3' utr derived from cytochrome c oxidase family gene and application

Abstract

The application discloses a DNA molecule for promoting mRNA expression, and a 3'UTR in an mRNA molecule obtained by transcription according to the DNA molecule, wherein the DNA molecule is derived from COX5A, COX17 or COX7B of a cytochrome C oxidase family of human origin. The application also discloses application of a composition containing the 3'UTR in preparation of an RNA therapeutic drug or an mRNA vaccine. Compared with a control group alpha-globin, insertion of the 3'UTR fragment provided by the application increases the expression of sea renilla luciferase in Lenti-X 293T cells by 2.03-2.88 times, and increases the expression of sea renilla luciferase in HeLa cells by 3.62-6.69 times, which shows excellent prospects of the 3'UTR in application in the RNA therapeutic drug or the mRNA vaccine.

Description

Technical Field

[0001] This invention discloses a 3' UTR molecule, belonging to the field of nucleic acid technology. Background Technology

[0002] The genetic information carried by the human genome needs to be transcribed from DNA into RNA, and then translated into proteins to perform biological regulatory functions. Traditional small molecule drugs and antibody drugs target protein molecules, mainly exerting their therapeutic effects by regulating protein function. In recent years, nucleic acid drugs, represented by DNA and RNA, have gradually become a hot topic in precision medicine and disease treatment. Compared with traditional drugs, nucleic acid drugs have advantages such as abundant candidate targets, simple design, short development cycle, strong targeting specificity, high expression efficiency, and long duration of action. Nucleic acid drugs can be broadly divided into two categories: DNA drugs and RNA drugs. Compared with DNA drugs, RNA drugs have a lower risk of genomic integration and mutation induction, higher expression efficiency, and no risk of continuous cumulative toxicity, making them more promising for disease prevention and treatment.

[0003] RNA drugs, such as mRNA (messenger RNA) and siRNA (small interfering RNA), generally use RNA molecules as the base drug or vaccine for the treatment and prevention of diseases. As a type of RNA drug, mRNA drugs have broad application prospects and have played a significant role in the prevention of major infectious diseases and diseases such as the novel coronavirus, demonstrating extraordinary potential. mRNA is a type of single-stranded RNA transcribed from the antisense strand of DNA as a template. It can direct ribosomes in the cytoplasm to translate specific proteins. The basic principle of mRNA vaccines is to introduce mRNA molecules encoding disease-specific antigens into human cells through a specific delivery system. The protein antigens are then expressed by ribosomes within the cells, stimulating the body to produce a specific immune response, generating antigen-specific antibodies, and thus providing immune protection.

[0004] Currently, mRNA drugs are mainly used in protein replacement therapy, regenerative medicine, and vaccines for infectious diseases and personalized tumors. These drugs have the following advantages: 1) mRNA can be directly, rapidly, and efficiently translated into functional protein molecules within the cytoplasm. Because it does not need to enter the cell nucleus, there is no risk of integration into the genome; 2) mRNA has low immunogenicity, which can be further reduced through base modification; 3) mRNA is degraded by normal cellular metabolic processes, and its metabolites are all-natural, without the risk of continuous accumulation of toxicity; 4) mRNA has a richer pool of candidate targets. It can encode and express many proteins or intracellular protein molecules that are difficult to drug, and secrete them extracellularly to enter the circulatory system and target specific receptors. The mRNA vaccines produced and marketed by Moderna (mRNA-1273) and Pfizer (BNT162b2) have become among the most effective vaccines against the novel coronavirus, driving the rapid development of nucleic acid drugs represented by mRNA.

