Mrna encoding red fluorescent protein and its use

Abstract

The application belongs to the field of biotechnology and medicine, and more particularly relates to mRNA encoding red fluorescent protein and application thereof. The mRNA encoding red fluorescent protein comprises an ORF sequence of red fluorescent protein, and the nucleotide sequence is as shown in SEQ ID NO: 5 to 13. The mRNA encoding red fluorescent protein provided in the application has high translation efficiency by reasonably optimizing the nucleic acid sequence of the functional region, so that the output of red fluorescent protein is increased, and the mRNA encoding red fluorescent protein has wide application prospects in the field of gene drug development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine, and more particularly relates to mRNA encoding red fluorescent protein and its application. BACKGROUND

[0002] Firefly luciferase is a new fluorescent protein gene isolated from sea anemone, which can emit red fluorescence under the excitation of ultraviolet light. It is the natural luminescent fluorescent protein with the longest excitation and emission wavelengths ever discovered, and it has less background interference. It has been widely used as a reporter gene for gene expression in eukaryotic cells such as animals, plants and yeasts.

[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 approach includes encapsulating mRNA containing red fluorescent protein reporter gene into lipid nanoparticles (LNP), injecting into the animal body, and then detecting the luminescence intensity by flow cytometry or immunological imaging technology to evaluate its delivery efficiency, distribution and metabolism. In the experimental design, in order to ensure the experimental effect while reducing the cost of materials and meeting the animal ethics requirements, it is particularly important to choose the lowest possible LNP-mRNA dose. At the same time, ensuring the high safety of mRNA sequence encapsulated by lipid nanoparticle (LNP) carrier injected into the body is crucial to ensure the treatment effect and patient safety. This is related to reducing potential immune response and side effects, ensuring the long-term stability and biocompatibility of drug delivery systems in the body. Therefore, accurately evaluating and designing the safety of mRNA sequence is not only a necessary step for drug development, but also a key to improving efficacy and reducing treatment risks. On this basis, developing a red fluorescent protein mRNA sequence that can produce higher protein yield will greatly improve the economic benefit and scientific value of the experiment. SUMMARY

[0004] The present application provides a brand new mRNA encoding red fluorescent protein. By optimizing the nucleic acid sequence of the functional region, the mRNA encoding red fluorescent protein has a high translation efficiency, thereby increasing the output of red fluorescent protein. The mRNA encoding red fluorescent protein has a wide application prospect in the field of gene drug development. Therefore, the present application proposes the following technical solutions.

[0005] The present application provides an mRNA encoding a red fluorescent protein, comprising an ORF sequence encoding a red fluorescent protein, the nucleotide sequence of which is 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 identical to any one of SEQ ID NOs: 5-10; preferably, the nucleotide sequence of the ORF sequence is the nucleotide sequence shown in any one of SEQ ID NOs: 5-10.

[0006] Further, the mRNA further comprises a 5' UTR and / or a 3' UTR;

[0007] The nucleotide sequence of the 5' UTR is a nucleotide sequence or its complement sequence which is 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 identical to SEQ ID NO: 1, 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 or its complement sequence which is 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 identical to SEQ ID NO: 2 or 3, preferably, the nucleotide sequence of the 3' UTR is the nucleotide sequence shown in SEQ ID NO: 2 or 3.

[0008] Preferably, the mRNA comprises, in the order from 5' to 3', a 5' UTR shown in SEQ ID NO: 1, an ORF sequence shown in any one of SEQ ID NOs: 5-10, and a 3' UTR shown in SEQ ID NOs: 2-3; preferably, the mRNA comprises, in the order from 5' to 3', a 5' UTR shown in SEQ ID NO: 1, an ORF sequence shown in SEQ ID NO: 7 or 9, and a 3' UTR shown in SEQ ID NO: 2; or,

[0009] The mRNA comprises, in the order from 5' to 3', a 5' UTR shown in SEQ ID NO: 1, an ORF sequence shown in SEQ ID NO: 5, 6 or 8 or 10, and a 3' UTR shown in SEQ ID NO: 4.

[0010] Further preferred, 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 at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 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.

[0011] The present application provides a DNA molecule encoding the mRNA.

[0012] The present application provides a vector comprising the DNA molecule.

[0013] The present application further provides a cell comprising the mRNA, the DNA molecule, or the vector.

[0014] The present application also provides a method for preparing a red fluorescent protein by culturing the cell, obtaining firefly luciferase;

[0015] or preparing the mRNA, the DNA molecule, or the vector into a linearized plasmid to obtain a red fluorescent protein by in vitro transcription and expression.

