mRNA and method for producing the same, device for producing protein, and method for producing protein

Abstract

The present invention provides an mRNA for protein synthesis, comprising: a translation region including a start codon and a stop codon; and an untranslated region located at the 5' end side of the start codon, and at least a portion of the phosphate groups in the range from the 5' end of the untranslated region to 15 nt at the 3' end side of the start codon are replaced by thiophosphate groups. In addition, a method for producing mRNA is provided, comprising: a step of preparing a DNA template; a step of preparing unmodified NTPs including ATP, GTP, CTP and UTP, and a step of preparing modified NTPs in which the phosphate group of at least one of these unmodified NTPs is replaced by a thiophosphate group; and a step of performing a transcription reaction by RNA polymerase using the DNA template as a template and the unmodified NTP and the modified NTP as substrates.

Description

Technical Field

[0001] The present invention relates to an mRNA and a method for producing the same, as well as a device for producing a protein and a method for producing the same. Background Art

[0002] It is known that biological stability can be achieved by introducing a phosphorothioate group (phosphorothioate group: PS) into nucleic acids. Nucleic acids into which PS has been introduced have been used in the molecular structure of antisense nucleic acids and siRNA, and are also used in oligonucleic acid pharmaceuticals that have been marketed. mRNA modified with PS (hereinafter, sometimes referred to as "PS-mRNA") (hereinafter, sometimes referred to as "PS-mRNA") can be enzymatically prepared using T7 RNA polymerase or Escherichia coli RNA polymerase and a substrate nucleotide triphosphate (NTPαS) into which PS has been introduced at the α position of the triphosphate.

[0003] Although methods for synthesizing NTPαS have been developed, PS modification has rarely been used for mRNA, and there were only a few reports about 30 years ago (for example, see Non-Patent Documents 1 to 3). These reports involved protein synthesis using a cell-free translation system using Escherichia coli.

[0004] Non-patent document 1 discloses that PS-mRNA consisting of a poly-U sequence was synthesized and polyalanine was synthesized at an efficiency of 45% of the natural sequence (third paragraph from the bottom). In this document, the sequence is very limited and the product cannot be strictly considered a protein.

[0005] In Non-Patent Document 2, PS-mRNA was produced by in vitro transcription using T7 RNA polymerase using T7 phage DNA as a template. This document also reports that the translation activity of PS-mRNA was evaluated using a cell-free translation system of thermophilic bacteria and Escherichia coli, and that PS-mRNA in which one base was substituted with NTPαS showed a maximum of 2 times the protein synthesis compared to the non-substituted form in these cell-free systems (Fig. 3).

[0006] In non-patent document 3, PS-mRNA of Escherichia coli dihydrofolate reductase was prepared by in vitro transcription, and the amount of protein synthesis using it as a template was measured (Fig. 2). In this paper, it is reported that during transcription, PS-mRNA in which only one base was substituted with NTPαS and PS-mRNA in which all four bases were substituted with NTPαS were produced, and the former had about 2 to 3 times the protein synthesized compared with the mRNA without substitution (hereinafter, sometimes referred to as "PO-mRNA"). The inventors speculate that the improvement in the synthesis efficiency is caused by the stability of PS-mRNA. However, it is also reported in this document that the amount of protein synthesis in PS-mRNA in which all four bases were substituted with NTPαS was reduced compared with that in PS-mRNA without substitution (about 30% of PO-mRNA).

[0007] Prior art literature

[0008] Patent Literature

[0009] Non-patent document 1: Polyribonucleotides containing a phosphorothioate backbone. F. Eckstein, H. Gindl, Eur. J. Biochem. 13, 558-564, 1970;

[0010] Non-patent document 2: Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro. T. Ueda, H. Tohda, N.Chikazumi, F. Eckstein, K. Watanabe, Nucleic Acids Res. 19 (3), 547-552,1991;

[0011] Non-patent document 3: Efficient expression of E. coli dihydrofolate reductasegene by an in vitro translation system using phosphorothioate mRNA. H. Tohda, N. Chikazumi, T. Ueda, K. Nishikawa, K. Watanabe, J. Biotechnol. 34, 61-69,1994. Summary of the invention

[0012] Technical problem to be solved by the invention

[0013] The existing PS-mRNA has a high and low translation efficiency due to the type and number of NTPαS modifications, and it is not clear what kind of PS modification in mRNA can improve the translation efficiency. It should be noted that none of the non-patent documents 1 to 3 describes the introduction of PS into the untranslated region.

[0014] The object of the present invention is to provide an mRNA having a high ribosome translation efficiency for protein synthesis and a method for producing the same. In addition, another object of the present invention is to provide a protein production device and a protein production method using the mRNA to improve the translation efficiency.

[0015] Solutions to technical problems

[0016] The present inventors have conducted intensive research to solve the above technical problems. As a result, it was found that the amount of protein synthesis increased 22 times when PS was partially introduced into mRNA from the 5' end to the vicinity of the start codon, while the amount of protein synthesis decreased by 20% when PS was introduced into mRNA within 250 bases from the vicinity of the start codon to the 3' end. Based on this content, it was found that PS can significantly improve the translation efficiency from the 5' end of mRNA to the vicinity of the start codon, and the present invention was completed.

