Method for identifying translation activity of 5 '-end cap analogue of mRNA (messenger ribonucleic acid)

Abstract

The invention discloses a method for identifying the translation activity of a 5 '-end cap analogue of mRNA (messenger Ribonucleic Acid), which is characterized in that a luciferase expression plasmid containing a 5' UTR (Untranslated Region), a firefly luciferase open reading frame, a 3 'UTR and a PolyA tail is used as a transcription system, and the translation activity of the 5'-end cap analogue is evaluated according to the activity of firefly luciferase expressed by a reporter gene. According to the method, differentiated translation activities can be identified for the 5 '-end cap analogues with different structures, a methodological basis is provided for accurate configuration of mRNA translation, and the method can also be used for high-throughput screening of the 5'-end cap analogues with intracellular translation activities.

Description

Technical Field

[0001] This invention discloses a method for identifying the activity of nucleoside analogs, belonging to the field of nucleic acid technology. Background Technology

[0002] As is well known, the 5′ cap of mRNA is a crucial component of intact mRNA. The 5′ cap protects mRNA from cleavage by 5′-3′ exonucleases, thereby enhancing mRNA stability. Furthermore, the 5′ cap serves as a marker for pre-mRNA splicing, polyadenylation, and nucleoextrusion. The Cap Binding Complex (CBC) recognizes the 5′ cap and binds to the mRNA, subsequently recruiting relevant protein factors to perform splicing and polyadenylation, and assisting in mRNA nucleoextrusion. Once the mRNA enters the cytoplasm, eIF4F (Eukaryotic Initiation Factor 4F) recognizes the 5′ cap and initiates mRNA translation. In addition, mRNAs with abnormal 5′ cap structures can trigger antiviral responses in cells.

[0003] In in vitro transcription systems, two common capping methods are enzymatic capping and co-transcriptional capping. Enzymatic capping involves first synthesizing mRNA in vitro, purifying it, and then using a capping enzyme system to complete the capping reaction. Co-transcriptional capping refers to the addition of a cap-like nucleotide to the 5' end of the mRNA during the transcription reaction, a one-step process. Enzymatic capping significantly increases production costs and complicates the overall process by introducing vaccinia virus capping enzymes, 2′-O-methyltransferases, and the substrate SAM, adding to the QC (Quality Control) procedures. Co-transcriptional capping, on the other hand, avoids the introduction of additional proteins and substrates, simplifying the process. Therefore, co-transcriptional capping has become the mainstream method for mRNA synthesis. The cap analogue of the co-transcriptional substrate is crucial for mRNA stability, efficient translation, and reducing mRNA immunogenicity. Therefore, developing convenient and rapid methods to identify the intracellular expression activity of cap analogues is essential in the development and synthesis of cap analogues.

[0004] Luciferase, derived from bioluminescent organisms, is a collective term for a class of enzymes that catalyze the oxidation of luciferin to produce light. Currently, the luciferase gene derived from the North American firefly is the most widely used. It catalyzes the oxidation of luciferin to oxyluciferin, a process that produces bioluminescence. As a reporter gene, the luciferase gene is increasingly used in basic research and scientific practice. Compared to other detection methods, luciferase detection offers advantages such as convenience, speed, and high sensitivity.

[0005] The purpose of this invention is to provide a method for identifying the translational activity of mRNA 5′ cap analogs. Summary of the Invention

[0006] To achieve the above objectives, the present invention first provides a method for identifying the translational activity of 5′ cap analogues of mRNA, the method comprising the following steps: (1) Linearize the luciferase expression plasmid containing 5′ UTR, firefly luciferase open reading frame, 3′ UTR and PolyA tail; (2) In the presence of the 5′ cap analog of the mRNA to be identified, the linearized plasmid obtained in step (1) is used as a template to transcribe the mRNA. (3) Transfect host cells with the mRNA obtained in step (2); (4) Culture the host cells obtained in step (3) and detect the fluorescence of the host cells.