[0005] However, the stability, dosage, and delivery efficiency of mRNA drugs in vivo have always been key factors restricting their development and application. Improving mRNA stability, delivery efficiency, and translation efficiency are obstacles that must be overcome for mRNA therapy to reach clinical application. Mature mRNA molecules contain a 5' cap structure, a 5' UTR (five prime untranslated region), a coding region, a 3' UTR (three prime untranslated region), and a Poly A tail. The 5' UTR is known to be closely related to translation initiation, while the 3' UTR often directly affects mRNA stability and translation efficiency. Analysis of the human transcriptome has revealed a wide range of 3' UTR lengths, with an average length of 1278 nt, accounting for approximately 36% of the total mRNA length. Furthermore, the 3' UTR exhibits a degree of conservation across different species; approximately 15% to 20% of the 3' UTR sequence is conserved in humans, mice, rats, and chickens, suggesting that this conserved sequence may have important regulatory functions. The 3' UTR region of mRNA typically contains conserved cis-regulatory elements, such as AU-rich elements (AREs), which accelerate mRNA degradation, while CU or GA-rich elements increase mRNA stability. Studies have shown that RNA-binding proteins can interact with cis-regulatory elements in the 3' UTR region to regulate mRNA stability and translation. Furthermore, the 3' UTR region of mRNA often contains multiple miRNA target sites. Research indicates that miRNAs can regulate the translation and stability of the vast majority of mRNAs; this regulation is crucial for controlling gene expression levels and participates in regulating various processes such as cell development, differentiation, proliferation, and carcinogenesis.

[0006] Since the stability of mRNA within cells largely depends on the 3' UTR, screening for 3' UTR sequences that enhance mRNA stability is a crucial method for increasing antigen protein yield and final antibody titer, and has become one of the prerequisites for the development of mRNA drug platforms. Therefore, screening for 3' UTR sequences that stably express mRNA is of great significance for the application of mRNA therapy.

[0007] The purpose of this invention is to screen for 3' UTR sequences that can enhance mRNA stability and thus promote mRNA expression. Summary of the Invention

[0008] Based on the above-mentioned objectives, the present invention first provides a DNA molecule that promotes mRNA expression. The DNA molecule is derived from the COX5A gene of the cytochrome C oxidase family (COX), with the sequence shown in SEQ ID NO.1; the COX17 gene of the cytochrome C oxidase family (COX), with the sequence shown in SEQ ID NO.2; or the COX7B gene of the cytochrome C oxidase family (COX), with the sequence shown in SEQ ID NO.3.

[0009] Secondly, the present invention provides a DNA molecule that can be transcribed into mRNA, said DNA molecule comprising:

[0010] (1) A polypeptide coding region, and one or more DNA molecules, as described above, that promote mRNA expression located upstream and downstream of the polypeptide coding region. The DNA molecule may also include functional elements for transcription and translation, such as promoters, enhancers, polyadenylated tails, etc. For the promoter, in one specific embodiment of the present invention, the T7 promoter or the HSV-TK promoter is selected; other promoters used in eukaryotic expression systems can also be used in the present invention. For the polyadenylated tail, the SV40 polyadenylated tail can be used.

[0011] (2) DNA molecules that promote mRNA expression as described above, located downstream of the polypeptide coding region.

[0012] Third, the present invention provides a carrier containing the above-mentioned DNA molecules.

[0013] Fourth, the present invention provides a host cell containing the above-mentioned vector.

[0014] Fifth, the present invention provides a pharmaceutical composition comprising the DNA molecule that promotes mRNA expression as described above, the vector as described above, or the host cell as described above.

[0015] Sixth, the present invention provides an mRNA molecule, the mRNA molecule comprising:

[0016] (1) A polypeptide coding region, comprising one or more functional elements, as described above, that promote mRNA expression, located upstream and downstream of the polypeptide coding region. These functional elements include elements for transcription and translation, such as promoters, enhancers, and polyadenylated tails. For the promoter, in one specific embodiment of the present invention, the T7 promoter or the HSV-TK promoter is selected; other promoters used in eukaryotic expression systems can also be used in the present invention. For the polyadenylated tail, the SV40 polyadenylated tail can be used.