[0017] That is, the present invention is an mRNA used for protein synthesis, characterized in that the mRNA includes: a translation region including a start codon and a stop codon; an untranslated region located on the 5' end side of the start codon, and at least a portion of the phosphate groups within the range from the 5' end of the untranslated region to 15 nt on the 3' end side of the start codon are substituted with thiophosphate groups.

[0018] In this case, preferably, the phosphate groups at two or more sites are the thiophosphate groups.

[0019] Further in this case, the phosphate groups of at least a portion of the untranslated region and the phosphate groups of at least a portion of the translated region are the phosphorothioate groups.

[0020] Alternatively, preferably, two or more nucleotides among adenosine monophosphate, guanosine monophosphate, cytidine monophosphate and uridine monophosphate constituting mRNA are phosphorothioate groups.

[0021] In any of the above cases, preferably, the untranslated region comprises a Shine-Dalgarno sequence.

[0022] In this case, at least 10 or more phosphate groups within the range from the 5' end of the untranslated region to 15 nt from the 3' end of the start codon are substituted with phosphorothioate groups, and both the Shine-Dalgarno sequence and the translated region contain the phosphorothioate groups.

[0023] The present invention is a method for producing mRNA as described above, characterized in that it includes: a step of configuring a DNA template complementary to the mRNA comprising the untranslated region and the translated region; a step of preparing unmodified NTPs comprising adenosine triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate, and a step of preparing modified NTPs in which at least one phosphate group of the unmodified NTPs is replaced by a thiophosphate group; and a step of performing a transcription reaction by RNA polymerase using the DNA template as a template and the unmodified NTPs and the modified NTPs as substrates.

[0024] In this case, preferably, the method comprises: a DNA cutting step of cutting a point in the sequence corresponding to the range from the 5' end of the non-translated region to 15 nt from the 3' end of the start codon in the DNA template to obtain a 5' end DNA fragment and a 3' end DNA fragment; a modified mRNA preparation step, wherein a transcription reaction is carried out using the 5' end DNA fragment as a template and the unmodified NTP and the modified NTP as substrates to obtain a 5' end modified mRNA fragment; an unmodified mRNA preparation step, wherein a transcription reaction is carried out using the 3' end DNA fragment as a template and only the unmodified NTP as a substrate to obtain a 3' end unmodified mRNA fragment; and a ligation step, wherein the 5' end modified mRNA fragment is ligated with the 3' end unmodified mRNA fragment.

[0025] Furthermore, the present invention is a protein production device comprising: the mRNA as described above, wherein the mRNA has a sequence encoding a target protein in the translation region; and a translation system comprising at least ribosomes, NTPs, tRNAs, amino acids, and aminoacyl-tRNA synthetases.

[0026] In the above, the translation system can be exemplified by a recombinant Escherichia coli translation system.

[0027] In this case, preferably, a part of the phosphate group of at least one of cytidine monophosphate and uridine monophosphate constituting the mRNA is a phosphorothioate group.

[0028] Alternatively, the present invention is a method for producing a protein, characterized in that it comprises: a step of preparing the mRNA described in any one of the above items, wherein the mRNA has a sequence encoding the target protein in the translation region; and a translation step, wherein the mRNA is added to a translation system comprising at least ribosomes, NTPs, tRNA, amino acids, and aminoacyl-tRNA synthetase, and the target protein is translated by the ribosomes.

[0029] In the above, the translation system can be exemplified by a recombinant Escherichia coli translation system.

[0030] In this case, preferably, a portion of at least one of the phosphate groups of cytidine monophosphate and uridine monophosphate constituting the mRNA is a phosphorothioate group.

[0031] Effects of the Invention

[0032] According to the present invention, an mRNA and a method for producing the same that are used in the synthesis of a protein and that have a high ribosome translation efficiency can be provided. In addition, another object of the present invention is to provide a device for producing a protein and a method for producing the protein that use the mRNA to improve the translation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a diagram showing phosphorothioate (PS) modification of the mRNA of the present invention.

[0034] Figure 2 This is a diagram showing the effect of PS modification on mRNA translation in a prokaryotic system.

[0035] Figure 3 This is a diagram showing the results of reverse phase HPLC analysis of NTPαSs used in Examples.

[0036] Figure 4 The figure shows the results of Western blot analysis of the translation of phosphorothioated mRNA in the PURE system.

[0037] Figure 5 is a graph showing the stability of phosphorothioated mRNA in the PURE translation mix.

[0038] Figure 6 This is a diagram showing the results of a single-turnover translation reaction of FLAG-EGF-Pro3 mRNA in the PURE system.

[0039] Figure 7 It is a diagram showing the difference in the position of translation activity of PS-modified RNA.

[0040] Figure 8 is a diagram showing a synthesis experiment of chimeric RNAs including different types of PS modifications.

[0041] Fig. 9 This is a graph showing the results of plotting the fluorescence and temperature curves of the RiboGreen reagent mixed with FLAG-EGF mRNA.

[0042] Fig.10 This is a diagram showing the transcription / translation coupling reaction in the PURE system using NTPαSs. DETAILED DESCRIPTION

[0043] 1. mRNA

[0044] The mRNA of the present invention is described below. The mRNA of the present invention is used for the synthesis of the target protein and includes a translation region and a non-translation region. The translation region includes a start codon and a stop codon and is a region (coding region) encoding the target protein. The non-translation region is located on the 5' end side of the start codon and is a region (non-coding region) that does not translate the protein. The non-translation region includes a sequence that binds to the ribosome (the so-called "consensus sequence"), and as such a sequence, the Shine-Dalgarno sequence (SD sequence) in prokaryotes can be cited. It should be noted that, as long as it does not hinder the effect of the present invention, there may be other non-translation regions downstream of the 3' end side of the stop codon.

