), and Luc-dA30hairpin-mRNA / pMV (4380bp), and recombinant template plasmids capable of transcribing eGFP mRNA: eGFP-dA100-mRNA / pMV (3426bp), eGFP-dA15pureC-mRNA / pMV (3426bp), and eGFP-dA30hairpin-mRNA / pMV (3447bp). The 1.2% agarose gel electrophoresis results are shown below. Figure 5 As shown.

[0111] Table 4. DNA sequences of Luciferase and eGFP in the example CDS region

[0112]

[0113]

[0114] Example 5: In vitro synthesis of mRNA via co-transcription

[0115] Step 1: Extraction and linearization of mRNA template plasmids

[0116] Using a plasmid extraction kit (Tiangen, #DP117), the six recombinant mRNA template plasmids capable of translating Luciferase and eGFP were extracted according to the manufacturer's instructions. The purified plasmids were then digested overnight at 37°C in a system containing 10×Digestionbuffer II and BsaI restriction enzymes (Novizan, #DD4303). Finally, the digestion products were purified (Zymo Research, #D4033) to obtain six linearized plasmid DNAs. Detection by 1.2% agarose gel electrophoresis showed that the template plasmids Luc-dA100-mRNA / pMV, Luc-dA15pureC-mRNA / pMV, Luc-dA30hairpin-mRNA / pMV, eGFP-dA100-mRNA / pMV, eGFP-dA15pureC-mRNA / pMV, and eGFP-dA30hairpin-mRNA / pMV were all completely linearized, with no residual circular DNA. Figure 6 ).

[0117] Step 2: mRNA transcription and synthesis

[0118] Following the instructions of the TranscriptAid T7 High-Yield Transcription Kit (Thermo Fisher Scientific, #K0441), cap analogs m7(3'OMeG)(5')ppp(5')(2'OMeA)pG were added to the IVT reaction system. Using the six linearized plasmid DNAs mentioned above as templates, in vitro transcription was performed to synthesize the corresponding complete mRNAs. The mRNA products obtained from in vitro transcription were then digested with DNAseI (Novizan, #EN401) and DNase I was inactivated by chloroform extraction. Finally, an equal volume of 5M LiCl solution was added to the mRNA solution, mixed, and allowed to stand at -20℃ for 1 h for precipitation. The mixture was then centrifuged at 14000 rpm at 4℃ for 20 min, the supernatant was discarded, and the purified mRNA precipitate was dissolved in an appropriate amount of Nuclease-Free Water to obtain the purified mRNA solution. The results were detected by 1.2% agarose gel electrophoresis. Figure 7 The results showed that the six mRNAs obtained—Luc-mA100-mRNA (1979nt), Luc-mA15pureC-mRNA (1979nt), Luc-mA30hairpin-mRNA (2000nt), eGFP-mA100-mRNA (1046nt), eGFP-mA15pureC-mRNA (1046nt), and eGFP-mA30hairpin-mRNA (1067nt)—had high purity and good uniformity, making them suitable for subsequent transfection and expression experiments.

[0119] Example 6: Validation of in vitro expression of mRNA

[0120] Step 1: In vitro expression of Luciferase mRNA

[0121] Hek293T cells in logarithmic phase were divided into groups of 2 × 10⁻⁶. 4 Cells were seeded at a rate of 100 cells / well in 96-well plates, with each well containing 0.1 mL of DMEM medium containing 10% FBS. After overnight incubation at 37°C and 5% CO2, Hek293T cells were transfected with 0.1 μg of Luc-mA100-mRNA, Luc-mA15pureC-mRNA, and Luc-mA30hairpin-mRNA using Lipofectamine Messenger MAX reagent (Invitrogen, #LMRNA015). Untreated cells served as a negative control. 24 h after transfection, the mean luminous intensity (MFI) of each well was measured using a microplate reader (BioTek, SYNERGY H1) according to the instructions of the Luciferase Assay System (Promega, #E1500) to determine the translation efficiency of the Luciferase-encoding mRNA. Figure 8 The MFI of luciferase detected in each mRNA transfection group is shown. The results indicate that compared to the Luc-mA100-mRNA group, the expression levels of Luciferase in cells from the Luc-mA15pureC-mRNA and Luc-mA30hairpin-mRNA groups were significantly increased, with the Luc-mA30hairpin-mRNA group exhibiting the highest Luciferase expression level. These results suggest that the optimized poly(A) tail sequences A15pureC and A30hairpin not only significantly improve the stable passage of mRNA template plasmids but also significantly promote mRNA translation efficiency; among them, A30hairpin shows a more significant promoting effect.

[0122] Step 2: In vitro expression of eGFP mRNA

[0123] Cells were seeded in 96-well plates under the same conditions as described above. Under the same transfection conditions, 0.1 μg of eGFP-mA100-mRNA, eGFP-mA15pureC-mRNA, and eGFP-mA30hairpin-mRNA were transfected into Hek293T cells. Untreated cells served as the negative control group. Twenty-four hours after transfection, eGFP expression and MFI were observed using an inverted microscope (Nikon, ECLIPSE Ti2) to determine the translation efficiency of the eGFP-encoding mRNA. Figure 9 The results show the eGFP expression and MFI observed in each mRNA transfection group. The results indicate that compared to the eGFP-mA100-mRNA group, the eGFP-mA15pureC-mRNA and eGFP-mA30hairpin-mRNA groups showed significantly increased eGFP expression levels, with the eGFP-mA30hairpin-mRNA group exhibiting the highest eGFP expression level. These results further demonstrate that the optimized ploy(A) tail sequences A15pureC and A30hairpin not only significantly improve the stable passage of mRNA template plasmids but also significantly promote mRNA translation efficiency; among them, A30hairpin showed the best promoting effect.

