Element for improving cap-free and tail-free linear mRNA translation efficiency

Abstract

The invention belongs to the technical field of nucleic acid, particularly relates to a cap-free and tail-free linear translation system mRNA (messenger ribonucleic acid) and an element for improving the translation efficiency of the cap-free and tail-free linear mRNA, and mainly solves the problems of stability, translability, preparation difficulty, preparation efficiency and preparation cost of the mRNA. The cap-free and tail-free linear mRNA translation system mainly comprises four elements: a 5'stem-loop structural element for replacing an original 5 'cap cap of mRNA, a 5' IRES structural element for replacing an original 5 'UTR of mRNA, a coding region CDS and a 3' stem-loop structural element for replacing an original 3 'poly (A) of mRNA. The element is a strong-stability nucleic acid sequence and structure with more than three stem-loop structures. Strong-stability stem-loop sequences and structures are respectively introduced to 5'and 3 'ends of linear mRNA, a 5' IRES structural element is introduced, mature, stable and effectively translated mRNA can be conveniently obtained directly through one-step transcription without capping, tailing or cyclizing treatment of the mRNA, and the mRNA with the translation element and system can be rapidly, simply, economically and efficiently prepared.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid technology, and specifically relates to a cap-less and tail-less linear translation system mRNA, and a component for improving the translation efficiency of the cap-less and tail-less linear mRNA. Background Art

[0002] The application field of mRNA is very broad. It can be used in many fields such as vaccine development, cancer treatment, genetic disease treatment, immunomodulators, gene editing, protein replacement drugs and personalized treatment (Nature, 2020, 586 (7830): 589-593; Mol Cancer, 2021, 20 (1): 48-56). There are two main types of eukaryotic mRNA. They are classical linear mRNA and circular mRNA. Classical linear mRNA is the main form of mRNA in eukaryotic cells. Mature classical linear mRNA consists of a 5' cap (5' cap), a 5' untranslated region (5' UTR), a coding region (CDS), a 3' untranslated region (3' UTR) and a poly A (poly A) tail structure (Nat Rev Drug Discov, 2018, 17 (4): 261-279) ( Figure 1 ). Each element has an important function. The function of the 5'cap structure is to initiate translation and improve mRNA stability. There are two ways to cap in vitro synthesized mRNA, including co-transcription and post-transcriptional capping. Both capping methods are time-consuming, labor-intensive and uneconomical (Curr Protoc, 2021, 2(8): 552-560.). The function of the poly (A) tail is to increase the half-life and translation efficiency of mRNA in vivo (Nat Rev Mol Cell Biol, 2022, 23(2): 93-106.). Circular mRNA (circRNA) is a covalently closed RNA molecule. Since it has no free ends, it can resist degradation by RNA exonucleases and has higher stability. As an expression vector, circular RNA must be introduced into the internal ribosome entry site (IRES). Circular mRNA recruits ribosomes for translation through IRES (Nat Commun, 2018, 9(1): 2629-2639). Circularization efficiency is one of the key factors for the effective translation of circular mRNA. The circularization efficiency will gradually decrease as the length of the mRNA to be circularized increases, which also limits its widespread use.

[0003] In order to make up for the shortcomings of the above two types of mRNA, it is urgent to develop a fast, effective, simple and economical mRNA translation type. Summary of the invention

[0004] Conventional linear mRNA 5'cap structure has two functions: improving the stability of mRNA and initiating translation. Circular mRNA also has these two characteristics. Circular mRNA has no free ends, so it can resist exonuclease degradation and improve stability. In addition, it initiates translation through the IRES structure. Therefore, circular mRNA can also be effectively translated. However, adding 5'cap to classic linear mRNA is time-consuming, labor-intensive and uneconomical. Similarly, circular RNA is complicated to operate and has problems with cyclization efficiency. In order to avoid the difficulties encountered in the preparation of these two types of mRNA, the present invention combines the characteristics of conventional linear mRNA and circular mRNA to innovate a capless and tailless linear translation system mRNA, as well as an element for improving the translation efficiency of capless and tailless linear mRNA ( Figure 2 ). By introducing a strong stable stem-loop structure sequence at the 5' and 3' of mRNA, mature, stable and effectively translatable mRNA can be conveniently obtained by direct one-step transcription without capping, tailing or cyclization. mRNA with this translation element and system can be prepared quickly, simply and cost-effectively.