[0017] (2) 3' UTR obtained by transcription of DNA molecules that promote mRNA expression as described above, located downstream of the coding region.

[0018] Seventh, the present invention provides a vector containing the above-mentioned mRNA molecule.

[0019] Eighth, the present invention provides a host cell containing the above-mentioned carrier.

[0020] Ninth, the present invention provides a pharmaceutical composition comprising the mRNA molecule described above, the vector described above, or the host cell described above.

[0021] Finally, this invention provides the application of the above-mentioned mRNA molecules in the preparation of RNA therapeutic drugs or mRNA vaccines, wherein the 3' UTR described in this invention serves as an enhancer for RNA molecule expression in RNA therapeutic drugs or mRNA vaccines.

[0022] The 3' UTR provided by this invention has excellent mRNA expression-promoting function. Compared with the α-globin 3' UTR sequence used by Moderna, the insertion of the 3' UTR fragment from the COX5A gene provided by this invention increased the expression level of Renal luciferase in Lenti-X 293T cells by 2.88 times and the expression level of Renal luciferase in HeLa cells by 6.69 times; the insertion of the 3' UTR fragment from the COX17 gene increased the expression level of Renal luciferase in Lenti-X 293T cells by 2.76 times and the expression level of Renal luciferase in HeLa cells by 6.41 times; and the insertion of the 3' UTR fragment from the COX7B gene increased the expression level of Renal luciferase in Lenti-X 293T cells by 2.03 times and the expression level of Renal luciferase in HeLa cells by 3.62 times. The 3' UTR fragment provided by this invention plays a translation-promoting role in different types of host cells, exhibiting broad-spectrum effects and demonstrating its application potential in the preparation of RNA therapeutics or mRNA vaccines. The 3' UTR serves as an enhancer for RNA molecule expression in RNA therapeutics or mRNA vaccines. Attached Figure Description

[0023] Figure 1A flowchart illustrating the relationship between whole-transcriptome half-life analysis and mRNA stability using 3' UTR sequences. First, publicly available whole-transcriptome half-life data from human HeLa cells were obtained. Bioinformatics analysis identified 775 highly stable mRNA molecules with half-lives exceeding 24 hours. Next, the conservation scores of these long-half-lived 3' UTR sequences were assessed, yielding 122 highly conserved 3' UTR sequences with a conservation score greater than 0.5. Further screening of these long-half-lived and highly conserved 3' UTR sequences resulted in the retention of only those shorter than 300 bases. Ultimately, 52 candidate 3' UTR sequences were obtained.

[0024] Figure 2 Using high conservation, short length, and long half-life as indicators, a total of 52 potential 3' UTR sequences (triangles) related to mRNA stability were screened.

[0025] Figure 3 The experimental flowchart for verifying the effect of 3' UTR on gene expression using a dual-luciferase reporter gene assay system is as follows: First, DNA fragments of the selected 3' UTR sequences were obtained using PCR or artificial synthesis. Next, these 3' UTR sequences were ligated into the psiCHECK-2 vector of the dual-luciferase reporter gene expression system, which was digested with restriction endonucleases XhoⅠ and NotⅠ. Finally, 50 psiCHECK-2 plasmids containing 3' UTR sequences, 3 plasmids containing control 3' UTR sequences (Moderna α-globin 3' UTR sequence, Pfizer / BioNTech mtRNR1+AES 3' UTR sequence, and c-fos gene 3' UTR sequence), and an empty vector were transfected into Lenti-X 293T cells to test the effect of these 3' UTR sequences on luciferase expression levels.

[0026] Figure 4 Flowchart for constructing plasmids for the dual-luciferase reporter gene detection system. First, the psiCHECK-2 vector of the dual-luciferase reporter gene expression system was digested with restriction endonucleases XhoⅠ and NotⅠ at 37℃ for 5-6 hours. Next, the 3' UTR sequences were ligated into the psiCHECK-2 vector of the dual-luciferase reporter gene detection system, which had been digested with restriction endonucleases XhoⅠ and NotⅠ, using homologous recombination or restriction enzyme ligation methods.