[0045] The mRNA of the present invention has at least a portion of the phosphate group in the range from the 5' end of the untranslated region to 15 nt (nucleotide: base) on the 3' end side of the start codon (hereinafter, sometimes referred to as the "PS modification range") replaced by a thiophosphate group. The 15 nt refers to the number of nucleotides counted from the nucleotide on the 5' side in the triplet of the start codon (for example, when the start codon is "AUG", it refers to "A"). The phosphate group mentioned here refers to the phosphate group of the nucleotide that is a unit constituting the mRNA, and the thiophosphate group is a group in which the oxygen double-bonded to phosphorus of the phosphate group is replaced by sulfur. Figure 1 (a) shows the chemical structure of RNA. When X is O, it represents a phosphate group (unmodified nucleotide, i.e., natural nucleotide), and when X is S, it represents a thiophosphate group (modified nucleotide).

[0046] Figure 1(b) is a schematic diagram conceptually showing the mechanism by which the translation efficiency is improved by using the mRNA of the present invention. The upper half of the figure shows the state before the ribosome binds to the mRNA. As shown in the figure, the mRNA (PS-mRNA) substituted with a thiophosphate group is PS-modified at multiple sites within the PS modification range. The ribosome binds to the consensus sequence (SD sequence, etc.) of the untranslated region of the mRNA to form a complex, and translation begins. At this time, the mRNA (PS-mRNA) modified by PS within the PS modification range has a faster translation start speed than the unmodified mRNA (natural mRNA), and the translation efficiency is improved.

[0047] As shown in the following examples, a single-turnover analysis was performed to stop the ribosome during the translation reaction. The results showed that the speed of the peptide extension reaction performed by the ribosome of PS-mRNA and natural mRNA was the same. However, the translation start speed (the speed of ribosome complex formation) was faster for the PS-mRNA template than for the natural type. Generally, in the translation cycle, it is believed that the rate-limiting step exists in the translation start stage, so it can be considered that the reason why the translation efficiency of PS-mRNA is higher is that PS-mRNA accelerates the complex formation process of the ribosome. This result will become a new strategy to use non-natural mRNA to improve translation ability.

[0048] In addition, as shown in the following examples, the amount of mRNA synthesized by partially introducing PS in the translation region from the 5' end to the vicinity of the start codon (34 bases from the 5' end = 11 bases from the start codon) increased by 22 times. However, the amount of mRNA synthesized by introducing PS in the 250 bases from the vicinity of the start codon to the 3' end decreased by 20%. From this, it can be seen that from the perspective of translation efficiency, the site for introducing PS is not any site of mRNA, but is limited to the translation region from the 5' end to the vicinity of the start codon (20 bases downstream of the start codon).

[0049] Although in the mRNA of the present invention, more than one phosphate group is replaced by a thiophosphate group, it is preferred that more than two phosphate groups are thiophosphate groups. When the substitution position is less, the translation efficiency is easily reduced. The substitution position of PS is preferably more than 5, more preferably more than 10. Although there is no specific upper limit for the substitution position, it is preferably less than 20.

[0050] In addition, within the PS modification range, preferably, both the phosphate groups of at least a part of the untranslated region and the phosphate groups of at least a part of the translated region are replaced by thiophosphate groups. In addition, in the present invention, as long as the PS modification is carried out at least within the PS modification range, in the case where the translation region at the 3' end side of the PS translation region and the 3' end side of the stop codon have other untranslated regions, the untranslated region may also be PS modified. However, as shown in the following embodiments, only having PS modification within the PS modification range is more efficient than the case where PS modification is also carried out within the range other than the PS modification range, and therefore it is preferred that PS modification is carried out only within the PS modification range.

[0051] In the mRNA of the present invention, only any one of adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP) and uridine monophosphate (UMP) constituting the mRNA is a thiophosphate group. Alternatively, two or more of these nucleotides are thiophosphate groups. As to which of these nucleotides can be modified by PS to improve the translation efficiency, it also depends on the secondary structure of the mRNA, etc., and therefore cannot be generalized, and can be appropriately designed according to the sequence of the unmodified region, the translation system, etc.

[0052] 2. Methods for producing mRNA

[0053] Next, the method for producing mRNA is described. In the method for producing mRNA, first, a DNA template complementary to mRNA including an untranslated region and a translated region is prepared (DNA configuration step). The portion complementary to the untranslated region of mRNA in the DNA template includes: a promoter sequence, a sequence complementary to the above-mentioned SD sequence, and the like.

[0054] In addition, (unmodified) NTPs including adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) are prepared. Then, modified NTPs (NTPαSs) are prepared, including modified adenosine triphosphate (ATPαSs), modified guanosine triphosphate (GTPαSs), modified cytidine triphosphate (CTPαSs), and modified uridine triphosphate (UTPαSs) in which the phosphate group of NTP is replaced with a thiophosphate group. A reagent containing these NTPs and NTPαSs is prepared. Then, the transcription reaction is carried out by RNA polymerase using the above-mentioned DNA template as a template and the NTP and NTPαSs contained in the reagent as a substrate. Examples of RNA polymerase include T7 RNA polymerase. The ratio of NTP to NTPαSs depends on the amount of PS to be introduced into mRNA, and can be appropriately designed within the range of 1:100 to 100:1. The reaction temperature and reaction time of the transcription reaction can also be appropriately designed. For example, the reaction temperature is 20 to 50° C., and the reaction time is 30 minutes to 6 hours.

