MRNA (messenger ribonucleic acid) for coding red fluorescent protein and application thereof

Abstract

The invention belongs to the field of biotechnology and medicine, and particularly relates to mRNA (messenger Ribonucleic Acid) for coding red fluorescent protein and application thereof. The mRNA for coding the red fluorescent protein comprises an ORF sequence of the red fluorescent protein, and the nucleotide sequences of the red fluorescent protein are shown as SEQ ID NO: 5-13. According to the mRNA for coding the red fluorescent protein provided by the invention, the mRNA for coding the red fluorescent protein has relatively high translation efficiency by reasonably optimizing the nucleic acid sequence of the functional region, so that the output of the red fluorescent protein is increased, and the mRNA for coding the red fluorescent protein has a wide application prospect in the field of gene drug development.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medicine, and more specifically relates to mRNA encoding red fluorescent protein and application thereof. Background Art

[0002] Red fluorescent protein (firefly luciferase) is a new fluorescent protein gene isolated from sea anemones. It can emit red fluorescence under the excitation of ultraviolet light. It is the naturally luminous fluorescent protein with the longest excitation wavelength and emission wavelength discovered so far, and its background interference is relatively small. It has been widely used as a reporter gene for gene expression in eukaryotic cells such as animals, plants and yeast.

[0003] In recent years, mRNA technology and lipid nanoparticle (LNP) delivery systems have shown unprecedented potential in the field of biomedicine, especially in vaccine development and genetic disease treatment. In the process of developing and exploring the functions of carrier materials such as lipid nanoparticles, in vivo tracing experiments are often widely carried out to indicate the delivery efficiency, distribution and metabolism of lipid nanoparticles. The usual practice includes encapsulating mRNA containing a red fluorescent protein reporter gene into lipid nanoparticles (LNPs), injecting them into animals, and then evaluating their delivery efficiency, distribution and metabolic status by detecting the luminescence intensity using flow cytometry or immunoimaging technology. In the experimental design, in order to ensure the experimental effect while reducing material costs and meeting animal ethics requirements, it is particularly important to choose the lowest possible LNP-mRNA dose. At the same time, ensuring that the mRNA sequence encapsulated by the lipid nanoparticle (LNP) carrier injected into the body has high safety is crucial to ensuring the therapeutic effect and patient safety. This is related to reducing potential immune responses and side effects, and ensuring the long-term stability and biocompatibility of the drug delivery system in the body. Therefore, accurately evaluating and designing the safety of mRNA sequences is not only a necessary step in drug development, but also the key to improving efficacy and reducing treatment risks. On this basis, developing a red fluorescent protein mRNA sequence that can produce higher protein production will greatly enhance the economic benefits and scientific value of the experiment. Summary of the invention

[0004] The present invention provides a new mRNA encoding red fluorescent protein, which has a higher translation efficiency by rationally optimizing the nucleic acid sequence of the functional region, thereby increasing the output of red fluorescent protein, and making the mRNA encoding red fluorescent protein have a wide application prospect in the field of gene drug development. Therefore, the present invention provides the following technical solution.

[0005] The present invention provides an mRNA encoding a red fluorescent protein, comprising an ORF sequence encoding a red fluorescent protein, wherein the nucleotide sequence of the mRNA is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with any one of SEQ ID NOs: 5 to 10; preferably, the nucleotide sequence of the ORF sequence is a nucleotide sequence shown in any one of SEQ ID NOs: 5 to 10.

[0006] Further, the mRNA also includes a 5'UTR and / or a 3'UTR; The nucleotide sequence of the 5'UTR is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 1, or its complementary sequence. Preferably, the nucleotide sequence of the 5'UTR is the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence of the 3'UTR element is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 2 or 3, or its complementary sequence. Preferably, the nucleotide sequence of the 3'UTR is the nucleotide sequence shown in SEQ ID NO: 2 or 3.

[0007] Preferably, the mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in any one of SEQ ID NOs: 5-10, and the 3'UTR shown in SEQ ID NOs: 2-3; preferably, the mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in SEQ ID NO: 7 or 9, and the 3'UTR shown in SEQ ID NO: 2; or, The mRNA comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 5, 6 or 8 or 10, and a 3'UTR as shown in SEQ ID NO: 4.

[0008] Further preferably, the mRNA further comprises a 5' cap structure and / or a poly A sequence and / or at least one chemical modification; the 5' cap structure is selected from a Cap0 cap structure, a Cap1 cap structure or a Cap2 cap structure; the poly A sequence comprises 20-500 adenine nucleotides; the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiouridine, At least one of 1-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine.