[0005] A capless and tailless linear translation system mRNA, and an efficiency verification of an element for improving the translation efficiency of the capless and tailless linear mRNA, comprising the following steps:

[0006] (1) Construction of capless and tailless linear mRNA vector;

[0007] (2) using plasmids to prepare DNA templates;

[0008] (3) transcribe mRNA fragments;

[0009] (4) Transfect cells to detect the expression effect of capless and tailless linear mRNA;

[0010] Furthermore, in step (1), the capless and tailless linear mRNA vector is obtained by PCR amplification technology and homologous recombination technology. The above method is well known and will not be described here.

[0011] Furthermore, in step (2), the cap-less and tail-less linear mRNA template is obtained by PCR amplification technology or plasmid enzyme digestion. The above method is known and will not be described here.

[0012] Furthermore, the capless and tailless linear mRNA in step (3) is obtained by in vitro transcription. The above method is known and will not be described in detail here.

[0013] Furthermore, the mRNA transcribed in step (4) is precipitated with lithium chloride and then adjusted to a concentration of 40 ng / μL, and then Lipofectamine 3000 is used as a vector to deliver the mRNA into HEK-293T cells for fluorescence expression detection. The above method is known and will not be described in detail here.

[0014] Beneficial effects achieved in the present invention:

[0015] (1) Economical and practical: There is no need to add expensive 5' caps, and the transcribed mRNA can be directly translated.

[0016] (2) Simple and easy to prepare: Mature and effective mRNA can be obtained through one-step transcription, without the need for circularization and complex purification processes similar to circular RNA.

[0017] The invention is expected to accelerate mRNA research and therapeutic applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the classic linear mRNA structure.

[0019] Figure 2 Schematic diagram of the structure of capless and tailless linear mRNA and its translation efficiency elements.

[0020] Figure 3 Translation of capless and tailless linear mRNA and mRNA with translation efficiency elements. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to specific embodiments and accompanying drawings.

[0022] The primers used in the following examples (Table 1) were all synthesized by chemical methods and synthesized by Sangon (Shanghai) Technology Co., Ltd.

[0023] The vector construction backbone fragments and insert sequence DNA fragments and transcription template DNA used in the following examples were all prepared from plasmids by PCR. Briefly, 0.01 ng / μL DNA plasmid, 1× PrimeSTAR Max Premix (Takara Bio) and 0.8 μM primers were used to synthesize transcription templates and vector construction related fragments by PCR, and the obtained products were analyzed and purified by agarose electrophoresis and Cycle Pure Kit (OMEGA), respectively.

[0024] The vectors used in the following examples were constructed using the principle of homologous recombination. The kit used was the Uniclone One Step Seamless Cloning Kit produced by Beijing Jinsha Biotechnology Co., Ltd., and the construction method was carried out according to the instructions.

[0025] The mRNA used in the following examples was prepared by in vitro transcription (IVT) using a fragment obtained by plasmid PCR as a template. Briefly, the in vitro transcription reaction was carried out at 37°C for 2 hours with 1 μg DNA transcription template, NTPs (5 mM each), IVT buffer [40 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; pH 8.5), 40 mM dithiothreitol (DTT), 10 mM NaOAc, 75 mM Mg(OAc)2 and 0.2 mM spermidine], and T7 RNA polymerase (0.1 mg / mL). The crude transcript was treated with DNase I (2 U per 1 μg DNA; NEB) for 15 minutes at 37°C to digest the DNA template, and then the RNA was precipitated with lithium chloride (LiCl, 2.5 M) to obtain a pure RNA substrate for ligation.