[0007] In a preferred embodiment, the host cell is selected from any one of 293 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, monkey kidney (COS) cells, and A549 cells. In a specific embodiment of the invention, the host cell is a cell that has been passaged up to 30 times; more specifically, the cell is 293 cells.

[0008] In another preferred embodiment, the sequences of the 5′ UTR, 3′ UTR, and firefly luciferase open reading frames are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0009] In yet another preferred embodiment, the transfection reagent in step (3) is a liposome.

[0010] In a preferred embodiment, the luciferase expression plasmid is constructed using pEF1 / Myc-His A with the T7 promoter.

[0011] In another preferred embodiment, from the time of transfection in step (3), the transfection ratio of mRNA to host cells is 1.2-3.6 ng mRNA per 50,000 cells.

[0012] More preferably, the transfection ratio of mRNA to host cells is 1.6 ng or 3.2 ng mRNA per 50,000 cells.

[0013] In yet another preferred embodiment, the mRNA obtained in step (2) further includes a purification step.

[0014] More preferably, the purification step is one or more of column purification, magnetic bead purification, phenol / chloroform purification, and lithium chloride precipitation purification.

[0015] Secondly, the present invention provides a kit for identifying the translational activity of mRNA 5′ cap analogs based on the above method. The kit includes: a luciferase expression plasmid containing a 5′ UTR, a firefly luciferase open reading frame, a 3′ UTR and a PolyA tail, a transcription reagent for transcribing DNA into mRNA, a host cell, and a reagent for transfecting mRNA into the host cell.

[0016] This invention, based on the quantitatively detectable Luc reporter gene and combined with in vitro transcription and cell culture techniques, provides a method for identifying the translational activity of 5′ cap analogs for mRNA. This method overcomes the technical problem of existing identification methods failing to differentiate the translational activity of 5′ cap analogs with different structures, making it difficult to discover and utilize 5′ cap analogs with excellent translational activity. The method provided by this invention can identify 5′ cap analogs with excellent intracellular translational activity among seven different 5′ cap analogs with similar translational activity in conventional identification methods, providing a methodological basis for precise configuration of mRNA translation. This method can also be used in conjunction with the synthesis and development of cap analogs for high-throughput screening of 5′ cap analogs with intracellular translational activity. Attached Figure Description

[0017] Figure 1 Firefly luciferase expression plasmid structure; Figure 2 Linearized electrophoresis pattern of firefly luciferase expression plasmid; Figure 3 Electrophoretic pattern of Fluc mRNA purified by lithium chloride precipitation; Figure 4 Determining the amount of Fluc mRNA transfected; Figure 5Results of luciferase activity assay after transfection of 293 cells with 1.6 ng Fluc mRNA; Figure 6 Results of luciferase activity assay after transfection of 1ug Fluc mRNA into 293 cells.

[0018] Figure 6 middle: 1.Cap1-GAG m7G(5′)ppp(5′)(2′OMeA)pG; 2.Cap1-GAG(3′OMe) m7(3′OMeG)(5′)ppp(5′)(2′OMeA)pG; 3.CAP GGG m7G(5′)ppp(5′)(2′OMeG)pG; 4.CAP GGG(3′Ome) m7(3′OMeG)(5′)ppp(5′)(2′OMeG)pG; 5. LZCap® AG (3'Acm); 6. LZCap ® AU(3'Acm); 7.m7G(5′)ppp(5′)(2′OMeA)pG. Detailed Implementation

[0019] 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.