[0009] The present invention provides a DNA molecule encoding the mRNA.

[0010] The invention provides a vector comprising the DNA molecule.

[0011] The present invention further provides a cell, which comprises the mRNA, the DNA molecule, or the vector.

[0012] The present invention also provides a method for preparing red fluorescent protein, which comprises culturing the cells to obtain firefly luciferase; Alternatively, the mRNA, the DNA molecule, or the vector is prepared into a linearized plasmid, and the red fluorescent protein is obtained by in vitro transcription and expression.

[0013] The present invention also provides the use of the mRNA or the vector as a reporter gene.

[0014] A lipid nanoparticle is further provided, which comprises the mRNA, the DNA molecule, or the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is an exemplary capillary electrophoresis detection result of red fluorescent protein mRNA molecule.

[0016] Figure 2 is the fluorescence intensity of red fluorescent protein expressed by the candidate mRNA molecule at 24h.

[0017] Figure 3 It is a test of the ratio of candidate red fluorescent protein mRNA aggregates. DETAILED DESCRIPTION

[0018] I. Definition and Explanation In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0019] As used herein, the terms "comprises," "includes," "contains," and "has," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to the process, method, product, or device.

[0020] The term "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0021] "Nucleotide", "nucleotide molecule", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is purine (A or G), "Y" is pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "I" is inosine, and "N" is any nucleotide. Although nucleotide sequences herein may be expressed as DNA sequences (including T), when RNA is referred to, one skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0022] The term "mRNA" refers to a single-stranded RNA molecule that carries genetic information and directs protein synthesis within a cell.

[0023] The term "protein" refers to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term "protein" may also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0024] The term "Open Reading Frame" (ORF) refers to the normal nucleotide sequence of a structural gene, which has the potential to encode a protein or polypeptide, starting from the start codon and ending at the stop codon, without a stop codon in between to interrupt translation. On an mRNA chain, the ribosome starts translating from the start codon, synthesizes the polypeptide chain along the RNA sequence and continues to extend. When encountering the stop codon, the extension reaction of the polypeptide chain stops. The terms "ORF" or "ORF sequence" can be used interchangeably.

[0025] The term "identity" refers to the ratio of mismatched nucleotides to the total number of nucleotides in a homologous region expressed as a percentage. For example, a 20 base oligonucleotide that hybridizes to a homologous region (site) in a target genome with two mismatches is said to have 90% identity with that region.

[0026] The term "complementary sequence" refers to a nucleic acid sequence that forms hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types.

[0027] The term "5'UTR" refers to the sequence from the 5' end of the mRNA molecule to the translation start codon, which is able to recruit the ribosome complex and initiate the translation of the mRNA. The 5'UTR includes the 5'UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. The 5'UTR regulates post-transcriptional modification, the formation and stability of the translation initiation complex, etc. by interacting with transcription factors, ribosomes, and other transcription regulatory proteins.

[0028] The term "3'UTR" refers to the sequence between the stop codon of the polypeptide coding sequence in mRNA and the poly(A) sequence. 3'UTR can regulate the translation of mRNA by interacting with mRNA binding proteins, miRNA, etc. 3'UTR includes the 3'UTR region structure on mRNA or the structure corresponds to the coding sequence on the DNA template. It is closely related to post-transcriptional modification and mRNA stability. The sequence and structural characteristics of 3'UTR can affect the stability of mRNA, the scanning of ribosomes and the formation of translation termination complexes, etc., thereby affecting the expression level of protein.

[0029] The term "poly A sequence" includes a poly A sequence structure on mRNA or the structure corresponds to a coding sequence on a DNA template. The addition of a poly A sequence contributes to the stability and transport of mRNA, prevents its degradation, and plays an important role in the post-transcriptional modification process. The poly A sequence can be a continuous chain of pure adenine nucleotides or can contain non-adenine nucleotides. In any form, as long as its function is equivalent to that of a traditional poly A sequence, that is, it can provide similar biological functions as traditional poly A sequences, such as affecting the stability of mRNA, translation efficiency, or ribosome binding, the sequence is identified as a poly A sequence. This includes but is not limited to known variants such as the human growth hormone (hGH) poly A sequence and the simian virus 40 (SV40) poly A sequence, which may differ in nucleotide composition but are functionally identified as equivalent to traditional poly A sequences.