[0027] Figure 5The effect of 3' UTRs on reporter gene expression was verified using a dual-luciferase reporter gene assay system in Lenti-X 293T cells. The dotted-line plot bars represent positive control group 1 and positive control group 2, namely the α-globin 3' UTR sequence used in Moderna's mRNA-1273 vaccine and the mtRNR1+AES 3' UTR sequence used in Pfizer / BioNTech's BNT162b2 vaccine, respectively. The diagonal-line plot bars represent the effect of 50 3' UTR sequences tested in the experimental groups on reporter gene expression compared to the α-globin 3' UTR in positive control group 1. Insertion of 9 3' UTR fragments increased the expression of the Renali luciferase reporter gene by more than 2-fold; insertion of 4 3' UTR fragments increased the expression by 1.5-2-fold; and insertion of 22 3' UTR fragments resulted in expression of the Renali luciferase reporter gene being comparable to the control group.

[0028] Figure 6 This study uses a dual-luciferase reporter gene assay system to verify the translation-promoting effects of nine selected 3' UTR sequences that enhance mRNA translation in HeLa cells. The dotted-line graph represents positive control group 1 and positive control group 2, namely the α-globin 3' UTR sequence used in Moderna's mRNA-1273 vaccine and the mtRNR1+AES 3' UTR sequence used in Pfizer / BioNTech's BNT162b2 vaccine, respectively. The diagonal-line graph represents the effect of the nine 3' UTR sequences tested in the experimental groups on reporter gene expression compared to the α-globin 3' UTR in positive control group 1. The results show that the insertion of these nine 3' UTR sequences increased the expression of the Renalis luciferase reporter gene by 3.62–11.67 times. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0030] Example 1. Design and preparation of 3' UTR molecules

[0031] 1.1 Screening of 3' UTR sequences

[0032] The stability of mRNA is mainly reflected in the length of its half-life. After blocking transcription using the transcription inhibitor actinomycin D, RNA was collected at different time points of blocking, and then whole transcriptome sequencing was performed to systematically quantify the half-life of each mRNA molecule. This invention obtained whole transcriptome half-life data of human cells (using HeLa cells as an example) from (Tani H, Mizutani R, Salam KA, et al. (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome research, 22(5): 947-956), and screened 775 highly stable mRNA molecules with a half-life exceeding 24 hours through bioinformatics analysis. The 3' UTR of these mRNAs may have a stabilizing effect, and this invention further extracted these sequences for further screening (e.g., Figure 1 (As shown). In the course of evolution, highly conserved 3' UTR sequences often have more important regulatory functions. To obtain highly conserved 3' UTR sequences, this invention extracted homologous 3' UTR sequences from 100 species and used PhastCons to score the conservation of the obtained 3' UTR sequences with long half-lives, obtaining 122 highly conserved 3' UTR sequences with a conservation score greater than 0.5 (e.g., as shown). Figure 1 (As shown).

[0033] The length of RNA sequences limits their industrial production efficiency. If the length exceeds a certain threshold, the industrial synthesis rate of RNA decreases, byproducts increase, and downstream purification becomes more difficult. Therefore, for the 3' UTR sequences with long half-lives and high conservation identified above, this invention further screened them, retaining only those shorter than 300 bases. Ultimately, 52 candidate 3' UTR sequences were obtained and sorted from highest to lowest conservation (e.g., ...). Figure 1 , Figure 2 (As shown).

[0034] Table 1. Detailed information on 52 potential 3' UTR sequences related to mRNA stability (sorted by 3' UTR length).

[0035] .