[0055] Next, a method for producing PS-mRNA in which only the PS modification region is PS-modified (mRNA in which no PS modification is made except for the PS modification region) will be described.

[0056] First, a site in the sequence corresponding to the range from the 5' end of the untranslated region to 15 nt from the 3' end of the start codon (PS modification range) in the DNA template is cut. Thus, a 5' end DNA fragment and a 3' end DNA fragment are obtained (DNA cutting step). Cutting of the DNA template is performed using a restriction enzyme or the like.

[0057] Next, a transcription reaction is performed using the 5' terminal DNA fragment as a template and both NTP and NTPαSs as substrates to obtain a 5' terminal modified mRNA fragment (modified mRNA configuration step). In addition, a transcription reaction is performed using the 3' terminal DNA fragment as a template and only NTPαSs as a substrate to obtain a 3' terminal unmodified mRNA fragment (unmodified mRNA configuration step). In the step of obtaining these mRNA fragments, T7 RNA polymerase or the like can be used, and the above-mentioned reaction conditions can be applied.

[0058] Finally, the 5' terminal modified mRNA fragment is ligated to the 3' terminal unmodified mRNA fragment (ligation step). In this step, an enzyme such as T4 RNA ligase can be used for ligation. Thus, a chimeric mRNA in which the 5' terminal modified mRNA fragment and the 3' terminal unmodified mRNA fragment are linked can be produced.

[0059] 3. Protein production device and production method

[0060] Next, the protein manufacturing device and manufacturing method of the present invention are described. The mRNA of the present invention can be used to manufacture the target protein. First, an mRNA having a sequence encoding the target protein in the translation region is prepared (mRNA preparation step). The preparation of mRNA can be carried out using the above-mentioned DNA template containing the sequence encoding the target protein.

[0061] Next, the mRNA is added to the translation system for translation (translation step). The translation system uses a translation system that at least includes ribosomes, NTPs, tRNAs, amino acids, and aminoacyl-tRNA synthetases. The translation system is preferably a recombinant Escherichia coli translation system (the so-called PURE system). The PURE system is obtained by recombining the translation system of Escherichia coli in a test tube. The PURE system includes: initiation factors (IF1, IF2, IF3), elongation factors (EF-Tu, EF-Ts, EF-G), termination (release) factors (RF1, RF2, RF3), ribosome regeneration factors (RRF), aminoacyl-tRNA synthetases (ARS), methionine tRNA formyltransferases, ribosomes, T7 RNA polymerases, amino acids, NTPs, tRNAs, etc. By adding mRNA to the system for translation, the target protein can be obtained. The reaction temperature and reaction time can be appropriately set, for example, the reaction temperature can be set to 20 to 50°C, and the reaction time can be set to 1 to 24 hours. In the PURE system, ATP and GTP as substrates are easily decomposed in the system, and therefore CTPαSs and / or UTPαSs are preferably used.

[0062] Example

[0063] Hereinafter, the present invention will be specifically described based on examples, but the purpose of the present invention is not limited thereto. In the following examples, "%" represents a mass basis (mass percentage) unless otherwise indicated.

[0064] 1. Experimental Example 1: Sequence Design and Synthesis of PS-mRNA and Protein Synthesis (FLAG-EGF) Using E. coli Cell-free Translation System

[0065] Figure 2 The upper half of (a) shows a schematic diagram of FLAG-EGF mRNA transcription from the corresponding dsDNA as a template. In this experiment, first, a sequence containing the Shine-Dalgarno (SD) sequence, three FLAG octapeptide coding sequences, and an epidermal growth factor (EGF) coding sequence was designed as a model sequence for mRNA FLAG-EGF.

[0066] FLAG-EGF mRNA was prepared by in vitro transcription reaction using T7 RNA polymerase and dsDNA template obtained by PCR amplification. PS-mRNA was prepared by replacing one or more NTPs with the corresponding NTPαS. PS-mRNA with 16 possible combinations of unmodified NTPs and NTPαS was synthesized.

[0067] Figure 2 The lower part of (a) shows the results of modified PAGE analysis of in vitro transcription using NTPαSs and T7 RNA polymerase. One or more NTPs replaced by the corresponding NTPαSs are indicated by + in the figure. The gel was visualized by SYBR Green II staining. Transcription reactions were performed for all 16 combinations, and the transcripts were analyzed by modified polyacrylamide gel electrophoresis (PAGE). The gel was stained and the relative amount of transcribed RNA was calculated.

[0068] The results showed that the target RNA was observed in all combinations, and the relative yield of mRNA decreased as the number of NTPs after substitution increased.

[0069] Next, the translation reactions of 16 types of FLAG-EGF mRNA with different PS modification patterns were compared at the same concentration in the PURE system. The PURE system was provided by Dr. Yoshihiro Shimizu of the RIKEN Institute of Physical and Chemical Research. Its production method is described in the following paper and other publications.