[0030] The term "5' cap structure" means a 5' cap structure and its analogs present on natural mRNA. The 5' cap structure on natural mRNA refers to a methylated guanylate connected to the 5' terminal nucleotide of RNA via pyrophosphate to form a 5', 5'-triphosphate connection (5', 5'-triphosphate linkage). There are three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), respectively referred to as Cap0, Cap1 and Cap2. Cap0 refers to the unmethylated ribose of the terminal nucleotide, Cap1 refers to the methylation of the ribose of one terminal nucleotide, and Cap2 refers to the methylation of the ribose of the two terminal nucleotides. Methods for capping mRNA molecules are known in the art. The 5' cap structure of the aforementioned mRNA molecule can be added by enzymatic reaction after obtaining the mRNA molecule by chemical synthesis or in vitro transcription (e.g., by a commercial kit comprising vaccinia capping enzyme and mRNA cap structure 2'-O-methyltransferase). However, capped mRNA can also be produced by incorporating a capping nucleotide analog directly as the first nucleotide into the transcript during in vitro transcription.

[0031] The term "chemical modification" or "chemically modified" refers to the modification of one or more of the position, pattern, percentage or population of adenosine (A), guanosine (G), uridine (U) or cytidine (C) ribonucleosides or deoxyribonucleosides. In this article, these terms are not intended to refer to ribonucleotide modifications in naturally occurring 5' cap structures.

[0032] The term "vector" refers to a DNA extracted from a virus, plasmid or a cell of a higher organism, into which a foreign DNA fragment can be inserted or has been inserted for cloning and / or expression purposes. In certain embodiments, the vector can be stably maintained in an organism. The vector may include, for example, an origin of replication, a selective marker or a reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc.

[0033] The terms "cell" and "host cell" are used interchangeably in the present invention and refer to cells that express or are capable of expressing a sequence to be expressed. The host cells of the present invention express polynucleotides encoding polypeptides or RNAs with a variety of uses, including biotechnology, molecular biology, and clinical applications. Host cells include prokaryotic cells or eukaryotic cells, and examples of suitable host cells in the present invention include, but are not limited to, bacteria, yeast cells, insect cells, animal cells, and mammalian cells.

[0034] The term "lipid nanoparticle" refers to particles containing lipid components and having nanometer-scale dimensions.

[0035] The term "ionizable cationic lipid" refers to a lipid molecule that can be positively charged under physiological pH conditions. For example, the ionizable cationic lipid is an amino lipid.

[0036] The term "neutral lipid" refers to lipid molecules that are uncharged under specific pH conditions, such as physiological pH conditions.

[0037] The term “structural lipid” refers to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids.

[0038] The term "polymer lipid" refers to a molecule containing a polymer portion and a lipid portion. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid.

[0039] II. Detailed description of specific implementation plan The present invention provides an mRNA encoding a red fluorescent protein, comprising an ORF sequence encoding a red fluorescent protein, wherein the nucleotide sequence of the ORF sequence is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with any one of SEQ ID NOs: 5 to 10. Preferably, the nucleotide sequence of the ORF sequence is a nucleotide sequence shown in any one of SEQ ID NOs: 5 to 10.

[0040] In some embodiments, the mRNA further comprises a 5'UTR and / or a 3'UTR; The nucleotide sequence of the 5'UTR is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 1, or a complementary sequence thereof; preferably, the 5'UTR is the nucleotide sequence shown in SEQ ID NO: 1. The nucleotide sequence of the 3'UTR element is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 2 or 3, or a complementary sequence thereof; preferably, the 3'UTR is the nucleotide sequence shown in SEQ ID NO: 2 or 3.

[0041] In some embodiments, the mRNA comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in any one of SEQ ID NOs: 5-10, and a 3'UTR as shown in SEQ ID NOs: 2-3; preferably, the mRNA comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 7 or 9, and a 3'UTR as shown in SEQ ID NO: 2; or, the mRNA comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NOs: 5, 6 or 8 or 10, and a 3'UTR as shown in SEQ ID NO: 3.

[0042] In some embodiments, the mRNA further comprises a 5' cap structure and / or a poly A sequence and / or at least one chemical modification. The 5' cap structure is selected from a Cap0 cap structure, a Cap1 cap structure or a Cap2 cap structure; preferably, the 5' cap structure is selected from a Cap1 cap structure. The poly A sequence comprises 20-500 adenine nucleotides; preferably, the poly A sequence comprises 25, 50, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450 or 500 adenine nucleotides; more preferably, the poly A sequence comprises 70 adenine nucleotides. The chemical modification is selected from at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine.