[0036] 1.2 Preparation of 3' UTR molecules

[0037] For candidate 3' UTRs longer than 100 bp selected through screening, this invention designed specific primers and amplified them using polymerase chain reaction (PCR) with extracted HeLa cell human genomic DNA as a template. The PCR reagent used was PowerPol 2×PCR Mix with Dye from ABclonal (catalog number RK20719). The specific PCR reaction system was as follows: 100 ng genomic DNA, 1 µl each of forward and reverse primers (10 µM), 25 µl PowerPol 2×PCR Mix, and finally, Nuclease-free water to a final volume of 50 µl. After mixing the PCR reaction solution, it was placed in a PCR instrument, and the PCR reaction was performed according to the conditions described in the ABclonal manual. The specific annealing temperature and extension time depended on the primer sequence and the length of the 3' UTR sequence, and a total of 30 cycles were performed. The obtained PCR products were separated using a 2% agarose gel (agarose dissolved in 1×TBE buffer to a final concentration of 2% (W / V), the solvent of 1×TBE buffer was water, and the solutes and concentrations were as follows: 89 mmol / L Tris-boric acid, 2 mmol / L EDTA), and purified and recovered according to the band size. The purification and recovery kit was Universal DNA Purification Kit, catalog number DP214-03.

[0038] For candidate 3' UTRs with a length of 100 bp or less, this invention first artificially synthesizes two DNA single strands, and then anneals them to form double strands (the preferred reagent used is 10×T4 DNA Ligase Reaction Buffer from New England Biolabs, catalog number M0202L). The 20 µl annealing reaction system consists of: 4 µl each of the two DNA single strands (10 µM), 2 µl of 10×T4 DNA Ligase Reaction Buffer, and Nuclease-free Water to a final volume of 20 µl. After mixing, the mixture is placed in a PCR instrument. The annealing conditions are: 95 °C for 2 minutes, then slowly cooled to 22 °C at a rate of −0.1 °C / cycle, and finally reacted at 22 °C for 5 minutes. After the reaction, the sample is immediately placed on ice for at least 2 minutes.

[0039] This invention successfully obtained 50 candidate 3' UTR sequences through screening. Two of these 3' UTR sequences, listed as 11 and 51 in Table 1, did not yield DNA fragments. To obtain the α-globin 3' UTR sequence used in Moderna's COVID-19 vaccine mRNA-1273 (sequence shown in SEQ ID NO.4, upstream and downstream amplification primers shown in SEQ ID NO.13 and SEQ ID NO.14) as a positive control, we synthesized two primers with partially complementary regions, which served as PCR templates for each other. The specific steps were as follows: 2 µl each of 10 µM forward primer and 10 µM reverse primer were added to a 200 µl PCR tube, along with 25 µl of PowerPol 2×PCR Mix. The volume was then brought to 50 µl with Nuclease-free Water. After mixing, the tube was placed in a PCR instrument and the PCR reaction was performed according to the conditions described in the ABclonal instructions: annealing temperature 60°C, extension time 15 seconds, for a total of 25 cycles. In addition, we subcloned the mtRNR1+AES 3' UTR (sequence shown in SEQ ID NO. 5, upstream and downstream amplification primers shown in SEQ ID NO. 15 and SEQ ID NO. 16) used in the Pfizer / BioNTech COVID-19 vaccine BNT162b2 (Orlandini von Niessen, AG, et al. (2019). "Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening." Molecular Therapy 27(4): 824-836.) as another positive control. As a negative control, the 3' UTR sequence of the c-fos gene was also amplified from human genomic DNA by PCR (sequence shown in SEQ ID NO. 6, upstream and downstream amplification primers shown in SEQ ID NO. 17 and SEQ ID NO. 18), and the reaction system and reaction conditions were consistent with those of the screened 3' UTR sequence.

