A method for sequence optimization to enhance mRNA protein expression by using RNA-binding proteins

Abstract

The present invention discloses a sequence optimization method for enhancing mRNA protein expression by using RNA-binding proteins. This method utilizes the RNA-binding protein HuR that is widely expressed in various cells. By inserting a sequence specifically recognized by the HuR protein into the 3' UTR of the mRNA molecule, the HuR protein specifically binds to the mRNA molecule, enhances the mRNA stability, and improves the protein expression. By adopting the above sequence optimization method for enhancing mRNA protein expression by using RNA-binding proteins, the present invention can solve the problem that the mRNA molecule is unstable and easily degraded, resulting in low protein expression levels.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a sequence optimization method for enhancing mRNA protein expression by using RNA-binding proteins. Background Art

[0002] mRNA vaccines are the third-generation human vaccine technology developed on the basis of the first-generation attenuated / inactivated vaccines and the second-generation subunit vaccines. The mRNA production process is simple, rapid in synthesis, and low in cost; and it is translated in the cytoplasm without entering the nucleus, and there is no risk of integrating into the host genome; at the same time, as a nucleic acid itself, it has an adjuvant effect of activating the immune response. From the aspects of the inherent characteristics of mRNA, the immune response it induces, and the favorable conditions for large-scale vaccine production, mRNA vaccines have advantages that cannot be compared with other vaccines.

[0003] However, mRNA is extremely unstable and is easily degraded by RNase in tissues and blood and rapidly recognized and cleared by the immune system. The application of mRNA vaccines needs to solve the problems of poor stability, easy degradation, and low protein expression. mRNA as a vaccine is usually obtained by in vitro transcription using linearized DNA as a template. Researchers can design its DNA template and transcription raw materials to obtain an mRNA product with precise molecular-level design. The composition of mRNA contains several necessary elements, including a cap structure (Cap), a 5' untranslated region (5'UTR), an open reading frame (ORF) encoding an antigen protein, a 3' untranslated region (3'UTR), and a polyadenylate tail (Poly(A) tail) structure. At present, the research methods for improving the stability of mRNA mainly involve sequence design and optimization of the above elements on the DNA template, so as to improve the stability and translation efficiency of mRNA.

[0004] RNA binding proteins (RBPs) are a class of important proteins in cells. RBPs interact with RNA by recognizing specific RNA sequences and are widely involved in multiple post-transcriptional regulation processes such as RNA splicing, transport, sequence editing, intracellular localization, and translation control. The present invention proposes inserting a sequence specifically bound by an RNA binding protein into the 3'UTR. This sequence is a short sequence rich in adenine and uracil, simply referred to as the ARE (AU-rich element) element. HuR is an important RNA binding protein widely expressed in various tissues of the body. HuR enhances the stability of mRNA molecules and increases protein expression by binding to the ARE element. In terms of increasing mRNA protein expression, the present invention is a novel method for optimizing mRNA sequences using intracellular RNA binding proteins. Summary of the Invention

[0005] The object of the present invention is to provide a sequence optimization method for enhancing mRNA protein expression using RNA binding proteins, and to solve the problem of low protein expression caused by the instability and easy degradation of mRNA molecules.

[0006] To achieve the above object, the present invention provides an ARE for increasing protein expression and enhancing mRNA stability, including the following:

[0007] ARE-0, whose nucleotide sequence is shown in SEQ ID NO.44;

[0008] ARE-5, whose nucleotide sequence is shown in SEQ ID NO.8;

[0009] ARE-9, whose nucleotide sequence is shown in SEQ ID NO.12;

[0010] ARE-V8, whose nucleotide sequence is ATTTATTTA;

[0011] ARE-T8, whose nucleotide sequence is shown in SEQ ID NO.31.

[0012] The present invention also provides an mRNA, which is obtained by inserting the ARE described in claim 1 in front of, in the middle of, or behind the 3'UTR of its DNA template sequence.

[0013] The present invention also provides a plasmid vector containing the above ARE.

[0014] The present invention also provides the application of ARE in increasing protein expression and enhancing mRNA stability.

