Sequence construction of immunostimulatory sequence with truncated poly-a tail and use thereof

Abstract

Provided in the present invention is a nucleic acid molecule, which comprises a first nucleic acid sequence that can be transcribed or can be used to introduce a transcribable nucleic acid sequence; and a second nucleic acid sequence. The second nucleic acid sequence comprises at least two A elements, wherein each of the A elements is defined as a nucleotide sequence consisting of 30 to 80 T nucleotides, and at least one B element, wherein each of the B elements is a sequence that can activate PRRs after transcription. The total number of the A elements is one more than the total number of the B element, and any two of the A elements are separated by one B element. An RNA transcribed from the nucleic acid molecule has a higher immunogenicity and basically does not affect the expression of the protein thereof.

Description

Construction and application of a sequence with truncated poly A tail of immunostimulatory sequence Technical Field

[0001] The present invention relates to the field of molecular construction technology, and in particular to a nucleic acid molecule that can be transcribed into mRNA Background Art

[0002] mRNA technology is a promising tool, for example, to complete the synthesis of therapeutic proteins in cells. The advantage of using mRNA molecules is that the mRNA only needs to be introduced into the cytoplasm for protein translation. Compared with the use of the corresponding DNA sequence contained in an appropriate vector (such as a plasmid), the production process of mRNA molecules is standardized and scalable, and avoids the risk of changing chromosomal DNA when the plasmid or part of the plasmid is incorporated into the genome. The use of, for example, chemically modified nucleotides has successfully solved the initial challenges associated with the introduction of mRNA molecules into cells, such as the instability of mRNA molecules and the immune response. So far, several determinants of mRNA efficiency have been identified and studied, including the efficiency of 5' capping and the composition of the 5' cap, the nature of the untranslated region, codon optimization of the protein-encoding sequence, and 3'polyA.

[0003] In order to improve the efficiency of mRNA-based methods, optimizing polyA is crucial. However, existing technologies have difficulty in directly producing mRNA molecules with a defined polyA length on a large scale. The mRNA molecules used to synthesize proteins in vaccine / therapeutic applications are mainly generated by in vitro transcription. Such methods are based on DNA templates, in which polyA is already encoded and amplified by cloning or polymerase chain reaction (PCR). Compared with enzymatically adding polyA to the generated mRNA, DNA templates theoretically have the advantage of providing polyA of a defined and reproducible length, which can be used for large-scale production of polyA mRNA molecules, and the plasmid production cost is relatively low.

[0004] However, one of the challenges of using plasmids to amplify template DNA sequences in bacteria is that homopolymeric sequences (such as those encoding polyA) can recombine during bacterial amplification of plasmid DNA, so the sequence encoding polyA will shorten in an unpredictable manner over time. For example, compared to the sequence encoding polyA consisting of at least 100 nucleotides, the sequence encoding 70 nucleotides long polyA remains stable. Therefore, further optimization is required to address the shortening of polyA due to recombination. The 3'polyA sequence of RNA plays an important role in nuclear export, RNA stability, and mRNA translation efficiency. Optimization of polyA should not affect RNA stability and translation efficiency.

[0005] Immunity is divided into innate immunity and acquired immunity, which work together to form an effective and sustained defense against pathogens. "Pattern recognition receptors" (PRRs) are the main components of innate immunity. PRRs are located in various subcellular compartments and can detect specific pathogen-associated molecular patterns (PAMPs) that are unique to pathogens and not found in the host. Several PRRs, including toll-like receptors (TLRs), nod-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize different microbial components and directly activate immune cells. TLRs are transmembrane receptors, while NLRs and RLRs are intracellular molecules. Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades, rapidly inducing the expression of a variety of overlapping and unique genes involved in inflammatory and immune responses.

[0006] TLRs are type I transmembrane receptors that are evolutionarily conserved between insects and humans. Ten TLRs (TLRs 1-10) have been discovered. TLRs share similar extracellular and intracellular domains. The extracellular domain contains leucine-rich repeats, while the intracellular domain is similar to the intracellular domain of the interleukin-1 receptor (IL-1R). The intracellular domain of TLRs can interact with the adaptor protein Myd88, leading to the activation of cytokines such as NF-κB. TLRs are primarily expressed on antigen-presenting cells (e.g., dendritic cells and macrophages). Activation of dendritic cells by stimulation of TLRs leads to their maturation and the production of inflammatory cytokines such as IL-12. TLRs agonists mainly come from bacteria and viruses. Each TLRs subtype recognizes a different PAMP, for example, TLR2 recognizes peptidoglycan, TLR3 recognizes dsRNA, TLR4 recognizes Gram-negative bacterial lipopolysaccharide (LPS), TLR5 recognizes bacterial flagellin, TLR7 / 8 recognizes imidazoquinoline and ssRNA, and TLR9 recognizes CpG DNA of bacteria, viruses and protists, as well as hemozoin.