[0043] The present invention provides a DNA molecule encoding the mRNA.

[0044] The invention provides a vector comprising the DNA molecule.

[0045] In some embodiments, the vector is an expression vector, more specifically, an expression vector suitable for animal cells.

[0046] In some embodiments, the vector includes but is not limited to the following types: linear DNA fragments (such as PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BAC) or yeast artificial chromosomes (YAC), etc. Preferably, the vector is a plasmid vector or a viral vector.

[0047] In some embodiments, the carrier comprises an RNA polymerase promoter sequence, an RNA coding sequence, and / or a restriction endonuclease site and a selective marker. The RNA polymerase promoter sequence is operably connected to the RNA coding sequence, which allows RNA molecules to be transcribed in vivo and / or in vitro. The RNA polymerase promoter can be a polytype, includes but is not limited to T7 RNA polymerase promoter, T6 viral RNA polymerase promoter, SP6 viral RNA polymerase promoter, T3 viral RNA polymerase promoter or T4 viral RNA polymerase promoter.

[0048] In some embodiments, the vector comprises a restriction endonuclease site, such as a type IIS restriction endonuclease site, at the 3' flank of the coding sequence of the mRNA molecule. Suitable restriction endonucleases include, but are not limited to, BsmBI, BsaI, or SapI, etc. The aforementioned restriction endonuclease site can be used to linearize the vector for in vitro transcription.

[0049] Methods for obtaining RNA molecules by in vitro transcription from nucleic acid vectors are known in the art. For example, commercial kits can be used for in vitro transcription.

[0050] The present invention provides a cell, which comprises the mRNA, the DNA molecule, or the vector.

[0051] In some embodiments, the cell comprises a prokaryotic cell or a eukaryotic cell. Preferably, the cell is selected from the group consisting of Escherichia coli, yeast cells, or animal cells. More preferably, the cell is a mammalian cell, including but not limited to rodent cells such as mouse cells or rat cells, etc.; primate cells such as monkey cells or human cells, etc. Further preferably, the cell is a human cell.

[0052] The present invention provides a method for preparing red fluorescent protein, which comprises culturing the cells to obtain red luciferase; Alternatively, the mRNA, nucleotide molecule, or vector is prepared into a linearized plasmid, and the red fluorescent protein is obtained by in vitro transcription and expression.

[0053] The present invention provides the use of the mRNA or the DNA molecule or the vector as a reporter gene.

[0054] In some embodiments, the above-mentioned mRNA or the above-mentioned DNA molecule or the above-mentioned vector is used in detecting gene expression levels, in vivo bioluminescence imaging, in vitro bioluminescence imaging cell tracing, high-throughput screening, safety and toxicology testing or environmental monitoring.

[0055] The invention provides a lipid nanoparticle, which comprises the mRNA, the DNA molecule or the vector.

[0056] In some preferred embodiments, the lipid nanoparticles further comprise one or more lipid moieties, wherein the one or more lipid moieties are selected from the group consisting of ionizable cationic lipids, structural lipids, neutral lipids or polymer lipids.

[0057] The present invention is described below through specific implementation modes to provide a better understanding of the present invention, but the present invention is not limited thereto.

[0058] Example 1: Construction of a vector containing a red fluorescent protein mRNA sequence In order to achieve the effect of increasing the production of red fluorescent protein, the UTR combination that can effectively improve the translation efficiency was selected: 5'UTR (SEQ ID NO: 1) and 3'UTR (SEQ ID NO: 2-3) for sequence design and screening. For the red fluorescent protein determined by the amino acid sequence (SEQ ID NO: 11), the conservative codon sequence was screened out through sequence alignment, and the technology in the field of natural language processing and RNA secondary structure prediction models (such as CDSfold, Ribotree) were further used to assign weights and identify stable mRNA sequences. The two parameters of CAI and MFE were balanced and combined to screen out a batch of mRNA sequences that may achieve higher protein production. The ORF sequence of red fluorescent protein in Cloning vector pGBT-R14 was used as a control sequence. Two types of UTR pairs were added and named as RFP-ctrl 1 (5'UTR sequence is SEQ ID NO: 1, ORF sequence is SEQ ID NO: 4, 3'UTR sequence is SEQ ID NO: 2) and RFP-ctrl 2 (5'UTR sequence is SEQ ID NO: 1, ORF sequence is SEQ ID NO: 4, 3'UTR sequence is SEQ ID NO: 3). These mRNA sequences were obtained by gene synthesis, in vitro transcription template preparation, plasmid linearization, IVT mRNA synthesis, purification and quality inspection, etc., and used for in vitro cell experiments. Finally, 10 candidate red fluorescent protein mRNA sequences with advantages were obtained (see Table 2).