[0124] pMV plasmid DNA full-length sequence (SEQ ID NO:16), 2220bp:

[0125]

Claims

1. A polyA tail element, characterized in that, The polyA tail element contains the sequence "GCUCUUCAC" or "GUGAAGAGC" after any of the 27th to 33rd positions of consecutive A in the 5' to 3' direction, and contains a sequence after any of the 85th to 91st positions of A that can complement the sequence contained after any of the 27th to 33rd positions of A. The polyA tail element contains the sequence "GCUCUUCAC" or "GUGAAGAGC" after any of the 85th to 91st positions of consecutive A's in the 5' to 3' direction, and contains a sequence after any of the 27th to 33rd positions of A that is complementary to the sequence after any of the 85th to 91st positions of A; or... The polyA tail element has C at multiples of the 12th, 13th, 14th or 15th positions in the 5' to 3' direction; Preferably, the polyA tail element satisfies one or more of the following conditions: (1) The polyA tail element comprises 90 to 120 A's; (2) The sequence "GCUCUUCAC" is contained after any A in the 27th to 33rd positions; (3) The sequence "GUGAAGAGC" is contained after any A in positions 85 to 91; (4) Any one of the 27th to 33rd positions is the 30th position; (5) Any one of the 85th to 91st positions is the 88th position; and, (6) The 3' end of the polyA tail element contains 12 to 15 consecutive A's.

2. A polyA tail element, characterized in that, The polyA tail element comprises a nucleotide sequence as shown in SEQ ID NO:5 or SEQ ID NO:

4.

3. An isolated nucleic acid that transcribes the polyA tail element as described in claim 1 or 2; Preferably, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO:10 or SEQ ID NO:

9.

4. A construct of an mRNA transcription template, characterized in that, The construct comprises, from 5' to 3', the following nucleic acid transcribed in the 5'UTR region, the nucleic acid transcribed in the 3'UTR region, and the nucleic acid as described in claim 3; optionally, a multiple cloning site and / or a CDS sequence are also included between the nucleic acid transcribed in the 5'UTR region and the nucleic acid transcribed in the 3'UTR region; Preferably, the 5'UTR region and / or the 3'UTR region are derived from the UTR sequence of human endogenous β-globin; and / or, the multiple cloning site includes one or more restriction enzyme sites selected from SacⅠ, NcoⅠ, NotⅠ and XhoⅠ; More preferably, the sequence of the nucleotide transcribed in the 5'UTR region is as shown in SEQ ID NO:6 and / or the sequence of the nucleotide transcribed in the 3'UTR region is as shown in SEQ ID NO:7; and / or, the nucleotide sequence of the multiple cloning site is as shown in SEQ ID NO:

13.

5. An isolated nucleic acid, which is mRNA, characterized in that, The nucleic acid comprises, from 5' to 3', a 5'UTR region, a 3'UTR region, and a polyA tail element as described in claim 1 or 2; optionally, between the 5'UTR region and the 3'UTR region, mRNA complementary to the multiple cloning site and / or CDS sequence is also included; Preferably, the 5'UTR region and / or the 3'UTR region are derived from the UTR sequence of human endogenous β-globin; and / or, the multiple cloning site includes one or more restriction enzyme sites selected from SacⅠ, NcoⅠ, NotⅠ and XhoⅠ; More preferably, the nucleotide sequence of the 5'UTR region is as shown in SEQ ID NO:1 and / or the nucleotide sequence of the 3'UTR region is as shown in SEQ ID NO:2; and / or, the nucleotide sequence of the multiple cloning site is as shown in SEQ ID NO:13; More preferably, the nucleic acid also contains 5'Cap, such as m7(2'OMeG)(5')ppp(5')(2'OMeA)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 3 or the construct as described in claim 4; Preferably, the backbone of the recombinant expression vector is pMV; and / or, it further includes an enzyme cleavage site, such as BsaⅠ, after the polyA tail element.

7. A transformant, characterized in that, The transformant comprises the polyA tail element as described in claim 1 or 2, the nucleic acid as described in claim 3, the construct as described in claim 4, the nucleic acid as described in claim 5, or the recombinant expression vector as described in claim 6; the host cell of the transformant is a eukaryote or a prokaryote; Preferably, the prokaryote is Escherichia coli, such as Stbl2.

8. A reagent kit, characterized in that, The kit comprises the nucleic acid as described in claim 3, the construct as described in claim 4, the recombinant expression vector as described in claim 6, or the transformant as described in claim 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nucleic acid as described in claim 5, and a pharmaceutically acceptable carrier; Preferably, the CDS sequence encodes a drug protein or polypeptide.

10. The use of the polyA tail element of claim 1 or 2, the nucleic acid of claim 3, the construct of claim 4, the nucleic acid of claim 5, the recombinant expression vector of claim 6, the transformant of claim 7, the kit of claim 8, or the pharmaceutical composition of claim 9 in the preparation of mRNA or products containing the thereof; Preferably, the product is an mRNA vaccine or an mRNA therapeutic agent.

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