[0007] The retinoic acid-inducible gene I (RIG-I) receptor, the melanoma differentiation-associated protein 5 (MDA5) receptor, and the LGP2 receptor are the three RLRs identified to date. All RLRs possess a highly conserved DExD / H-box (Asp-Glu-Ala-Asp) helicase domain and a C-terminal domain (CTD). RIG-I and MDA5 also possess a caspase activation and recruitment domain (CARD), which is crucial for signal transduction. RIG-I and MDA5 are intracellular viral sensors. Their helicase domains recognize viral palindromic dsRNA containing 5'ppp and long dsRNA, respectively, inducing ATP-dependent conformational changes that lead to the formation of filamentous oligomers on the RNA. Their N-terminal CARDs form "lock-washer" tetramers with K63-linked ubiquitin chains, which then form signaling-active oligomers with the CARD-containing MAVS, ultimately activating several transcription factors, including IFN regulatory factor 3, IFN regulatory factor 7, and NF-κB, to induce type I interferons and proinflammatory cytokines. LGP2, which lacks a CARD and instead possesses a DExD / H-box helicase domain, has been reported to act as a negative regulator, particularly in RIG-I and MDA5-mediated pathways.

[0008] Targeting TLRs / RLRs is being studied as potential vaccine adjuvants. One benefit of using immunostimulatory RNA as an adjuvant compared to traditional adjuvants is the broad and well-defined immune response it elicits. Recent advances in nucleic acid technology have enabled the emergence of in vitro transcribed (IVT) mRNA as an alternative platform for vaccine development. Compared to other vaccine strategies, mRNA vaccines are easy to manufacture and have a favorable safety profile. To be an effective vaccine, mRNA vaccines must fulfill two essential tasks: express the antigen and elicit a strong immune response to the expressed antigen. However, mRNA stability, protein expression, and a balanced and effective immune response remain challenges. Summary of the Invention

[0009] In view of this, the present invention provides a nucleic acid molecule that can encode an mRNA molecule and a modified polyA, wherein the nucleic acid molecule comprises 1) a first nucleic acid sequence that can be transcribed or used to introduce a transcribable nucleic acid sequence; and 2) a second nucleic acid sequence, the second nucleotide sequence consisting of: a) at least two A elements, each of the A elements being defined as a nucleotide sequence consisting of 40 to 65 T nucleotides, and b) at least one B element, each of the B elements being a sequence that can activate PRRs after transcription or a combination thereof; wherein the total number of A elements is one more than the total number of B elements, and wherein any two A elements are separated by one B element.

[0010] The nucleic acid molecule shows reduced recombination during amplification in bacterial host cells, and the mRNA transcribed from the nucleic acid molecule has higher immunogenicity and does not substantially affect the expression of its protein.

[0011] The term "vaccine" in the present invention refers to a composition suitable for use in animals (including humans) that induces an immune response after administration, the strength of which is sufficient to minimally help prevent, improve or cure clinical diseases caused by microbial infection.

[0012] As used herein, the term "nucleotide" refers to unmodified nucleotides or modified nucleotides. Unmodified nucleotides are A, C, G, T and U nucleotides. Modified nucleotides refer to any naturally occurring or chemically synthesized isomers of A, C, G, T and U nucleotides, and refer to any naturally occurring or chemically synthesized analogs, alternatives or modified nucleotides or isomers with, for example, chemical modifications or substituted residues. Modified nucleotides can have base modifications and / or sugar modifications. Modified nucleotides can also have, for example, modifications of the phosphate group of five main caps of the polyribonucleotides comprising the sequence of the encoded protein. Modified nucleotides also include nucleotides synthesized after transcription by the covalent modification of nucleotides. In addition, any suitable mixture of unmodified and modified nucleotides is possible.