[0059] Table 1. UTR sequence information

[0060] Table 2. Structural composition of candidate red fluorescent protein mRNA sequences

[0061] To test candidate mRNA sequences, a nucleic acid fragment containing a T7 promoter, 5′UTR, an ORF sequence encoding red fluorescent protein ( Table 2 ), a 3′UTR, a poly(A) sequence containing 70 A nucleotide residues, and a type IIS restriction endonuclease cleavage site (corresponding to RFP-1 to RFP-6) was synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgeneplasmid #101362).

[0062] Example 2: mRNA molecule length and integrity detection The vector obtained in Example 1 was linearized, and T7-RNA polymerase was used to perform in vitro transcription to produce mRNA molecules, and a 5' cap structure was added at the same time. The 5' cap structure is capped by co-transcription, and the cap analog is incorporated into the transcript as the first nucleotide during in vitro transcription to directly produce an mRNA molecule with a Cap1 structure. The mRNA molecules obtained in this way were purified and resuspended in water. The mRNA molecules were quality checked using a 5200 fragment analyzer (Agilent) to detect whether the length and integrity of the mRNA molecules (the values ​​are derived from the area under the curve of the expected length fragments) met the requirements (see Figure 1 ), which can be used for subsequent testing of different candidate sequences.

[0063] Example 3: Verification of in vitro expression level of red fluorescent protein mRNA sequence The candidate red fluorescent protein mRNA sequences generated by the algorithm were tested in vitro to see whether they had higher protein output, as follows: The same amount of the candidate mRNA molecules and green fluorescent protein mRNA molecules were co-transfected into mammalian cells 293T. The fluorescence value of red fluorescent protein was detected 24 hours after transfection, representing its protein expression; the fluorescence value of green fluorescent protein represented the transfection efficiency of the group. The relative fluorescence ratio of the group was obtained by dividing the fluorescence value reading of red fluorescent protein by the fluorescence value of green fluorescent protein. Each candidate mRNA molecule was processed to the corresponding relative fluorescence value in this experiment, which can represent the protein expression level of the candidate red fluorescent protein mRNA molecule.

[0064] The specific experimental procedure for determining the red fluorescent protein fluorescence value of translation efficiency is as follows: 293T human embryonic kidney cells were cultured at 5×10 4The cells were seeded in a 48-well plate at a density of 10 cells / well. The next day, the cells were washed in Opti-MEM, and then 2 μl of Lipofectamine2000, 200 ng / well of the candidate mRNA molecule encoding red fluorescent protein, and 100 ng / well of mRNA molecule encoding green fluorescent protein were added to Opti-MEM for co-transfection. Cells without any RNA molecules were used as background groups. After 6 hours of transfection, the mixed culture medium was aspirated and replaced with complete culture medium.

[0065] 24 hours after transfection, the medium was removed, 50 μL of 0.2% trypsin was added to ensure that all cells were infiltrated, digested at 37°C for 1 minute, and the digestion was terminated by adding serum-containing medium. The cells were pipetted into a sterile 1.5 ml centrifuge tube, centrifuged at 2000 rpm for 5 minutes, washed twice with PBS, and the red and green fluorescence values ​​were measured in a flow cytometer.

[0066] The relative fluorescence values ​​of different candidate red fluorescent protein mRNA molecules at 24 h were analyzed and plotted, as shown in Figure 2. Figure 2 shown.

[0067] from Figure 2 It can be seen that 24 hours after transfection, candidate red fluorescent protein mRNAs such as RFP-1, RFP-2, RFP-3, RFP-4, RFP-5, and RFP-6 have higher protein production than the two control sequences.

[0068] Example 4: Detection of the ratio of candidate red fluorescent protein mRNA aggregates The aggregate ratio of the candidate red fluorescent protein mRNA sequence was analyzed in detail by size exclusion chromatography (SEC). While the data reflects the aggregation state of the candidate mRNA, it also indicates the stability and availability of the candidate mRNA sequence in practical applications, thereby determining its efficiency and safety in biomedical applications. Larger aggregates may affect the bioavailability and safety of mRNA, reducing the safety and effectiveness of mRNA therapy by causing nonspecific immune responses or affecting delivery efficiency. Size exclusion chromatography is a technique commonly used to separate large molecules or particles, and is particularly suitable for evaluating the distribution of monomers and aggregates in RNA samples.