[0016] The present application also provides the use of the mRNA or the vector as a reporter gene.

[0017] Further provided is a lipid nanoparticle comprising the mRNA, the DNA molecule, or the vector. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the result of capillary electrophoresis detection of exemplary red fluorescent protein mRNA molecules.

[0019] Figure 2 is the fluorescence intensity of candidate mRNA molecules expressing red fluorescent protein at 24h.

[0020] Figure 3 is the detection of the proportion of candidate red fluorescent protein mRNA aggregates. DETAILED DESCRIPTION

[0021] I. Definitions and Descriptions

[0022] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and techniques employed herein relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunological procedures and laboratory procedures are those well known and commonly used in the corresponding art.

[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", "characterized by" and the like can be used interchangeably with the term "comprising", unless otherwise indicated.

[0024] The term "and / or", describes an associated relationship with associated objects, which means that there can be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist together, and B exists alone. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0025] "Nucleotides", "nucleotide molecules", "nucleotide sequences", or "nucleic acid fragments" are used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally can contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their individual letter designations: "A" for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. Although nucleotide sequences herein can be represented in the DNA sequence (containing T), the corresponding RNA sequence (i.e., replacing T with U) can be easily determined by those skilled in the art when referring to RNA.

[0026] The term "mRNA" refers to a single-stranded ribonucleic acid molecule that carries genetic information capable of directing protein synthesis in a cell.

[0027] 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 are artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term "protein" can also include modified forms, including but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0028] The term "Open Reading Frame" (ORF) refers to the normal nucleotide sequence of a structural gene, with the potential to encode a protein or polypeptide, beginning with an initiation codon and ending with a termination codon, without intervening termination codons that would disrupt translation. On a strand of mRNA, the ribosome begins translation at the initiation codon, synthesizing the polypeptide chain along the RNA sequence and extending until it encounters a termination codon. The terms "ORF" or "ORF sequence" are used interchangeably.

[0029] The term "identity" refers to the ratio, expressed as a percentage, of mismatched nucleotides to the total number of nucleotides in a homologous region. 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 to that region.

[0030] The term "complement" refers to a nucleic acid that forms hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types.

[0031] The term "5' UTR" refers to the sequence of an mRNA molecule between the 5' end and the translation initiation codon, which is capable of recruiting ribosome complexes and initiating translation of the mRNA. The 5' UTR includes the 5' UTR region structure on the mRNA or the structure corresponding to the coding sequence on the DNA template. The 5' UTR regulates processes such as post-transcriptional modification, formation and stability of the translation initiation complex, etc. by interacting with transcription factors, ribosomes and other transcriptional regulatory proteins.

[0032] The term "3' UTR" refers to the sequence of an mRNA between the stop codon of the polypeptide coding sequence and the poly(A) sequence. The 3' UTR can regulate translation of the mRNA by interacting with mRNA binding proteins, miRNAs, etc. The 3' UTR includes the 3' UTR region structure on the mRNA or the structure corresponding to the coding sequence on the DNA template. It is closely related to post-transcriptional modification and mRNA stability. The sequence and structural features of the 3' UTR can affect the stability of the mRNA, the scanning of the ribosome and the formation of the translation termination complex, etc., thereby affecting the expression level of the protein.

[0033] The term "poly-A sequence" includes the poly-A sequence structure on mRNA or the structure corresponding to the coding sequence on the DNA template. The addition of the poly-A sequence helps the stability and transport of the 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 include non-adenine nucleotides. In any form, as long as it is functionally equivalent to the traditional poly-A sequence, i.e., it can provide similar biological functions to the traditional poly-A sequence, such as affecting the stability, translation efficiency or ribosome binding of mRNA, etc., the sequence is recognized as a poly-A sequence. This includes but is not limited to known variants such as human growth hormone (hGH) poly-A sequence and monkey virus 40 (SV40) poly-A sequence, which can differ in nucleotide composition but are functionally equivalent to the traditional poly-A sequence.

[0034] The term "5' cap structure" includes the 5' cap structure present on natural mRNA and its analogs. The 5' cap structure on natural mRNA refers to the methylation of guanosine nucleotide connected to the 5' terminal nucleotide of RNA via pyrophosphate, forming a 5', 5'-triphosphate linkage. The 5' cap structure usually has three types (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), respectively called Cap0, Cap1 and Cap2. Cap0 refers to the ribose of the terminal nucleotide being unmethylated, Cap1 refers to the ribose of one nucleotide at the end being methylated, and Cap2 refers to the riboses of both nucleotides at the end being methylated. The method of capping mRNA molecules is known in the art. The 5' cap structure of the aforementioned mRNA molecules can be added after the mRNA molecules are obtained by chemical synthesis or in vitro transcription using an enzymatic reaction (for example, by using a commercial kit containing vaccinia capping enzyme and mRNA cap structure 2'-O-methyltransferase). However, mRNA with a cap structure can also be produced by directly incorporating nucleotide analogs with a cap structure as the first nucleotide into the transcript during in vitro transcription.