[0070] Cell-free translation reconstituted with purified components. Y.Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Nat. Biotechnol. 2001, 19, 751-755; b) The PURE System for ProteinProduction. Y. Shimizu, Y. Kuruma, T. Kanamori, T. Ueda, in Cell-Free ProteinSynthesis: Methods and Protocols, Vol. 1118 (Eds.: K. Alexandrov, WA Johnston), Springer Nature Switzerland AG, 2014, pp. 275-284.

[0071] Figure 2 (b) is a diagram showing the results of translating FLAG-EGF mRNA in the PURE system and performing protein blotting analysis. After transcription, the purified RNA (0.2 μM) was incubated in the system at 37°C for 2 hours. The modification type of RNA during transcription is indicated by the + symbol representing the substituted NTPαSs. The translation product was detected using anti-FLAG M2 antibody. The relative amount of the translation product obtained from PS-mRNA and the translation product obtained from PO-mRNA was calculated by protein blotting.

[0072] The results showed that all PS-mRNAs showed higher translation levels than PO-mRNAs. The highest relative ratio was 11 times when both UTPαS and CTPαS were used, and the second highest relative ratio was 8.6 times when only ATPαS was used.

[0073] Next, isomers contained in NTPαS were analyzed. Figure 3 , is a diagram showing the results of reverse phase HPLC analysis of NTPαSs used in this experiment. The results of HPLC analysis showed that the NTPαS purchased from TriLink BioTechnologies was an equal mixture of Rp isomers and Sp isomers. It should be noted that T7 RNA polymerase only uses the Sp type as a substrate, and the Rp type does not hinder the transcription reaction.

[0074] 2. Experimental Example 2: Protein synthesis of other sequences (AcGFP, Spider-FLAG)

[0075] Next, in order to determine whether the improvement in translation efficiency caused by PS modification is somewhat universal, two other sequences (AcGFP and Spider-FLAG) different from FLAG-EGF in Experimental Example 1 were used for investigation.

[0076] Figure 4 , is a diagram showing a Western blot analysis of the translation of phosphorothioated mRNA in the PURE system. AcGFP mRNA (a) and Spider-FLAG mRNA (b) are obtained by replacing ATP or GTP with ATPαSs or GTPαSs, respectively, and then transcribed. After transcription, the purified RNA (0.2 μM) was incubated at 37°C for 2 hours in the PURE system. The translation product was detected using an anti-GFP antibody (Santa Cruz Biotech, sc-9996) for AcGFP mRNA and an anti-FLAG M2 antibody (Sigma-Aldrich, F1804) for Spider-FLAG mRNA.

[0077] As a result, ATPαS or GTPαS was introduced into Spider-FLAG mRNA and AcGFP mRNA, and the translation amount of PS-mRNA was compared with PO-mRNA. As a result, the translation amount of any PS-mRNA was increased compared with PO-mRNA. Furthermore, the relationship between the position of PS introduction and the translation amount of EGF-FLAG mRNA was analyzed by bioinformatics, but no relationship was found. It is possible that the distribution of PS in mRNA, rather than the type of substituted base, is the factor that affects the translation ability.

[0078] 3. Experimental Example 3: Stability of phosphorothioated mRNA in PURE Translation Mix

[0079] Next, we explored why introducing PS into mRNA can improve translation efficiency. One possible reason is the stability of PS-mRNA. The stability of FLAG-EGF mRNA, i.e., PS(A,C)-mRNA, which is transcribed by replacing ATP and CTP with ATPαS and CTPαS, respectively, was compared with the stability of PO-mRNA by culturing in a translation solution and then quantifying it using RT-PCR.

[0080] Figure 5 This is a graph showing the stability of phosphorothioated mRNA in the PURE translation mixture (50mM HEPES-KOH (pH7.6), 100mM potassium glutamate, 2mM spermidine, 1mM DTT, 20mM creatine phosphate sodium, 0.1mM each of 20 amino acids). PO-mRNA and PS (A, C)-mRNA of the FLAG-EGF sequence were incubated at a concentration of 0.05pmol / μL at 37°C. After 10 minutes and 30 minutes, an aliquot (2μL) was taken out from the mixture, mixed with 148μL of water, and stored at -30°C. The RNA concentration in these solutions was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was performed using Prime Script of One Step TBGreen (registered trademark) TM RT-PCR Kit (Takara Bio) was used and implemented in the CFX Connect Real-Time PCR Detection System (Bio-Rad).

[0081] The primer sequences used were FLAG-EGF_F: 5'-CAATATACATGCACACACCG-3', FLAG-EGF_R: 5'-GGGATCCGAAGGAGATATATCC-3'. Renilla reniformis luciferase mRNA (prepared using pRL-TK from BioRad) was used, and the primer set (Rluc_F: 5'-GTTGGACGACGAACTTCACC-3'Rluc_R: 5'-ATCATGGGATGAATGGCCTG-3') was used as an internal control group for qRT-PCR. Reaction conditions: 42°C, 5 minutes → 95°C, 10 seconds → (95°C, 5 seconds → 60°C, 30 seconds) × 40 cycles.

[0082] As a result, no difference was observed in the stability of the two mRNAs. It is believed that since the PURE system is a recombinant E. coli translation system and does not contain nuclease, RNA degradation reactions are unlikely to occur. Therefore, it can be judged that the stability of PS-mRNA is not the main reason for the improvement of its translation efficiency.