[0040] To insert the obtained 3' UTR sequence into the dual-luciferase reporter vector psiCHECK-2 (Su, R., Fan, LH., Cao, C. et al. Global profiling of RNA-binding protein target sites by LACE-seq. Nat Cell Biol 23, 664–675 (2021).), we first double-digested the plasmid with restriction endonucleases XhoⅠ and NotⅠ (preferably products from New England Biolabs, catalog numbers R0146L and R3189L, respectively). The digestion system was: 5 µg psiCHECK-2 plasmid, 5 µl 10×rCutSmart™ Buffer, 0.5 µl each of restriction endonucleases XhoⅠ and NotⅠ, and ddH2O to a final volume of 50 µl. After mixing, the mixture was incubated in a PCR instrument at 37°C for 5–6 hours. After the digestion reaction, the linearized psiCHECK-2 plasmid was purified and recovered using a 1% agarose gel (e.g., Figure 3 and Figure 4 (As shown). Figure 4 In the diagram, 1 represents the T7 promoter; 2 represents the Renida luciferase reporter gene (hRluc); 3 represents the multiple cloning region, flanked by restriction endonuclease sites XhoⅠ and NotⅠ. After construction, the multiple cloning region was cloned into the 3' UTR sequence; 4 represents the artificially synthesized polyadenylated tail; 5 represents the HSV-TK promoter; and 6 represents the firefly luciferase reporter gene (hRluc). + ); 7 represents the SV40 polyadenylated tail.

[0041] For 3' UTR fragments larger than 100 bp, this invention utilizes homologous recombination (preferably using NEBuilder® HiFi DNA Assembly Master Mix from New England Biolabs, catalog number E2621L) to insert them into the linearized psiCHECK-2 plasmid. The specific reaction system is as follows: 100 ng of linearized psiCHECK-2 plasmid, 0.2 pmol of the 3' UTR fragment, 2 µl of NEBuilder® HiFi DNA Assembly Master Mix, and Nuclease-free Water to a final volume of 4 µl. After mixing, the mixture is incubated at 50°C for 30 minutes.

[0042] For 3' UTR fragments of 100 bp or less, this invention employs an enzyme digestion and ligation method (preferably using ThermoFisher's Rapid DNA Ligation Kit, catalog number K1423) to ligate them with linearized psiCHECK-2 plasmid. The specific method is as follows: 100 ng of linearized psiCHECK-2 vector, 0.2 pmol of 3' UTR fragment, 2 µl of 5×Rapid Ligation Buffer, 0.5 µl of T4 DNA Ligase (400,000 units / ml), and Nuclease-free Water to a final volume of 10 µl. After mixing, the mixture is incubated at 22°C for 10 minutes.

[0043] The homologous recombination and ligation reaction samples were added to 70 µl of DH5α competent cells (the competent cells were from Qingke Biotechnology, catalog number TSC-C14), and chemical transformation was performed according to Qingke Biotechnology's instructions. After culturing in 500 µl of LB medium for 1 h, the cells were centrifuged at 3000 rpm for 5 min, resuspended in 100 µl of LB medium, and 50 µl of the bacterial suspension was evenly spread on 100 µg / ml ampicillin-resistant LB agar plates (solvent was water, and the solutes and concentrations were as follows: 1% (w / v) Tryptone, 0.5% (w / v) Yeast Extract, 1% (w / v) NaCl, 15 g / L Agar). After incubating the plates at 37°C for approximately 14 hours, single colonies were picked and placed in LB medium containing 100 µg / ml ampicillin resistance (solvent: water, solute concentrations: 1% (w / v) Tryptone, 0.5% (w / v) Yeast Extract, 1% (w / v) NaCl). The medium was then incubated overnight at 37°C on a shaker at 220 rpm. Subsequently, plasmids were extracted using the HiPure Plasmid EF Mini Kit (MegBio, catalog number P1112-02) and sequenced. Correctly sequenced plasmids will be used in subsequent experiments.