[0035] The term "chemical modification" or "chemically modified" refers to modification of one or more of the position, pattern, percentage, or population of an adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleoside or deoxyribonucleoside. In this context, these terms are not intended to refer to the ribonucleotide modifications in the naturally occurring 5' cap structure.

[0036] The term "vector" refers to a piece of DNA extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA segment can be or has been inserted for the purpose of cloning and / or expression. In certain embodiments, a vector can be stably maintained in an organism. Vectors can comprise, for example, an origin of replication, a selectable marker or 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, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0037] The terms "cell" and "host cell" are used interchangeably herein to refer to a cell that expresses or is capable of expressing a sequence to be expressed. Host cells of the present application express polynucleotides that encode polypeptides or RNAs having a variety of uses, including biotechnological, molecular biological, and clinical applications. Host cells include prokaryotic or eukaryotic cells, and examples of suitable host cells in the present application include, but are not limited to, bacterial, yeast cells, insect cells, animal cells, and mammalian cells.

[0038] The term "lipid nanoparticle" refers to a particle that contains a lipid component and has a nanoscale size.

[0039] The term "ionizable cationic lipid" refers to a lipid molecule that is capable of carrying a positive charge under physiological pH conditions. As an example, an ionizable cationic lipid is an amino lipid.

[0040] The term "neutral lipid" refers to a lipid molecule that does not carry a charge under a particular pH condition, such as physiological pH conditions.

[0041] The term "structural lipid" refers to a lipid that enhances the stability of a nanoparticle by filling the gaps between lipids, such as a sterol, for example.

[0042] The term "polymer lipid" refers to a molecule that contains a polymer moiety and a lipid moiety. In some embodiments, a polymer lipid is a polyethylene glycol (PEG) lipid.

[0043] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0044] The present application provides an mRNA encoding a red fluorescent protein, which comprises 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 to any one of SEQ ID NOs: 5-10. Preferably, the nucleotide sequence of the ORF sequence is the nucleotide sequence set forth in any one of SEQ ID NOs: 5-10.

[0045] In some embodiments, the mRNA further comprises a 5' UTR and / or a 3' UTR;

[0046] 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 complement thereof; preferably, the 5' UTR is the nucleotide sequence set forth 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 complement thereof; preferably, the 3' UTR is the nucleotide sequence set forth in SEQ ID NO: 2 or 3.

[0047] In some embodiments, the mRNA comprises, in the 5' to 3' order: a 5' UTR set forth in SEQ ID NO: 1, an ORF sequence set forth in any one of SEQ ID NOs: 5-10, and a 3' UTR set forth in SEQ ID NOs: 2-3; preferably, the mRNA comprises, in the 5' to 3' order: a 5' UTR set forth in SEQ ID NO: 1, an ORF sequence set forth in SEQ ID NO: 7 or 9, and a 3' UTR set forth in SEQ ID NO: 2; or, the mRNA comprises, in the 5' to 3' order: a 5' UTR set forth in SEQ ID NO: 1, an ORF sequence set forth in SEQ ID NO: 5, 6, or 8 or 10, and a 3' UTR set forth in SEQ ID NO: 3.

[0048] 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-methylpseudouridine, N1-ethylpseudouridine, 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.

[0049] The present application provides a DNA molecule encoding the mRNA.

[0050] The present application provides a vector comprising the DNA molecule.

[0051] In some embodiments, the vector is an expression vector, more particularly, an expression vector suitable for animal cells.

[0052] 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 (BACs) or yeast artificial chromosomes (YACs), etc. Preferably, the vector is a plasmid vector or a viral vector.

[0053] In some embodiments, the vector comprises a RNA polymerase promoter sequence, a RNA coding sequence, and / or a restriction endonuclease site and a selection marker. The RNA polymerase promoter sequence is operably linked to the RNA coding sequence, which allows the RNA molecule to be transcribed in vivo and / or in vitro. The RNA polymerase promoter can be of various types, including but not limited to a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, a SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0054] In some embodiments, the vector comprises a restriction endonuclease site, such as a type IIS restriction endonuclease site, flanking the 3' 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 sites can be used to linearize the vector for in vitro transcription.