[0083] 4. Experimental Example 4: Single-turnover analysis of the translation reaction of FLAG-EGF-Pro3 mRNA in the PURE system

[0084] Next, we will focus on the mechanism of translation reaction. The translation reaction of ribosomes is usually divided into three stages: initiation, elongation, and termination. Among them, the initiation stage, that is, the stage where the ribosome binds to the mRNA, takes the most time and is considered to be the rate-limiting stage. Therefore, the hypothesis that the introduction of PS into mRNA increases the binding rate of the ribosome and thus improves the translation efficiency is proposed. In order to verify this hypothesis, the initial velocity of the translation reaction is analyzed. In order to obtain the initial velocity of the translation reaction, the experimental conditions of a single conversion in which the ribosome does not dissociate are set ( Figure 6 (a)). Figure 6 , is a diagram showing the results of a single-turnover analysis of the translation reaction of FLAG-EGF-Pro3 mRNA in the PURE system, Figure 6 (a) shows an outline of the experimental plan.

[0085] It is known that by introducing a repeating sequence of three or more proline (Pro) at the end of a protein sequence, the ribosome will not detach and stop at that site. Therefore, FLAG-EGF-Pro3 mRNA with three Pro sequences introduced at the end of FLAG-EGF mRNA was designed and synthesized, and a translation reaction was performed. Using a combination of ATPαS and CTPαS, which showed an increase in translation in previous experimental results, PS (A, C)-mRNA was prepared by transcription. After the translation reaction was carried out for 2 to 7 minutes, the amount of protein synthesis was analyzed by Western blotting ( Figure 6 ).

[0086] Figure 6 (b) shows the time course of the translation reaction of thiophosphorylated mRNA (PS(A,C)-mRNA+Pro3) or unmodified mRNA (PO-mRNA+Pro3) analyzed by Western blotting.

[0087] In the translation reaction of FLAG-EGF mRNA, a band of the product was formed at the position of ~15 kDa, and in the translation reaction of FLAG-EGF-Pro3 mRNA, a band of the product was formed at around 37 kDa ( Figure 6 (b) This is because the ribosome cannot elongate the peptide on the repeated Pro and no termination reaction occurs, so the reaction is terminated in the state where tRNA is bound, and tRNA is harvested as a translation product.

[0088] Figure 6 (c) shows the results of Western blot analysis of the translation product after treatment with RNase A. After mRNA (PO-mRNA+Pro3) was translated in the system at 37°C for 240 minutes, RNase was added to the mixture.

[0089] As a result, it was found that when the translation product was actually treated with RNase A, which is an RNA degrading enzyme, tRNA was degraded and a product was observed around 15 kDa. This result confirmed that this system can analyze the single turnover reaction of the ribosome.

[0090] Figure 6 (d) shows a reaction time conversion curve. The signal intensity of the band appearing between the 25-kDa marker and the 37-kDa marker is plotted.

[0091] As a result, the time-dependent changes in the amount of products around 37 kDa produced by natural mRNA and PS-mRNA were plotted into a curve, and it was found that the x-intercepts of the two straight lines showed approximately the same value at approximately 100 seconds. This indicates that the extension speeds in the translation reactions of natural mRNA and PS-mRNA are approximately the same. The slope of each graph represents the initial speed of the translation reaction, from which it can be seen that PS-mRNA is approximately 4 times faster than PO-mRNA. Since the rate-limiting stage of the translation reaction is the initial stage (ribosome binding), it can be shown that PS modification can promote the binding of ribosomes to mRNA, which is the main reason for the increase in translation activity.

[0092] 5. Example and Comparative Example: Effects of different PS modification introduction positions on translation reactions

[0093] Next, two chimeric mRNAs were designed based on the FLAG-EGF sequence. For 5'-PS-mRNA, PS was introduced into the A and C bases of 34 nt (11 nt from the start codon) from the 5' end, which is mainly composed of UTR (untranslated region). For 3'-PS-mRNA, PS was introduced into the A and C bases of the 250 bases from the 3' end ( Figure 7 , Figure 8 These two are obtained by connecting two RNA chains using T4 RNA ligase 2 ( Figure 8 ).

[0094] Figure 7 , is a diagram showing the difference in the position of translation activity of PS-modified RNA (difference in modification position). Figure 7 (a), shows the synthesis scheme of FLAG-EGF mRNA with different PS modification types. Figure 7 (b) shows the nucleotide sequence of 5'-PS-mRNA. Figure 7 (c) shows the results of Western blot analysis of four mRNAs in the PURE system. Figure 7 (d) shows the Figure 7 (c) Quantification results of relative expression levels of four RNAs based on Western blot. Error bars indicate standard deviation (n=3). P values ​​were calculated based on unpaired t-test.

[0095] Figure 8 , is a diagram showing the results of experiments to synthesize chimeric RNAs containing different types of PS modifications. Figure 8 (a) shows the results of MALDI-TOF MS analysis of the 34 nt RNA 5'-fragment. The major peak corresponds to the 5' triphosphorylated 34 nt transcript, and the minor peak corresponds to the 5' pyrophosphorylated transcript. Figure 8(b) shows the results of 6% modified PAGE analysis of the ligation reaction used to prepare chimeric RNA. Figure 8 (c) shows the analysis results of the purified RNA fractions from the PAGE. The gel was visualized by SYBR Green II staining. Figure 8 In (c), 0.5 pmol of each RNA was analyzed.