[0044] Example 2. Effect of 3' UTR molecules on the expression level of Renal luciferase in Lenti-X 293T cells

[0045] Next, this invention will use 50 psiCHECK-2 plasmids containing 3' UTR sequences (sequences other than 11 and 51 in Table 1), 3 plasmids containing control group 3' UTR sequences (Moderna α-globin 3' UTR sequence (SEQ ID NO. 4), Pfizer / BioNTech mtRNR1+AES 3' UTR sequence (SEQ ID NO. 5), c-fos gene 3' UTR sequence (SEQ ID NO. 6), and empty vectors to transfect Lenti-X 293T cells to test the effect of these 3' UTR sequences on luciferase expression levels.

[0046] First, Lenti-X 293T cells were counted and evenly seeded into 24-well plates treated with 50 g / ml PDL (Poly-D-lysine hydrobromide, which promotes cell adhesion). The cell density at transfection was 70%-80%, with three replicates per group. 200 ng of plasmid was transfected into each well (transfection reagent was GeneTwin from Biomed). TM Gene transfection reagent, catalog number TG101-01; plasmid DNA (μg) and transfection reagent GeneTwin TMThe ratio of 1:2 (μl) was used. Twenty-four hours after transfection, the original culture medium was discarded, and the cells were washed twice with PBS buffer (pH 7.4; solvent: water, solute and concentration as follows: NaCl 137 mmol / L, KCl 2.7 mmol / L, Na2HPO4 10 mmol / L, KH2PO4 2 mmol / L). Cells were lysed in each well with 100 μl of 1× Passive Lysis Buffer at room temperature for 15 minutes. Then, 10 μl of the lysis supernatant from each well was added to a 96-well microplate, and luciferase activity was detected using a GloMax 96-well microplate luminescence detector (lysis buffer and both reaction substrates were Promega Dual-Luciferase® Reporter Assay System, catalog number E1910). The specific reaction conditions are as follows: Add 50 μl of LAR II to each well. After 5 seconds of reaction, begin reading the fluorescence value generated by the reaction between *Rhizophora luciferase* and the substrate. After 10 seconds, add 50 μl of Stop & Glo® Reagent to each well. After another 5 seconds, begin reading the fluorescence value generated by the reaction between firefly luciferase and the substrate, and read for 10 seconds. After reading, divide the *Rhizophora luciferase* reading by the internal control firefly luciferase reading to obtain the corresponding ratio (R = Rluc / Fluc) for each 3' UTR reporter plasmid. Compared with the ratio corresponding to the empty vector, if the reading of the 3' UTR reporter plasmid is larger, it means that the 3' UTR sequence has a role in promoting mRNA translation and stabilization.

[0047] The results showed that, compared with the α-globin 3' UTR sequence used by Moderna, the insertion of 9 out of the 50 3' UTR sequences detected increased the expression of Renalis luciferase reporter genes by more than 2-fold: TMSB10 (number 14) by 3.68-fold; NMU (number 22) by 2.12-fold; ATP5C1 (number 13) by 2.67-fold; COX5A (number 34) by 2.88-fold; COX17 (number 15) by 2.76-fold; MDH1 (number 16) by 2.49-fold; AGBL5 (number 32) by 2.63-fold; SERPINI1 (number 27) by 2.52-fold; and COX7B (number 49) by 2.03-fold.

[0048] Of the 3' UTRs mentioned above, the 3' UTR numbered 34 is derived from the gene COX5A encoding human cytochrome C oxidase. The sequence of the 3' UTR is shown in SEQ ID NO.1, and the primer sequences for amplifying the 3' UTR sequence of COX5A are shown in SEQ ID NO.7 and 8, respectively.

[0049] Of the 3' UTRs mentioned above, the 3' UTR numbered 15 is derived from the gene COX17 encoding human cytochrome C oxidase. The sequence of the 3' UTR is shown in SEQ ID NO.2. The primer sequences for amplifying the 3' UTR sequence of COX5A are shown in SEQ ID NO.9 and 10, respectively.

[0050] Of the 3' UTRs mentioned above, the 3' UTR numbered 49 is derived from the gene COX7B encoding human cytochrome C oxidase. The sequence of the 3' UTR is shown in SEQ ID NO.3. The primer sequences for amplifying the 3' UTR sequence of COX5A are shown in SEQ ID NO.11 and 12, respectively.