[0015] The present invention also provides a sequence optimization method for enhancing mRNA protein expression by using an RNA-binding protein, comprising the following steps:

[0016] S1. Design the DNA template sequence of the above mRNA, and construct a plasmid expressing luciferase;

[0017] S2. Design primers, apply PCR reaction to amplify the linear gene fragment of the vector from the plasmid, apply Gibson reaction to ligate the linear vector and ARE, insert ARE into the vector, and on the basis of the designed plasmid, apply site-directed mutagenesis to transform different AREs or truncate and design ARE;

[0018] S3. Extract the plasmid, linearize it by enzymatic digestion, and add raw materials such as enzymes, ribonucleases, and buffers to transcribe mRNA in vitro;

[0019] S4. Mix mRNA and liposomes in a certain ratio to synthesize lipid nanoparticles;

[0020] S5. Transfect cells, and the mRNA translates into proteins in the cells;

[0021] S6. Add a luciferin substrate reagent, fully lyse the cells, and detect chemiluminescence with a microplate reader;

[0022] S7. Verify whether ARE binds to the HuR protein by RNA pull down.

[0023] Preferably, the volume ratio of the mRNA to the liposomes is 3:1.

[0024] Preferably, the molar ratio of each component in the liposomes is cationic liposome: co-lipid: cholesterol: polyethylene glycol = 50:10:38.5:1.5.

[0025] The advantages and positive effects of the sequence optimization method for enhancing mRNA protein expression by using an RNA-binding protein according to the present invention are as follows:

[0026] 1. The present invention provides a novel method for optimizing mRNA sequences, that is, by using RNA-binding proteins expressed in cells to bind to specific sequence elements to up-regulate the stability of mRNA molecules, thereby increasing the protein amount of the open reading frame and achieving an increase in translation ability and bioavailability.

[0027] 2. Through RNA pull downs experiments combined with western blot detection means, compared with the negative control, the HuR protein binds to the ARE element, and there is a significant difference in the expression level of luciferase.

[0028] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the insertion positions of the ARE element before, in the middle, and after in the embodiments of the present invention;

[0030] Figure 2 Electrophoresis diagram of the DNA template into which the ARE element is inserted in the embodiments of the present invention;

[0031] Figure 3 Cell transfection result diagrams of ARE-F, ARE-M, and ARE-R in the embodiments of the present invention;

[0032] Figure 4 Cell transfection result diagrams of ARE1-13 in the embodiments of the present invention;

[0033] Figure 5 Cell transfection result diagrams of AREV1-V10 in the embodiments of the present invention;

[0034] Figure 6 Cell transfection result diagrams of ARET1-T2 and ARET4-T8 in the embodiments of the present invention;

[0035] Figure 7 Western blot result diagram in the embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] The technical solutions of the present invention are further described below through the drawings and embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0037] Taking luciferase, whose protein expression level is easy to detect, as an example, first, the ARE element is inserted at different positions of the 3'UTR of the DNA template, and compared with the sequence Non-ARE without the inserted ARE element, it shows that the RNA-binding protein binds to the ARE element to enhance the protein expression level, and the most significant enhancement effect is when the ARE is in front of the 3'UTR.

[0038] Secondly, on the basis of determining that the ARE in front of the 3'UTR is the optimal position, 13 different ARE element sequences are replaced by site-directed mutagenesis. The 13 different ARE elements all come from the natural gene library to explore the optimal sequence of the ARE element.

[0039] Finally, on the basis of screening from the optimal sequence and the natural gene library, the shortest and longest functional sequences that ensure the function of the ARE element are explored. Therefore, the present invention adopts a sequence optimization method that uses RNA-binding proteins to enhance mRNA protein expression, which can solve the problem of low protein expression level caused by the instability of mRNA molecules and their easy degradation.

[0040] Example

[0041] I. Design of DNA sequence: Using PUC57-Kan as the plasmid vector, XbaL I and Pci I were selected as the restriction enzyme sites on both sides of the target gene. The target gene sequence was successively the T7 promoter sequence, 5'UTR sequence, optimized luciferase sequence, 3'UTR sequence, and polyA sequence.

[0042] 1. Optimal position of ARE element: The insertion position of the ARE element is as Figure 1 shown. When the ARE element is inserted in front of the 3'UTR sequence, it is marked as ARE-F; when inserted in the middle of the 3'UTR sequence, it is marked as ARE-M; when inserted behind the 3'UTR sequence, it is marked as ARE-R. The plasmid without the inserted ARE element is named Non-ARE.

[0043] 2. ARE elements screened from the natural gene pool: 13 sequences were selected from the natural gene pool. Except that the DNA sequence of the ARE element is different from that of ARE-F, the other genes on the plasmid are the same as those of ARE-F. The ARE elements in the 13 sequences are successively named as serial numbers ARE1-13, and their nucleotide sequences are shown in SEQ ID NO.4-16.