[0096] The translation activity of these chimeric mRNAs was evaluated. As a result, it was found that, compared with PO-mRNA without PS modification (Comparative Example 1), 5'-PS-mRNA (Example 1) showed a significant increase of 22 times, 3'-PS-mRNA (Comparative Example 2) showed a decrease of 0.8 times, and PS (A, C)-mRNA (Example 2) in which the entire mRNA was PS-modified showed a 5.8-fold increase ( Figure 7 (c) Figure 7 (d)). This result means that the introduction of PS from the 5' end to the vicinity of the start codon is the main reason for the increase in translation. In addition, considering that the translation amount of PS (A, C)-mRNA is lower than that of 5'-PS-mRNA, this means that the introduction of PS from the start codon to the 3' end has the effect of reducing the translation amount. Based on the results obtained so far, it can be judged that the introduction of PS from the 5' end to the vicinity of the start codon can promote the initiation of translation and increase the amount of translation product.

[0097] 5. Experimental Example 5: Evaluation of the double-strand formation ability and thermodynamic stability of PO-mRNA and PS (A, C)-mRNA

[0098] It is known that the secondary structure formation of mRNA affects the formation of the ribosome complex during the translation initiation stage. Figure 6 as well as Figure 7 The results shown here mean that the introduction of PS modification helps reduce the possibility of secondary structure formation around the 5'UTR region. When there are fewer secondary structures in the mRNA sequence from 5'UTR to the start codon, translation initiation is faster. However, there are reports that (Rp)-PS-RNA and PO-RNA form stable duplexes by thermodynamics. It is known that the duplex formation site in RNA emits fluorescence, and the double-strand formation ability and thermodynamic stability of PO-mRNA and PS (A, C)-mRNA were evaluated using RiboGreen reagent.

[0099] Fig. 9, is a graph showing the results of plotting the fluorescence and temperature curves of the RiboGreen reagent mixed with FLAG-EGF mRNA. RNA [0.2μM; PO-mRNA or PS(A,C)-mRNA] was mixed with Quant-iT RiboGreen RNA reagent (400-fold dilution, Thermo Fisher Scientific) in PURE system buffer [50mM HEPES-KOH (pH7.6), 100mM potassium glutamate, 2mM spermidine, 13mM Mg(OAc)2, 1mM DTT, 20mM creatine phosphate sodium, 0.1mM each of 20 amino acids, 2mM ATP, 2mM GTP, 1mM CTP, 1mM UTP]. The RNA in the solution (25 μL) was annealed by lowering the temperature from 95°C to 25°C, and then the temperature was raised from 25°C while recording fluorescence (Ex 450-490 nm, Em 515-530 nm) at 5°C intervals in the range of 25 to 95°C using the CFX Connect Real-Time PCR Detection System (Bio-Rad).

[0100] As a result, it was found that the secondary structure of PS (A, C)-mRNA is slightly more stable than that of PO-mRNA. The physical properties of these mRNAs cannot explain the increase in translation efficiency from the perspective of RNA higher-order structure formation. There is a possibility that other RNA higher-order structure formation or other factors are involved in promoting the ribosome initiation complex, which must be clarified in the future.

[0101] 6. Experimental Example 5: Conjugation reaction of transcription and translation using NTPαS

[0102] In experiments so far, the transcribed PS-mRNA was separated and purified before being used in the translation reaction. However, considering practicality, we studied whether adding NTPαS to the transcription-translation conjugate system would also improve the translation efficiency. However, ATP and GTP are substrates for GTPases (GTPases) that function in the translation system, such as aminoacylation reactions, initiation factor 2, and elongation factor G. Therefore, it is expected that substitution of ATPαS and GTPαS will affect the translation reaction. First, using Figure 2 The template DNA shown in a was subjected to a transcription-translation conjugation reaction by replacing one base NTP with NTPαS using the PURE system. The resulting translation product was analyzed by protein blotting (western method) ( Fig.10 (a)).

[0103] Fig.10 , is a diagram showing the transcription / translation coupling reaction in the PURE system using NTPαSs. Fig.10 (a) shows a Western blot analysis of the translation products obtained using a template DNA encoding FLAG-EGF mRNA substituted with one NTPαSs. The substituted NTPαSs are shown in the figure. Fig.10 The left side of (b) is a diagram showing the strategy for enhancing translation using NTPαSs in the PURE system. In addition to ATPαSs, one or more NTPs are replaced with corresponding NTPαSs. Fig.10 The right side of (b) shows a Western blot analysis of the reaction after 2 hours of incubation in which one or more NTPαSs were substituted in addition to ATPαSs using a template DNA encoding FLAG-EGF mRNA.

[0104] As a result, although the substitution with CTPαS, UTPαS, and GTPαS gave higher levels of translation products than the unsubstituted reaction, the substitution with ATPαS did not form a translation product. ATP is hydrolyzed into AMP by the action of aminoacyl-tRNA synthetase in the PURE system. Therefore, it is considered that there is a possibility that the substitution with ATPαS inhibits these enzyme reactions and, as a result, inhibits the entire translation reaction. In contrast, although GTP is hydrolyzed into GDP by several GTPase enzymes in the translation reaction, substitution with GTPαS does not affect the translation efficiency, and it is considered that GTPαS plays the same role as GTP. Therefore, the different effects of ATP and GTP substitution on translation efficiency may be caused by the difference between the hydrolysis products (AMP and GDP).