[0020] Example 1. Construction and enzyme digestion of firefly luciferase expression plasmid The luciferase expression plasmid pEF1-Luc was constructed by inserting a sequence containing a 5′ UTR (SEQ ID NO.1), a firefly luciferase open reading frame (SEQ ID NO.3, genbank no. AB762768.1), a 3′ UTR (SEQ ID NO.2), and a PolyA tail downstream of the T7 promoter of plasmid pEF1 / Myc-His A (Invitrogen). The pEF1-Luc plasmid was linearized by single digestion with BspQ I and recovered as a template. Figure 1 This is a schematic diagram of the constructed luciferase expression plasmid pEF1-Luc, with 5′ UTR, Luc, and 3′ UTR inserted sequentially at the multiple cloning site of the pEF1 / Myc-His A plasmid. Figure 2 The pEF1-Luc plasmid was linearized by single enzyme digestion, resulting in a band of approximately 7.8 kb.

[0021] Example 2. In vitro transcription of Fluc mRNA The transcription reaction system is shown in Table 1.

[0022] Table 1. Transcription reaction system

[0023] Note: During the experiment, the required volume of materials for the system was calculated first, and then samples were added. First, sterile, enzyme-free water was added to the system, followed by 10× buffer, NTPs, and cap analogues in sequence. After mixing, the mixture was gently centrifuged. Then, nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and linearized DNA template were added. After thorough mixing, the mixture was gently centrifuged and incubated at 37°C. After 2 hours, 1U of DNase I was added, and incubation was continued at 37°C for 30 minutes to remove the DNA template. RNA purification was then performed using lithium chloride precipitation. The purified mRNA was dissolved in sterile, enzyme-free water and then quantitatively detected using Nanodrop One. This invention detects and evaluates the translational activity of seven cap analogues (one from CoreBio, and two cap analogues each from Shenji, Yisheng, and Hengnuokang with different structures). 1. Cap1-GAG m7G(5')ppp(5')(2'OMeA)pG (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.)

[0024] 2.Cap1-GAG(3'OMe) m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (Yisheng Biotechnology (Shanghai) Co., Ltd.)

[0025] 3.CAP GGG m7G(5′)ppp(5′)(2′OMeG)pG (Jiangsu Shenji Biotechnology Co., Ltd.)

[0026] 4. CAP GGG(3'Ome) m7(3'OMeG)(5′)ppp(5′)(2'OMeG)pG (Jiangsu Shenji Biotechnology Co., Ltd.)

[0027] 5. LZCap® AG (3'Acm) (Guangzhou Hengnuokang Pharmaceutical Technology Co., Ltd.)

[0028] 6. LZCap®AU (3'Acm) (Guangzhou Hengnuokang Pharmaceutical Technology Co., Ltd.)

[0029] 7. m7G(5')ppp(5')(2'OMeA)pG (Core Biopharmaceutical Technology (Hangzhou) Co., Ltd.)

[0030] The above are labeled 1-7, representing the CleanCap® Reagent AG cap analogue (TriLink BioTechnologies). As a reference, Table 2 shows that all nucleoside cap analogs from various manufacturers can be transcribed into the corresponding target mRNA, and the yield of in vitro transcription products is not significantly different from that of the reference CleanCap® AG group (Table 2). Figure 3 ).

[0031] Table 2. In vitro transcription yield of mRNA.

[0032]

[0033] Example 3. Determination of mRNA transfection amount Using the linearized plasmid from Example 1 as a template, in vitro transcription was performed using a cap analogue as an initiator. The mRNA synthesized through in vitro transcription was transfected into 293 cells. 293 cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 100 μl / well in a 96-well plate. Cells up to passage 30 are recommended for transfection experiments. A cell density of 80% is ideal at transfection. Different amounts of mRNA were transfected per well (0, 1.6, 3.2, 6.4, 12.5, 25, 50, 100 ng). Lipofectamine 3000 Transfection Reagent (Invitrogen) was used as the transfection reagent, and its usage instructions were followed. Transfected cells were incubated at 37°C in a CO2 incubator. After 24 hours of incubation at 37°C in a CO2 incubator, the chemiluminescence values ​​were measured using the ONE-Glo™ Luciferase Assay System (Promega). Data were recorded and plotted. See [link to data]. Figure 4 When the transfection amount is 1.6-6.3 ng / well, the luminescence value of Luc is within the linear range. When the transfection amount reaches 12.5 ng / well, the luminescence value of Luc reaches the plateau phase. Therefore, in order to better distinguish the intracellular activity of different nucleoside cap analogs, the mRNA transfection amount in 96-well plates should be 1.6-6.3 ng / well, preferably 1.6 or 3.2 ng / well.