[0044] 3. Truncation and design of ARE elements: The ARE-0 element of ARE-F was truncated and designed. The truncated sequences were successively named ARE-V1 (SEQ ID NO.17), ARE-V2 (SEQ ID NO.18), ARE-V3 (SEQ ID NO.19), ARE-V4 (SEQ ID NO.20), ARE-V5 (SEQ ID NO.21), ARE-V6 (SEQ ID NO.22), ARE-V7 (SEQ IDNO.23), ARE-V8 (ATTTATTTA), ARE-V9 (SEQ ID NO.24), ARE-V10 (SEQ ID NO.25). The designed ARE elements were successively named ARE-T1, (SEQ ID NO.26) ARE-T2 (TATTTATTT), ARE T4-T8, and their nucleotide sequences are shown in SEQ ID NO.27-31.

[0045] II. Insertion of ARE elements: Primers were designed, and the vector with homologous ends and the ARE fragment of the inserted fragment were obtained by PCR. The plasmid containing the ARE element within the 3'UTR was assembled by Gibson. The primer sequences of the vector obtained by PCR reaction are shown in Table 1 (SEQ ID NO.32-37), and the PCR reaction parameters are shown in Table 2.

[0046] Primer Sequences of the Vector

[0047]

[0048] PCR Reaction Parameters of the Vector

[0049]

[0050]

[0051] III. The primer sequences for obtaining the ARE element through PCR reaction are shown in Table 3 (SEQ ID NO. 38 - 43), and the PCR reaction parameters are shown in Table 4.

[0052] Primer Sequences of the ARE Element

[0053]

[0054] PCR Reaction Parameters of the ARE Element

[0055]

[0056] IV. Purification of the PCR Reaction: Using a PCR reaction purification kit, following the operating steps in the instruction manual, the purified vector and ARE element are obtained. Qualitative and quantitative analysis of the DNA is performed using a ultra - micro ultraviolet spectrophotometer, and agarose gel electrophoresis is carried out to verify the integrity and purity of the DNA template. The electrophoresis pattern is as Figure 2 shown. Figure 2 In the left - hand figure, each band is in turn 5k marker (from largest to smallest: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 800bp, 500bp, 300bp), F - vector, F vector; in the middle figure, each band is in turn 5k, M - vector, R vector; in the right - hand figure, it is 2k marker (from largest to smallest: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp), ARE - F element, ARE - M element, ARE - R element. From Figure 2 this, we can see that through primer design and PCR reaction, all the vector bands are between 3000bp - 5000bp, and all the inserted ARE element bands are less than 100bp, and the band sizes all meet the expectations. Therefore, the vector with homologous ends and the ARE fragment of the inserted fragment are correctly obtained.

[0057] V. Gibson Assembly of the Vector and Insertion of the ARE Element

[0058] (1) Cloning Reaction System

[0059]

[0060] (2) Gently mix and incubate the sample at 50 °C for 1 h. After the reaction is completed, place the centrifuge tube on ice to cool for a few seconds. Store the sample at –20 °C or use it directly for subsequent transformation. The vector sequences of ARE-F into which the ARE-0 (SEQ ID NO.44) element has been inserted are shown in SEQ ID NO.1, those of ARE-M are shown in SEQ ID NO.2, and those of ARE-R are shown in SEQ ID NO.3.

[0061] VI. Site-directed mutagenesis of the ARE element

[0062] Based on the plasmid of ARE-F, site-directed mutagenesis of the ARE element was performed. During the site-directed mutagenesis process, in the first step, a mutant sequence was introduced by exponential amplification using a PCR enzyme. The second step was to treat with a special mixture containing kinase, ligase, and Dpn I enzyme to rapidly circularize the PCR product and remove the template. The third step was to efficiently transform into chemically competent cells. The fourth step was to extract the plasmid from the bacterial solution, sequence it, and obtain the correct mutant DNA sequence.

[0063] VII. Transformation of Gibson and site-directed mutagenesis recombinant products into competent cells.

[0064] (1) Thaw the E. coli DH5α competent cells on ice before use.

[0065] (2) Add 2 μL of the Gibson recombinant product to 50 μL of the cells and flick the tube wall gently to mix.