[0026] The main steps of the transfection experiment performed in the following example are as follows: HEK-293T cells (10,000 cells / well) were seeded in a 96-well plate. When the cell fusion rate exceeded 90%, 40 ng of uncapped linear mRNA was encapsulated and transfected into the cells using Lipofectamine 3000 (Invitrogen), and after incubation for 24 hours, the cells were observed under a fluorescence microscope and photographed.

[0027] The nucleotide sequences involved in the present invention are shown in Table 2

[0028] Table 1. Primer sequences used in this article

[0029]

[0030]

[0031] Table 2. Gene sequences used in this article

[0032]

[0033]

[0034]

[0035] Example

[0036] 1) Transcribe mRNA to be translated

[0037] EMCV-GFP and 5'-3'-SL-EMCV-GFP.

[0038] 2) Transfection of HEK-293T cells

[0039] HEK-293T cells (10,000 cells / well) were seeded in 96-well plates. When the cell fusion rate exceeded 90%, 40 ng of linear mRNA was encapsulated and transfected into the cells using Lipofectamine 3000 (Invitrogen), and the cells were observed under a fluorescence microscope and photographed after incubation for 24 hours.

[0040] Figure 3 The result is shown in FIG. The result shows that no green fluorescent protein expression was detected in the cap-less, tail-less and stem-loop-free linear mRNA EMCV-GFP. However, the cap-less, tail-less linear mRNA 5'-3'-SL-EMCV-GFP containing 5' and 3' stem-loop structures was observed to express green fluorescent protein. This shows that the present invention can effectively translate the cap-less and tail-less linear mRNA by introducing the stem-loop structure elements at the 5' and 3' ends of the mRNA.

Claims

1. A capless and tailless linear translation system mRNA having: (1) 5' stem-loop structural element; (2) 3' stem-loop structural element; (3) 5' IRES structural element; (4) Coding region CDS.

2. An element for improving the translation efficiency of capless and tailless linear mRNA, characterized in that: It can replace the 5' cap structure, can replace the 3' poly (A) tail structure, has a stem-loop structure, and the stem-loop structure is stable.

3. A capless and tailless linear translation system mRNA and an element for improving the translation efficiency of capless and tailless linear mRNA according to claims 1 and 2, characterized in that: The feature (1) uses a strong stabilizing 5' stem-loop structure element to replace the 5' cap, so that the linear mRNA can be translated without the need for a 5' cap structure.

4. The element for improving the translation efficiency of capless and tailless linear mRNA according to claim 3, characterized in that: The number of the characteristic stem-loop structures is greater than 3, the pairing length of the stem is 3-40 bp, the length of the loop is 2-10 nt, and the melting TM value is greater than 42°C.

5. A capless and tailless linear translation system mRNA and an element for improving the translation efficiency of capless and tailless linear mRNA according to claims 1 and 2, characterized in that: The feature (2) uses a strong stable 3' stem-loop structure element to replace the 3' poly (A) tail structure, so that the linear mRNA can be translated without the 3' poly (A) tail structure.

6. The element for improving the translation efficiency of capless and tailless linear mRNA according to claim 5, characterized in that: The number of the characteristic stem-loop structures is greater than 3, the pairing length of the stem is 3-40 bp, the length of the loop is 2-10 nt, and the melting TM value is greater than 42°C.

7. A capless and tailless linear translation system mRNA and an element for improving the translation efficiency of capless and tailless linear mRNA according to claims 1 and 2, characterized in that: The characteristic (3) 5'IRES element is an internal ribosome insertion site sequence, such as EMCV, CVB3 and HRV.

8. A capless and tailless linear translation system mRNA and an element for improving the translation efficiency of capless and tailless linear mRNA according to claims 1 and 2, characterized in that: The characteristic (4) coding region CDS can be any target protein coding sequence to be expressed.

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