[0013] In a first aspect of the present invention, a nucleic acid molecule comprises a DNA sequence comprising: 1) a first nucleotide sequence encoding an mRNA molecule, and 2) a second nucleotide sequence;

[0014] The specific second nucleotide sequence consists of the following:

[0015] a) at least two A elements, each of said A elements being defined as a nucleotide sequence consisting of 40 to 65 T nucleotides, and

[0016] b) at least one B element, each of which is an immunostimulatory sequence that can activate PRRs post-transcriptionally, or a combination thereof;

[0017] The total number of A elements is one more than the total number of B elements, and any two A elements are separated by one B element.

[0018] Specifically, the number of A elements is 2-6, more preferably, the number of A elements is two, three or four.

[0019] Specifically, the A element consists of 30-85 T nucleotides, the A element consists of 45-60 T nucleotides, more preferably, the A element consists of 50-60 T nucleotides, more preferably, the A element consists of 60 T nucleotides.

[0020] Specifically, the number of A elements is four, preferably, the nucleotide sequences of the four A elements together have a total length of 240 nucleotides, more preferably, each A element has a length of 60 nucleotides.

[0021] Specifically, the number of A elements is three, preferably, the nucleotide sequences of the three A elements together have a total length of 180 nucleotides, more preferably, each A element has a length of 60 nucleotides.

[0022] Specifically, the number of A elements is two, preferably, the nucleotide sequences of the two A elements together have a total length of 120 nucleotides, more preferably, each A element has a length of 60 nucleotides.

[0023] Specifically, the B element can activate PRRs after transcription, and preferably has a length of 5-250bp; 5-200bp; 5-150bp; 5-100bp; 5-80bp, 5-60bp, 5-50bp, 5-40bp, 5-30bp, 5-20bp, 5-20bp, 10-80bp, 10-60bp, 10-50bp, 10-40bp, 10-30bp, 10-20bp, 12-80bp, 12-60bp, 12-50bp, 12-40bp, 12-30bp, 12-25bp, 12-22bp.

[0024] Specifically, the B element is transcribed into a TLRs or RLRs, more preferably TLR7 / 8 and / or RIG-I / MAD-5 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, or 70% similarity thereto.

[0025] Specifically, the B element post-transcriptional sequence is a sequence that stimulates RIG-I / MAD-5 or a combination thereof or a sequence with 95%, 90%, 85%, 80%, 75%, or 70% similarity thereto.

[0026] Specifically, the post-transcriptional sequence of the B element is an immunostimulatory nucleotide sequence that stimulates TLR7 / 8, which contains a natural or modified base sequence, and the base sequence contains at least one natural or modified guanine and at least one natural or modified uracil, and its general formula is NaGbNc, preferably, wherein G is natural or modified guanine or uracil, more preferably, the sequence is any one of SEQ ID NO:1 to SEQ ID NO:20 or a combination thereof or a sequence with 95%, 90%, 85%, 80%, 75%, or 70% similarity thereto.

[0027] Specifically, N is a nucleic acid sequence having a length of about 4-50 nucleic acids, preferably 4-30 nucleotides, more preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides, each N being independently selected from natural or modified guanine, uracil, cytosine, or adenine.

[0028] Specifically, a and c are independently integers of 1-40, preferably 1-30, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, wherein when a or c is 1, N is guanine or uracil or an analog thereof, and when it is greater than 1, at least 50% of these nucleotides are guanine or uracil or an analog thereof; preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.

[0029] Specifically, b is an integer of 3-40, preferably 3-30, and more preferably 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

[0030] Specifically, the ratio of guanine to uracil in the RNA sequence is between 1G:100U and 30G:1U, preferably, between 1G:50U and 10G:1U, more preferably between 1G:40U and 2G:1U, or between 1G:30U and 10G:1U or 1G:20U to 5G:1U or 1G:10U to 1G:1U or 1G:5U to 1G:1U.

[0031] In a second aspect, the present invention provides a method for amplifying a nucleic acid molecule, comprising: (1) providing the nucleic acid molecule as described above; and (2) amplifying bacteria comprising the nucleic acid molecule.

[0032] The third aspect of the present invention provides a method for obtaining RNA, comprising amplifying the nucleic acid molecule according to the method described above, and transcribing the nucleic acid molecule into RNA in vitro using the nucleic acid molecule as a template.