[0055] Methods for obtaining RNA molecules from in vitro transcription of nucleic acid vectors are known in the art, for example, in vitro transcription can be performed using commercial kits.

[0056] The present application provides a cell comprising the mRNA, the DNA molecule, or the vector.

[0057] In some embodiments, the cell comprises a prokaryotic cell or a eukaryotic cell. Preferably, the aforementioned cell is selected from the group consisting of E. coli, a yeast cell, or an animal cell. More preferably, the cell is a mammalian cell, including but not limited to a rodent cell such as a mouse cell or a rat cell, etc.; a primate cell such as a monkey cell or a human cell, etc. Still more preferably, the cell is a human cell.

[0058] The present application provides a method for preparing a red fluorescent protein by culturing the cell, obtaining a red fluorescent protein;

[0059] or preparing the mRNA, the nucleotide molecule, or the vector into a linearized plasmid for expression by in vitro transcription to obtain a red fluorescent protein.

[0060] The present application provides the use of the mRNA or the DNA molecule or the vector as a reporter gene.

[0061] In some embodiments, the aforementioned mRNA or the DNA molecule or the vector is used for detecting the expression level of a gene, in vivo bioluminescent imaging, in vitro bioluminescent imaging cell tracking, high-throughput screening, safety and toxicology testing, or environmental monitoring.

[0062] The present application provides a kind of lipid nanoparticle, it includes the mRNA described, the DNA molecule described or the carrier described.

[0063] In some preferred embodiments, the above-mentioned lipid nanoparticle further comprises one or more lipid moieties selected from the group consisting of ionizable cationic lipids, structural lipids, neutral lipids or polymeric lipids.

[0064] The present application is described below by specific embodiments to better understand the present application, but does not constitute a limitation on the present application.

[0065] Example 1: Construction of a vector containing a red fluorescent protein mRNA sequence

[0066] In order to achieve the effect of improving the yield of red fluorescent protein, the UTR combination that can effectively improve the translation efficiency is 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 is screened out by sequence alignment, and further the technology in the field of natural language processing and the RNA secondary structure prediction model (such as CDSfold, Ribotree) is used to assign weights and identify stable mRNA sequences. And balance the two parameters of CAI and MFE, from which a batch of mRNA sequences that can achieve higher protein yield are screened out, and the ORF sequence of red fluorescent protein in the Cloning vector pGBT-R14 is used as a control sequence, and after being matched with two types of UTR pairs, it is named: 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 are obtained by gene synthesis, in vitro transcription template preparation, plasmid linearization, IVT mRNA synthesis, purification and quality inspection, etc. Steps to obtain mRNA molecules for in vitro cell experiments. Finally, 10 candidate red fluorescent protein mRNA sequences (see Table 2) are obtained.

[0067] Table 1. UTR sequence information

[0068]

[0069] Table 2. Candidate red fluorescent protein mRNA sequence structure composition

[0070]

[0071] To test the candidate mRNA sequences, nucleic acid fragments containing T7 promoter, 5' UTR, ORF sequence encoding red fluorescent protein (Table 2), 3' UTR, poly(A) sequence containing 70 A nucleotide residues, and IIS-type restriction endonuclease cleavage site (corresponding to RFP-1 to RFP-6) were synthesized in vitro and cloned into an in vitro transcription vector (pIVT Rup, Addgene plasmid #101362).

[0072] Example 2: mRNA molecule length and integrity detection

[0073] The vector obtained in Example 1 was linearized and used for in vitro transcription to produce mRNA molecules using T7-RNA polymerase, and at the same time, a 5' cap structure was added. The 5' cap structure was added by co-transcriptional capping, in which a cap analog was incorporated as the first nucleotide into the transcript during in vitro transcription, directly producing mRNA molecules with Cap1 structure. The mRNA molecules thus obtained were purified and resuspended in water. The mRNA molecules were quality tested using a 5200 Fragment Analyzer (Agilent) to detect the length and integrity of the mRNA molecules (the numerical value was derived from the area percentage of the curve of the expected length fragment) to meet the requirements (see Figure 1 ), which can be used for subsequent testing of different candidate sequences.