[0051] Four 3' UTR fragments were numbered 36, 35, 28 and 19, and their insertion increased the expression of the Renal luciferase reporter gene by 1.95-fold, 1.73-fold, 1.59-fold and 1.51-fold, respectively. There were 22 3' UTR fragments, numbered 24, 12, 21, 17, 29, 52, 1, 38, 37, 40, 30, 8, 23, 39, 9, 4, 31, 10, 18, 6, 5, and 43. Their insertion increased reporter gene expression by 1.48-fold, 1.47-fold, 1.45-fold, 1.44-fold, 1.41-fold, 1.39-fold, 1.34-fold, 1.32-fold, 1.27-fold, 1.27-fold, 1.25-fold, 1.24-fold, 1.23-fold, 1.23-fold, 1.22-fold, 1.18-fold, 1.17-fold, 1.12-fold, 1.08-fold, 1.07-fold, 1.04-fold, and 1.04-fold (e.g., ...) compared to the control group. Figure 5 (As shown). It is worth noting that the changes in reporter gene expression levels detected by the dual-luciferase reporter gene detection system do not match the ranking predicted by bioinformatics analysis. The conservation ranking of the 3' UTR sequences that can increase reporter gene expression by more than 2 times is 14, 34, 15, 13, 32, 27, 16, 22 and 49, which highlights the importance of experimental verification and also shows that the regulation of gene expression by 3' UTR sequences does not depend entirely on their conservation level.

[0052] Example 3. Effect of 3' UTR molecules on the expression level of Renal luciferase in HeLa cells.

[0053] To further examine whether the 3' UTR sequence, which can increase the expression of the *Rhizopus spp.* luciferase reporter gene by more than 2-fold, has the same function in other cell types, this invention transfected 24-well HeLa cells with nine selected 3'UTR reporter plasmids (numbered 14, 34, 15, 13, 32, 27, 16, 22, and 49) that promote efficient mRNA translation (increasing reporter gene expression by more than 2-fold in Lenti-X 293T cells), three control plasmids (α-globin 3' UTR, mtRNR1+AES 3' UTR, and c-fos 3' UTR), and an empty vector (transfection method was the same as for Lenti-X 293T cells). 200 ng of plasmid was transfected into each well. After 24 hours of transfection, cells were lysed, and the activities of *Rhizopus spp.* luciferase and the internal control firefly luciferase were quantified using a dual-luciferase reporter gene assay system. Compared to the α-globin 3' UTR sequence used by Moderna, the insertion of these nine 3' UTR sequences can increase reporter gene expression by 3.62–11.67 times (e.g., ...). Figure 6 (As shown). The 3' UTR sequences, ranked from highest to lowest conservation, are: TMSB10 (number 14) 11.67-fold increase, NMU (number 22) 7.43-fold increase, ATP5C1 (number 13) 7.35-fold increase, COX5A (number 34) 6.69-fold increase, COX17 (number 15) 6.41-fold increase, MDH1 (number 16) 5.77-fold increase, AGBL5 (number 32) 5.66-fold increase, SERPINI1 (number 27) 4.05-fold increase, and COX7B (number 49) 3.62-fold increase. The screened 3' UTR sequences are mostly housekeeping genes related to the cellular respiratory chain, suggesting that the 3' UTR sequences of housekeeping genes may have a stronger promoting effect on mRNA expression.

Claims

1. The application of an mRNA fragment as a 3' UTR in an mRNA molecule, characterized in that, The mRNA molecule includes: (1) The polypeptide coding region is the Renaissance luciferase coding region; (2) The sequence located downstream of the polypeptide coding region, as shown in SEQ ID NO.1, is an mRNA fragment obtained by transcription of DNA molecules derived from cytochrome C oxidase family genes that promotes mRNA expression.

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