[0033] Specifically, the RNA includes linear and circular forms; preferably, the linear form is conventional or self-replicating mRNA.

[0034] Specifically, the linear RNA further includes a 5' cap structure, a 5' non-coding region, and a 3' non-coding region.

[0035] In a fourth aspect of the present invention, the RNA provided can be applied to the field of preventive vaccines, and the protein encoded thereby can also have the potential to induce immunogenic responses, including but not limited to antigens in the following pathogens: novel coronavirus-related antigens, herpes simplex virus-related antigens, respiratory syncytial virus-related antigens, varicella-zoster virus-related antigens, influenza virus-related antigens, parainfluenza virus-related antigens, Epstein-Barr virus-related antigens, cytomegalovirus-related antigens, human metapneumovirus-related antigens, human papillomavirus-related antigens, rotavirus-related antigens, dengue virus-related antigens, malaria-related antigens, rabies virus-related antigens or monkeypox virus-related antigens.

[0036] Specifically, it can be applied to therapeutic fields, including but not limited to the encoding of shared antigens such as KRAS, BRAF, EGFR and their mutants in tumor treatment, as well as screened personalized antigens; encoding of tumor immune stimulators such as CD40L, CD70, TLR4, etc.; and systemic delivery of protein substitutes for rare monogenic diseases.

[0037] The fifth aspect of the present invention provides a method for obtaining a peptide or protein, comprising obtaining RNA according to the method described above, and translating the RNA into a peptide or protein sequence.

[0038] In a sixth aspect, the present invention provides a system for producing eukaryotic translatable mRNA, comprising 1) a vector or plasmid containing the nucleic acid molecule as described above; and 2) a host cell.

[0039] In a seventh aspect, the present invention provides a composition comprising RNA, peptide or protein obtained by the method described above.

[0040] Specifically, the composition includes one or more of a pharmaceutically acceptable excipient, carrier, buffer, protective agent, stabilizer, surfactant, osmotic pressure regulator, adjuvant, preservative, and inactivator. Compared to the prior art, the present invention has the following advantages: it addresses the need for an in vitro transcription template, exhibits reduced recombination of sequences encoding the poly(A) tail during amplification in bacterial cells, and enables the in vitro transcription template to encode an mRNA molecule with a defined poly(A) tail length; while also achieving highly effective immunogenicity without substantially affecting protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1: Effects of inserting the second nucleotide sequence B element at different sites on protein expression.

[0042] Figure 2: Effects of the combination of the second nucleotide sequence B element and A elements of different lengths on polyA recombination.

[0043] Figure 3: Effects of different B element lengths on polyA recombination.

[0044] Figure 4: HAI antibody titer detection.

[0045] Figure 5: ICS method was used to detect the frequency of CD4+ T cells that specifically secreted TNFα, IFNγ and IL-2.

[0046] Figure 6: ICS assay for the frequency of CD8+ T cells specifically secreting TNFα, IFNγ, and IL-2. DETAILED DESCRIPTION

[0047] To better understand the present invention, specific embodiments will be given to further illustrate the present invention. However, it should be understood that the embodiments described are exemplary embodiments and that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0048] Example 1 Effect of Insertion of the Second Nucleotide Sequence B Element at Different Sites on Protein Expression

[0049] To confirm the immune response effects of inserting the B element at different sites within the second nucleotide sequence, the following experiment was designed. A synthetic DNA vector was constructed. The vector contained a T7 promoter, a 5' UTR, a sequence encoding the EGFP protein, a 3' UTR, and a poly-A tail (a 100-bp T nucleotide sequence). The second nucleotide sequence B element (SEQ ID NO: 8) was inserted before the 5' UTR, after the 5' UTR, before the 3' UTR, after the 3' UTR, and 40 bp after the poly-A tail, respectively.

[0050] Transcribe using a T7 in vitro transcription kit to obtain uncapped mRNA. Digest the transcription template with DNase I and purify the mRNA using precipitation. Cap the mRNA using the Cap1 capping kit and purify the capped mRNA using an mRNA purification kit.