[0105] We then used CTPαS, UTPαS, and GTPαS to analyze the effects of substitution of more than two NTPαS ( Fig.10 b). All substitution combinations resulted in greater amounts of translation products compared to the results obtained using unmodified NTP. In particular, substitution with UTPαS or CTPαS had the best effect, with approximately 3 times the amount of translation product obtained. In a cell-free protein translation system, substitution with NTP alone can increase the amount of translation, so the method using NTPαS is a useful technique.

[0106] As described above, in this example, it is shown that the introduction of PS into mRNA can increase the protein synthesis amount of the E. coli cell-free translation system by up to 22 times. This means that the introduction of PS accelerates the binding rate of ribosomes at the start stage of the translation reaction. It is further clarified that the mRNA with PS introduced from the 5' end to the vicinity of the start codon is the most suitable molecular design, which is important for accelerating the start stage. In addition, it is known that the simultaneous reaction of transcription and translation using NTPαS was performed, and it was found that a higher translation amount was obtained compared with the case of using unmodified NTP. This method is a useful technology in the E. coli cell-free translation system.

[0107] The mRNA of the present invention can be applied not only to the translation system of prokaryotes, but also to the translation system of eukaryotes, and can improve the translation efficiency of the target protein. In addition, the mRNA of the present invention can efficiently translate the target protein, and therefore, is also useful not only in the industrial production of protein, but also in mRNA medicine and other aspects.

Claims

1. An mRNA for protein synthesis, characterized in that: include: the translation region containing the start and stop codons; as well as An untranslated region located at the 5' end of the start codon, and Only phosphate groups at more than 10 sites within the range from the 5' end of the untranslated region to 11 nt of the translated region are substituted with phosphorothioate groups, wherein the 11 nt refers to the number of nucleotides counted from the nucleotide on the 5' end side of the triplet of the start codon.

2. The mRNA according to claim 1, characterized in that Both of the phosphate groups in at least a portion of the untranslated region and the phosphate groups in at least a portion of the translated region are substituted with the phosphorothioate groups.

3. The mRNA according to claim 1, characterized in that Any two or more nucleotides among adenosine monophosphate, guanosine monophosphate, cytidine monophosphate and uridine monophosphate constituting mRNA are substituted with phosphorothioate groups.

4. The mRNA according to claim 1, characterized in that The untranslated region contains a Shine-Dalgarno sequence.

5. The mRNA according to claim 4, characterized in that Both the Shine-Dalgarno sequence and the translated region contain the phosphorothioate group.

6. A method for producing mRNA, which is the method for producing mRNA according to any one of claims 1 to 5, characterized in that: include: The step of preparing a DNA template complementary to the mRNA including the untranslated region and the translated region; A DNA cutting step, wherein a point in the sequence corresponding to the range from the 5' end of the untranslated region to 11 nt of the translated region in the DNA template is cut to obtain a 5' end side DNA fragment and a 3' end side DNA fragment, wherein the 11 nt refers to the number of nucleotides counted from the nucleotide on the 5' end side of the triplet of the start codon; The step of preparing unmodified NTPs including adenosine triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate, and modified NTPs in which the phosphate group of at least one of the unmodified NTPs is replaced by a thiophosphate group; a modified mRNA preparation step, wherein the 5' terminal side DNA fragment is used as a template, and the unmodified NTP and the modified NTP are used as substrates to perform a transcription reaction to obtain a 5' terminal side modified mRNA fragment; an unmodified mRNA preparation step, wherein the 3' terminal DNA fragment is used as a template and only the unmodified NTP is used as a substrate for a transcription reaction to obtain a 3' terminal unmodified mRNA fragment; and The ligation step comprises ligating the 5' terminal modified mRNA fragment and the 3' terminal unmodified mRNA fragment.

7. A protein production device, characterized in that: have: The mRNA according to any one of claims 1 to 5, wherein the mRNA has a sequence encoding a target protein in the translation region; and A translation system, which at least comprises: ribosome, NTP, tRNA, amino acid, and aminoacyl-tRNA synthetase.

8. The protein production device according to claim 7, characterized in that The translation system is a recombinant E. coli translation system obtained by recombining the translation system of E. coli in vitro.

9. The protein production device according to claim 8, characterized in that A portion of the phosphate group of at least one of cytidine monophosphate and uridine monophosphate constituting the mRNA is substituted with a phosphorothioate group.

10. A method for producing a protein, characterized in that: include: An mRNA preparation step, wherein the mRNA is the mRNA according to any one of claims 1 to 5, and has a sequence encoding a target protein in the translation region; and The translation step comprises adding the mRNA to a translation system comprising at least ribosomes, NTPs, tRNA, amino acids, and aminoacyl-tRNA synthetase, and translating the target protein by the ribosomes.

11. The method for producing a protein according to claim 10, wherein: The translation system is a recombinant E. coli translation system obtained by recombining the translation system of E. coli in vitro.

12. The method for producing a protein according to claim 11, wherein A portion of the phosphate group of at least one of cytidine monophosphate and uridine monophosphate constituting the mRNA is substituted with a phosphorothioate group.

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