[0034] Example 4. Detection of translation efficiency of Fluc mRNA produced using different cap analogues Based on the mRNA transfection dose determined in Example 3, 293T cells were transfected with 1.6 ng of mRNA and 1 μg of mRNA (the recommended dose from the transfection kit). Chemiluminescence values ​​were measured 24 hours after transfection, and the data were recorded and plotted. (See attached image.) Figure 5 , Figure 6 .Depend on Figure 6 As shown, when the mRNA transfection dose is the recommended 1 μg, the luminescence values ​​of Fluc mRNA produced by different cap analogs after cell transfection are quite similar (1-7 are 48706, 49339, 52875, 50377, 49253, 52557, and 49594, respectively), making it impossible to distinguish the intracellular activity of the cap analogs. However, when the transfection dose is 1.6 ng, the luminescence values ​​of Fluc mRNA produced by different cap analogs after cell transfection show significant differences. Figure 5 The values ​​1-7 are 359, 1597, 1597, 596, 593, 2090, and 1885 respectively, which can distinguish the intracellular activity of different cap analogs. Using CleanCap® AG as a reference, a significant decrease in RLU indicates poor activity, comparable activity indicates good activity, and a significant increase indicates excellent activity. Figure 5 The results showed that the activity of the 6th cap analog was comparable to that of CleanCap® AG; the intracellular activity of the other 6 cap analogs was significantly lower than that of CleanCap® AG.

Claims

1. A method for identifying the translational activity of a 5' cap analog of an mRNA, characterized in that, The method includes the following steps: (1) Linearize the luciferase expression plasmid containing 5′ UTR, firefly luciferase open reading frame, 3′ UTR and PolyA tail; (2) In the presence of the 5′ cap analog of the mRNA to be identified, the linearized plasmid obtained in step (1) is used as a template to transcribe the mRNA. (3) Transfect host cells with the mRNA obtained in step (2); (4) Culture the host cells obtained in step (3) and detect the fluorescence of the host cells.

2. The method of claim 1, wherein, The host cells are selected from any one of the following: 293 cells, 3T3 cells, Chinese hamster ovary cells, NSO, SP2 cells, HeLa cells, monkey kidney cells, and A549 cells.

3. The method of claim 1, wherein, The sequences of the 5′ UTR, 3′ UTR, and firefly luciferase open reading frames are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

4. The method of claim 1, wherein, The transfection reagent in step (3) is liposomes.

5. The method of claim 1, wherein, The luciferase expression plasmid was constructed using pEF1 / Myc-His A with the T7 promoter.

6. The method of claim 1, wherein, During the transfection in step (3), the transfection ratio of mRNA to host cells is 1.2-3.6 ng mRNA per 50,000 cells.

7. The method of claim 6, wherein, The mRNA to host cell transfection ratio is 1.6 ng or 3.2 ng mRNA per 50,000 cells.

8. The method of claim 1, wherein, The mRNA obtained in step (2) also includes a purification step.

9. The method of claim 8, wherein, The purification steps are one or more of column purification, magnetic bead purification, phenol / chloroform purification, and lithium chloride precipitation purification.

10. A kit for the identification of the translational activity of a 5' cap analog of an mRNA based on any of the methods of claims 1 to 8, characterized in that, The kit includes: a luciferase expression plasmid containing a 5′ UTR, a firefly luciferase open reading frame, a 3′ UTR, and a PolyA tail; a transcription reagent for transducing DNA into mRNA; host cells; and a reagent for transfecting mRNA into host cells.

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