[0066] (3) Incubate on ice for 30 min, quickly place in a 42 °C water bath for heat shock for 45 s, and then quickly transfer to ice and let stand for 2 min.

[0067] (4) Add SOC medium to make up the volume to 1 mL.

[0068] (5) Incubate with shaking at 37 °C for 1 hour.

[0069] (6) Concentrate: Centrifuge at 5000×g for 1 min, discard 900 μL of the supernatant, and pipette the remaining part to mix well.

[0070] (7) Spread 100 μL of the bacterial solution on an LB plate with 100 mg / L Kan resistance and incubate at 37 °C upright until the bacterial solution is absorbed.

[0071] (8) Incubate overnight at 37 °C in an inverted position.

[0072] (9) The next day, pick a single colony from the plate and inoculate it into an LB medium with Kan resistance, and incubate with shaking at 37 °C for 12–16 hours.

[0073] VIII. In vitro transcription of RNA: Extract the plasmid and linearize the DNA template by enzymatic digestion. According to the in vitro transcription kit, add transcription raw materials such as RNA polymerase, cap, nucleoside triphosphates, and DNA template in vitro, and incubate at 37 °C for 2 h to transcribe mRNA using DNA as the template.

[0074] IX. Preparation of liposomes: Prepare liposomes according to the following ratio. The molar ratio of cationic liposome: helper lipid: cholesterol: polyethylene glycol is 50:10:38.5:1.5. The cationic liposome is SM-102, the helper lipid is dioleoyl phosphatidylethanolamine, abbreviated as DOPE, and the polyethylene glycol is dimyristoyl glycerol-polyethylene glycol 2000, abbreviated as DMG-PEG 2000.

[0075] X. Synthesis of lipid nanoparticles: Prepare the required volume of nanoparticles according to the volume ratio of liposomes to mRNA of 3:1, gently shake and mix well to obtain the lipid nanoparticles required for cell transfection.

[0076] XI. Cell transfection: One day before transfection, plate the cells and observe the cell status. After the cell density grows to about 70%-90%, transfect ARE-F, ARE-M, ARE-R into Hela, HEK-293T, 4T1, A549 cells, and transfect ARE1-13, AREV1-V10, ARET1-T2, ARET4-T8 RNA into Hela cells. After transfection, gently shake and mix well, and place them in the cell culture incubator for culture.

[0077] XII. Detection of fluorescence intensity after 24 h: After 24 h of transfection, take out the well plate, equilibrate to room temperature, add the luciferin substrate, and allow the cells to lyse completely for 3 minutes, then detect the luminescence signal using a microplate reader. The transfection results of ARE-F, ARE-M, ARE-R are as Figure 3 shown, the transfection results of ARE1-13 are as Figure 4 shown, the transfection results of AREV1-V10 are as Figure 5 shown, and the transfection results of ARET1-T2, ARET4-T8 RNA are as Figure 6 shown.

[0078] It can be seen from Figure 3 that the transfection effect of ARE-F in the same cells is better. Therefore, a truncated design (AREV1-V10) was performed on ARE-F, and the transfection effect of the truncated sequence is as Figure 5 shown. It can be seen from Figure 5 that the transfection effect of sequence AREV8 is better, indicating that sequence AREV8 can significantly enhance the protein expression level.

[0079] Figure 4 are 13 sequences screened from the natural gene pool. It can be seen fromFigure 4 From the results, it can be seen that the transfection effects of ARE-5 and ARE-9 are close to that of ARE-F compared with Non-ARE, indicating that ARE-5 and ARE-9 can also significantly enhance the protein expression level.

[0080] Figure 6 Seven sequences were artificially designed and consisted of Figure 6 From the results, it can be seen that the transfection effect of ARET8 is excellent, indicating that ARET8 can significantly enhance the protein expression level.

[0081] Thirteen. RNA pull-down was used to confirm the ARE-binding protein HuR.

[0082] 1. Cell lysis

[0083] 1) Culture Hela cells in three 10-cm dishes.

[0084] 2) Discard the culture medium, wash three times with pre-cooled PBS, and aspirate as much supernatant as possible. Take 4 mL of RIPA and add 40 μL of PSMF, mix well and place on ice.

[0085] 3) Add 1230 μL of RIPA lysis buffer to each dish, pipette several times to allow the lysis buffer to fully contact the cells, and lyse on ice for 30 min.