[0051] The mRNA stock solution was transfected into HEK293 cells using Lip2000 transfection reagent, and EGFP protein expression was observed under a fluorescence microscope 24 hours later (as shown in Figure 1). The results showed that the fluorescence intensity was significantly weakened when the immunostimulatory sequence was inserted before or after the 5'UTR, while the fluorescence intensity was no significantly different when inserted after the 5'UTR, before the 3'UTR, or 40bp into the poly-A tail compared with the control group, indicating that the 5'UTR, the 3'UTR, and the 40bp into the poly-A tail are suitable sites for the insertion of immunostimulatory sequences, especially before the 3'UTR and the 40bp into the poly-A tail.

[0052] Example 2 Effects of the combination of the second nucleotide sequence B element and A elements of different lengths on polyA recombination

[0053] To test the effects of varying A element lengths on polyA recombination after transformation into E. coli, we engineered the open reading frame sequence of the GFP gene with four different polyA lengths into the pUC57-kanamycin vector (A elements were one 120A fragment, two 60A fragments, two 40A fragments, and three 40A fragments; the B element was either the stimulatory sequence SEQ ID NO:8 or the control sequence SEQ ID NO:21: GAUAUC). After transformation into E. coli, positive clones harboring both the target gene and polyA were selected. For each plasmid containing different polyA tails, 50 clones were selected, cultured, and the plasmids were extracted and sequenced. The recombination ratio refers to the percentage of clones harboring the unexpected polyA tail.

[0054] The results showed that compared with the absence of B elements, the insertion of B elements and 6 random nucleotide sequences (SEQ ID NO: 21) can significantly reduce the recombination of different A elements in E. coli; and the efficiency of reducing recombination by inserting B elements is significantly higher than that by inserting 6 random nucleotide sequences.

[0055] Example 3 Effects of B elements of different lengths on polyA recombination

[0056] To test the effects of plasmids with different B elements on polyA recombination after transformation into E. coli, we designed and inserted the open reading frame sequence of the GFP gene into the pUC57-kanamycin vector with polyA constructs of different B element lengths. The A element consisted of three 40A fragments, and the B element configuration was as shown in the table below.

[0057] The results showed that B elements of different lengths can effectively reduce the recombination ratio of A elements (taking the A element sequence 40A*3 as an example). When the B element length is 12-22bp, the recombination ratio of A elements is slightly higher, and when the B element length is 37bp or above, the recombination ratio of A elements is slightly lower.

[0058] Example 4 Effect of PolyA mRNA Containing Different B Element Sequences on Protein Expression after Transfection

[0059] To test whether polyA containing different B element sequences affects protein expression and mRNA stability after transcription, we designed DNA vectors for in vitro transcription of linear RNA. These vectors contain, in order, a T7 promoter, a 5' UTR, a sequence encoding the d2EGFP protein, a 3' UTR, and a polyA. Specifically, these are the 11 types mentioned in Example 3, resulting in a total of 11 DNA transcription templates.

[0060] Transcription was performed using a T7 in vitro transcription kit. Uncapped natural nucleoside mRNA and modified nucleoside mRNA were obtained by using either natural uracil or 1-methyl pseudouracil in the transcription reaction mixture. The transcription templates were digested with DNase I, and the mRNAs were purified by precipitation. The mRNAs were capped using the Cap1 capping kit and purified using an mRNA purification kit. The purified mRNAs were dissolved in acidic sodium citrate buffer and set aside.

[0061] HEK293 cells were cultured at 4 × 10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate and cultured for 24 hours before transfection with LIP2000 (250 ng / well mRNA). FACS analysis was performed. Cells were washed with PBS, trypsinized, and resuspended in FACS buffer. Propidium iodide staining was used to distinguish live from dead cells. d2EGFP fluorescence intensity was measured 4, 24, 48, and 72 hours after transfection.

[0062] The results showed that compared with the polyA-tailed mRNA sequence without added B elements, there was no significant difference in fluorescence intensity when the length of the inserted B element was 12-22bp, but when the length of the B element was 37bp or longer, the fluorescence intensity was significantly weakened after insertion, indicating that the length of the inserted B element will not affect protein expression when it is 12-22bp, but will reduce the protein expression level when it is greater than 37bp.

[0063] Example 5 Application of polyA mRNA containing different B element sequences in influenza vaccines

[0064] To test whether polyA-transcribed mRNA containing different B element sequences could enhance immune responses, we designed DNA vectors for in vitro transcription of linear RNA. These vectors sequentially contain a T7 promoter, a 5' untranslated region (UTR), a sequence encoding an influenza virus antigen protein, a 3' untranslated region (UTR), and polyA. Specifically, these were groups 1-9 mentioned in Example 3, resulting in a total of nine DNA transcription templates.