[0069] First, the candidate mRNA sequence was dissolved in a suitable buffer to ensure complete dissolution and dispersion of the mRNA. The sample containing the candidate mRNA molecule was diluted to 100-200 ng / ul with a buffer (100mM Tris-Ac, 2.5mM EDTA, pH 7.4) and purified by size exclusion chromatography on a Waters Arc HPLC instrument at a loading speed of 0.5ml / min. The selection of the chromatographic column was based on its ability to effectively distinguish different molecular sizes to ensure that monomers and aggregates of various sizes of mRNA can be distinguished. The chromatographic column used in this study was: Agilent Bio SEC-5 2000A 7.8*300mm, 5.0μm. During the process of the sample passing through the chromatographic column and being eluted, the absorption of RNA was monitored in real time using a UV detector. As the eluent flows, mRNA molecules of different sizes are separated due to their different retention times in the chromatographic column. The ratio of each component (monomer mRNA and different aggregates) is analyzed and determined based on the elution time and the area of ​​the elution peak of each molecule. Figure 3 And as shown in Table 3. Figure 3 Table 3 and Table 4 show the percentage of monomeric mRNA, primary aggregates (aggregate component 1), secondary aggregates (aggregate component 2), and tertiary aggregates (aggregate component 3) in each sample, and monomeric mRNA is also dominant in the mRNA molecules used as the control group. This provides a benchmark for comparison, showing that the candidate mRNA sequence is comparable to or even better than the industry standard in aggregate control.

[0070] Table 3. Detection results of candidate red fluorescent protein mRNA aggregate ratios

[0071] The proportion of monomeric mRNA in the candidate red fluorescent protein mRNA is much higher than that in the control group red fluorescent protein mRNA, indicating that the candidate red fluorescent protein mRNA has higher biological availability and safety.

Claims

1. An mRNA encoding a red fluorescent protein, characterized in that: It includes an ORF sequence encoding a red fluorescent protein, the nucleotide sequence of the ORF sequence is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with any one of SEQ ID NOs: 5 to 10; preferably, the nucleotide sequence of the ORF sequence is a nucleotide sequence shown in any one of SEQ ID NOs: 5 to 10.

2. The mRNA according to claim 1, characterized in that The mRNA further comprises a 5'UTR and / or a 3'UTR; The nucleotide sequence of the 5'UTR is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 1, or its complementary sequence. Preferably, the nucleotide sequence of the 5'UTR is the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence of the 3'UTR is a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 2 or 3, or its complementary sequence. Preferably, the nucleotide sequence of the 3'UTR is the nucleotide sequence shown in SEQ ID NO: 2 or 3.

3. The mRNA according to claim 2, characterized in that The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in any one of SEQ ID NOs: 5-10, and the 3'UTR shown in SEQ ID NOs: 2-3; preferably, the mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in SEQ ID NOs: 7 or 9 or 10, and the 3'UTR shown in SEQ ID NO: 2; or, The mRNA comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 5, 6 or 8 or 10, and a 3'UTR as shown in SEQ ID NO:

3.

4. The mRNA according to any one of claims 1 to 3, characterized in that The mRNA further comprises a 5' cap structure and / or a poly A sequence and / or at least one chemical modification; the 5' cap structure is selected from a Cap0 cap structure, a Cap1 cap structure or a Cap2 cap structure; the poly A sequence comprises 20-500 adenine nucleotides; the chemical modification is selected from pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1- At least one of methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine.

5. A DNA molecule encoding the mRNA according to any one of claims 1 to 4. A vector comprising the DNA molecule according to claim 5.

7. A cell comprising the mRNA according to any one of claims 1 to 4, the DNA molecule according to claim 5, or the vector according to claim 6.

8. A method for preparing red fluorescent protein, characterized in that: The method comprises culturing the cell according to claim 7 to obtain firefly luciferase; Alternatively, the mRNA according to any one of claims 1 to 4, the DNA molecule according to claim 5, or the vector according to claim 6 is prepared into a linearized plasmid, and the red fluorescent protein is obtained by in vitro transcription and expression.

9. Use of the mRNA according to any one of claims 1 to 4, the DNA molecule according to claim 5, or the vector according to claim 6 as a reporter gene.

10. A lipid nanoparticle comprising the mRNA according to any one of claims 1 to 4, the DNA molecule according to claim 5, or the vector according to claim 6.

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