[0086] 4) Scrape the cells with a cell scraper, pipette a few times with the lysis buffer, collect the lysed sample, centrifuge at 14000 g for 5 minutes, take the supernatant, and transfer 10 - 20 μL of the supernatant to a new tube as the experimental input group.

[0087] 5) Measure the protein concentration by BCA.

[0088] 2. Binding of magnetic beads and RNA

[0089] 1) Mix the magnetic beads

[0090] 2) Take three 50 μL aliquots and place them on a magnetic stand to remove the supernatant

[0091] 3) Wash once with 50 μL of 20 mM Tris (pH 7.5), resuspend the magnetic beads, place on the magnetic stand, and discard the supernatant.

[0092] 4) Repeat step 3) once.

[0093] 5) Add 50 μL of RNACapture Buffer to resuspend the magnetic beads.

[0094] 6) Add 10 μL of RNA (Biotin-ARE, Biotin-polyA, enzyme-free water) to the magnetic beads and gently mix with a pipette.

[0095] 7) Incubate with shaking at room temperature for 30 min.

[0096] 3. Binding of HuR protein to RNA-beads

[0097] 1) Place the bead and RNA mixture on a magnetic stand and discard the supernatant.

[0098] 2) Wash once with 50 μL of 20 mM Tris (pH 7.5), resuspend the beads, place on a magnetic stand, and discard the supernatant.

[0099] 3) Repeat step 3) once.

[0100] 4) Dilute 10× protein RNA binding buffer.

[0101] 5) Add 100 μL of 1× protein RNA binding buffer to the beads, mix well, place on a magnetic stand, and discard the supernatant.

[0102] 6) Prepare the cell lysate mixture.

[0103] 7) Add 100 μL of the cell lysate mixture to the RNA-bound beads and gently mix with a pipette.

[0104] 8) Incubate with shaking at 4 °C for 30 - 60 min.

[0105] 4. Elution of the RNA-binding protein complex

[0106] 1) Place the beads on a magnetic stand and discard the supernatant.

[0107] 2) Wash the beads with 100 μL of 1× wash buffer, place on a magnetic stand, and discard the supernatant.

[0108] 3) Repeat step 2) once.

[0109] 4) Add 50 μL of 1× SDS loading buffer and boil for 5 - 10 min.

[0110] 5) Then place on a magnetic stand and collect the supernatant for Western blot analysis. The remaining samples can be stored at -20 °C.

[0111] XIV. The results of Western blot are as Figure 7 shown.

[0112] From Figure 7 the results, it can be seen that ARE binds to the protein HuR, thus increasing the protein expression level.

[0113] Therefore, the present invention adopts the above-mentioned method for optimizing the sequence of enhancing mRNA protein expression by using RNA-binding proteins. The HuR protein specifically binds to mRNA through the ARE element, thereby increasing the protein expression level, and can solve the problem of low protein expression level caused by the instability of mRNA molecules and easy degradation.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for optimizing sequences to enhance mRNA protein expression using RNA-binding proteins, characterized in that, It includes the following steps: S1. Design the DNA template sequence of mRNA and construct a plasmid expressing luciferase; S2. Design primers, use PCR reaction to amplify the linear gene fragment of the vector from the plasmid, use Gibson reaction to connect the linear gene fragment and ARE, insert the linear gene fragment into ARE, and transform the plasmid into competent cells; wherein the nucleotide sequence of ARE is as shown in SEQ ID NO.12, and the insertion position of ARE is in front of 3'UTR; S3. Extract the plasmid, linearize it by enzyme digestion, and add enzymes, ribonucleases, and buffer raw materials to transcribe mRNA in vitro; S4. Mix mRNA and liposomes in a certain ratio to synthesize lipid nanoparticles; S5. Transfect cells, and mRNA translates into proteins in the cells; Add luciferin substrate reagent, fully lyse the cells, detect chemiluminescence with a microplate reader, and use RNA pull down to verify whether ARE binds to HuR protein; The above sequence optimization method is used to improve protein expression and enhance the stability of mRNA.

2. The sequence optimization method for enhancing mRNA protein expression using an RNA-binding protein according to claim 1, characterized in that: The volume ratio of the mRNA to the liposomes is 3:

1.

3. A method for optimizing a sequence to enhance mRNA protein expression using an RNA-binding protein according to claim 2, characterized in that: The molar ratio of each component in the liposomes is cationic liposome: co-lipid: cholesterol: polyethylene glycol = 50: 10：38.5：1.5。

Images