[0065] Transcription was performed using a T7 in vitro transcription kit. Uncapped natural nucleoside mRNA and modified nucleoside mRNA were obtained by using either natural uracil or 1-methyl pseudouracil in the transcription reaction mixture. The transcription templates were digested with DNase I, and the mRNA was purified by precipitation. The mRNA was capped using a Cap1 capping kit and purified using an mRNA purification kit. The purified mRNA was dissolved in acidic sodium citrate buffer and set aside. A cationic lipid, neutral phospholipid, steroidal lipid, and polyethylene glycol (PEG)-lipid were dissolved and mixed in ethanol at a molar ratio of 45:10:43:2. The nanomedicine manufacturing equipment was set at a total flow rate of 12 ml / min. The mRNA solution and lipid mixture were encapsulated at a flow rate ratio of 3:1. After encapsulation, the sample was collected by ultrafiltration using a tangential flow filtration system and sucrose solution was added to obtain mRNA-LNPs. BALB / c mice were randomly divided into groups of 8 and immunized with 5 μg of mRNA-LNP per mouse on days 0 and 14. Blood was collected on days 14 (before the second immunization) and 28 for antibody titer determination (Figure 4). Mice were sacrificed on day 28, and splenocytes were harvested and stimulated with a full-length influenza virus HA overlapping peptide library. The frequencies of corresponding antigen-specific CD4+ and CD8+ cytokine-secreting T cells were assessed by ICS (Figures 5 and 6).

[0066] The results showed that compared with the absence of the B element, the insertion of the B element increased antibody titers to varying degrees, induced a Th1-biased response, and significantly activated CD8+ T cell responses. When the B element is 20 bp in length, it can better exert its immune stimulation effect.

[0067] In summary, the nucleic acid molecules provided by the present invention can not only significantly reduce the proportion of polyA recombination, but also enhance humoral and cellular immune responses.

[0068] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0069] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes: 1) a first nucleic acid sequence that can be transcribed or used to introduce a transcribable nucleic acid sequence; and 2) a second nucleic acid sequence, the second nucleotide sequence including: a) at least two A elements, each A element being defined as a nucleotide sequence consisting of 30 to 80 T nucleotides, and b) at least one B element, each B element being a sequence that can activate PRRs post-transcriptionally; where the total number of A elements is one more than the total number of B elements, and where any two A elements are separated by one B element.

2. The nucleic acid molecule according to claim 1, wherein the number of A elements is 2 - 6, and more preferably, the number of A elements is two, three, or four.

3. The nucleic acid molecule according to claim 1, wherein the A element consists of 45 - 60 T nucleotides, more preferably, the A element consists of 50 - 60 T nucleotides, and more preferably, the A element consists of 60 T nucleotides.

4. The nucleic acid molecule according to any one of claims 1 - 2, wherein the number of A elements is four, and preferably, the nucleotide sequences of the four A elements together have a total length of 240 nucleotides, and more preferably, each A element has a length of 60 nucleotides.

5. The nucleic acid molecule according to any one of claims 1 - 2, wherein the number of A elements is three, and preferably, the nucleotide sequences of the three A elements together have a total length of 180 nucleotides, and more preferably, each A element has a length of 60 nucleotides.

6. The nucleic acid molecule according to any one of claims 1 - 2, wherein the number of A elements is two, and preferably, the nucleotide sequences of the two A elements together have a total length of 120 nucleotides, and more preferably, each A element has a length of 60 nucleotides.

7. The nucleic acid molecule according to claims 1 - 6, wherein the B element can activate PRRs post-transcriptionally, preferably with a length of 5 - 250bp; 5 - 200bp; 5 - 150bp; 5 - 100bp; 5 - 80bp, 5 - 60bp, 5 - 50bp, 5 - 40bp, 5 - 30bp, 5 - 20bp, 5 - 20bp, 10 - 80bp, 10 - 60bp, 10 - 50bp, 10 - 40bp, 10 - 30bp, 10 - 20bp, 12 - 80bp, 12 - 60bp, 12 - 50bp, 12 - 40bp, 12 - 30bp, 12 - 25bp, 12 - 22bp.

8. The nucleic acid molecule according to claims 1 - 7, wherein the B element is a TLRs or RLRs post-transcriptionally, and more preferably, is TLR7 / 8 and / or RIG-I / MAD-5 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

9. The nucleic acid molecule according to claims 1 - 8, wherein the post-transcriptional sequence of the B element is a sequence that stimulates RIG-I / MAD-5 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

10. The nucleic acid molecule according to any one of claims 1-9, wherein the post-transcriptional sequence of the B element is an immunostimulatory nucleotide sequence that stimulates TLR7 / 8, characterized in that, It contains a natural or modified base sequence, which contains at least one natural or modified guanine and at least one natural or modified uracil, and its general formula is N a G b N c , preferably, wherein G is a natural or modified guanine or uracil, more preferably, the sequence is any one of SEQ ID NO: 1 to SEQ ID NO: 20 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

11. The nucleic acid molecule according to claim 10, wherein N is a nucleic acid sequence having a length of about 4 to 50 nucleic acids, preferably 4 to 30 nucleotides, more preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 nucleotides, and each N is independently selected from natural or modified guanine, uracil, cytosine or adenine.

12. The nucleic acid molecule according to any one of claims 10-11, characterized in that, a and c are integers from 1 to 40 independent of each other, preferably 1 to 30, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. When a or c is 1, N is guanine or uracil or its analog; when it is greater than 1, at least 50% of these nucleotides are guanine or uracil or its analog; preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.

13. The nucleic acid molecule according to any one of claims 10 - 12, characterized in that, b is an integer from 3 to 40, preferably 3 to 30, more preferably 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

14. The nucleic acid molecule according to any one of claims 10 - 13, characterized in that, The ratio of guanine to uracil in the RNA sequence is between 1G:100U and 30G:1U, preferably between 1G:50U and 10G:1U, more preferably between 1G:40U and 2G:1U, or between 1G:30U and 10G:1U or between 1G:20U and 5G:1U or between 1G:10U and 1G:1U or between 1G:5U and 1G:1U.

15. A method for amplifying a nucleic acid molecule, comprising: (1) providing a nucleic acid molecule as described in any one of claims 1-14 (2) amplifying the bacterium comprising the nucleic acid molecule.

16. A method for obtaining RNA, comprising amplifying the nucleic acid molecule according to the method described in claim 15 and in vitro transcribing it into RNA using the nucleic acid molecule as a template.

17. The method for obtaining RNA according to claim 16, wherein, The RNA includes linear and circular forms; preferably, the linear form is a conventional or self-replicating mRNA.

18. The method for obtaining RNA according to claim 17, wherein The linear RNA also includes a 5' cap structure, a 5' untranslated region, and a 3' untranslated region.

19. Use of the RNA obtained by the method according to claims 16-18 in the preparation of a prophylactic vaccine, wherein the protein encoded by the RNA has the potential to induce an immunogenic response. Preferably, the antigen is selected from: antigens related to severe acute respiratory syndrome coronavirus 2, antigens related to herpes simplex virus, antigens related to respiratory syncytial virus, antigens related to varicella-zoster virus, antigens related to influenza virus, antigens related to parainfluenza virus, antigens related to Epstein-Barr virus, antigens related to cytomegalovirus, antigens related to human metapneumovirus, antigens related to human papillomavirus, antigens related to rotavirus, antigens related to dengue virus, antigens related to malaria, antigens related to rabies virus or antigens related to monkeypox virus. Use of RNA obtained by the method according to claims 16-18 in the preparation of a therapeutic drug. Preferably, the drug is a tumor therapeutic drug. More preferably, the tumor is lung cancer, pancreatic cancer or colon cancer. A method for obtaining a peptide or protein, comprising: obtaining RNA by the method according to any one of claims 16-18, and translating the RNA into a peptide or protein sequence.

22. A system for generating eukaryotic translatable mRNA, characterized in that, Comprising: 1) a vector or plasmid containing the nucleic acid molecule according to claims 1-14; 2) a host cell.

23. A composition, characterized in that, Comprising RNA obtained by the method according to any one of claims 16-18, or a peptide or protein obtained by the method according to claim 21.

24. The composition according to claim 23, wherein The composition comprises a pharmaceutically acceptable excipient. Preferably, the excipient is one or more of a carrier, a buffer, a protecting agent, a stabilizing agent, a surfactant, an osmotic pressure regulator, an adjuvant, a preservative or an inactivating agent.

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