[0074] Example 3: In vitro expression level verification of red fluorescent protein mRNA sequence

[0075] To test whether the candidate red fluorescent protein mRNA sequences generated by the algorithm have higher protein output, the following method was used:

[0076] The same amount of the above candidate mRNA molecules and green fluorescent protein mRNA molecules were co-transfected into mammalian cells 293T. The fluorescence value of the red fluorescent protein was detected 24 h after transfection, representing the protein expression amount; the fluorescence value of the green fluorescent protein represented the transfection efficiency of the group. The fluorescence value of the red fluorescent protein was divided by the fluorescence value of the green fluorescent protein to obtain the relative fluorescence ratio of the group. Each candidate mRNA molecule was detected to obtain the corresponding relative fluorescence value in this experiment, which could represent the protein expression level of the candidate red fluorescent protein mRNA molecule.

[0077] The specific experimental process for determining the red fluorescent protein fluorescence value for translation efficiency is as follows:

[0078] 293T human embryonic kidney cells were seeded at 5 x 105cells / well in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The cells were then transfected with 1 μg of the candidate mRNA molecules and 1 μg of the green fluorescent protein mRNA molecules using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. The cells were then collected and resuspended in PBS. The fluorescence value of the red fluorescent protein was detected using a flow cytometer (BD Biosciences). 4Cells were seeded at a density of 1 cell / well in 48-well plates. The next day, cells were washed in Opti-MEM and then co-transfected with 2 μl of Lipofectamine 2000, 200 ng / well of the candidate red fluorescent protein-encoding mRNA molecule and 100 ng / well of a green fluorescent protein-encoding mRNA molecule in Opti-MEM. Cells without any RNA molecule added were used as background group. Six hours after transfection, the mixed medium was aspirated and replaced with complete medium.

[0079] Twenty-four hours after transfection, the medium was aspirated and 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 stopped with serum-containing medium. The cells were aspirated into sterile 1.5 ml centrifuge tubes, centrifuged at 2000 rpm for 5 min, washed twice with PBS, and the red and green fluorescence values were measured in a flow cytometer.

[0080] The relative fluorescence values of different candidate red fluorescent protein mRNA molecules at 24h were analyzed and plotted as shown in Figure 2

[0081] As can be seen from Figure 2 , at 24 hours after transfection, RFP-1, RFP-2, RFP-3, RFP-4, RFP-5, RFP-6 and other candidate red fluorescent protein mRNA have high protein yield, and are higher than the two control sequences.

[0082] Example 4: Detection of the proportion of candidate red fluorescent protein mRNA aggregates

[0083] The proportion of aggregates of the candidate red fluorescent protein mRNA sequence was analyzed in detail by size exclusion chromatography (SEC). This data reflects the aggregation state of the candidate mRNA, but also indicates the stability and usability of the candidate mRNA sequence in practical applications, thereby determining its efficiency and safety in biomedical applications. Larger aggregates can affect the bioavailability and safety of mRNA, reducing the safety and effectiveness of mRNA therapy by causing non-specific immune reactions or affecting delivery efficiency. Size exclusion chromatography is a technique commonly used to separate macromolecules or particles, and is particularly suitable for assessing the distribution of monomers and aggregates in RNA samples.

[0084] ​First, the candidate mRNA sequence is dissolved in a suitable buffer to ensure complete dissolution and dispersion of the mRNA. The sample containing the candidate mRNA molecules is diluted to 100-200 ng / ul with a buffer (100 mM Tris-Ac, 2.5 mM EDTA, pH 7.4) and subjected to size exclusion chromatography purification on a Waters Arc HPLC instrument at a loading speed of 0.5 ml / min. The choice of chromatographic column is based on its ability to effectively distinguish different molecular sizes to ensure that monomers and various sizes of aggregates of mRNA can be distinguished. The chromatographic column used in this study is: Agilent Bio SEC-5 2000A 7.8*300mm, 5.0um. During the process of sample passing through the chromatographic column and being eluted, the absorption of RNA is monitored in real time using an ultraviolet light detector. As the eluent flows, different sizes of mRNA molecules are separated due to their different retention times in the chromatographic column. According to the elution time and elution peak area of each molecule, the proportion of each component (monomer mRNA and different aggregates) is analyzed and determined. The detection results are shown in Figure 3 and Table 3. Figure 3 Table 3 shows the percentage of monomer mRNA, primary aggregate (aggregate component 1), secondary aggregate (aggregate component 2) and tertiary aggregate (aggregate component 3) in each sample. The monomer mRNA in the mRNA molecules of the control group also dominates. This provides a benchmark for comparison, showing that the candidate mRNA sequence has comparable or even superior effect on aggregate control compared to industry standards.

[0085] Table 3. Detection results of the proportion of candidate red fluorescent protein mRNA aggregates

[0086]

[0087] The proportion of monomer mRNA in the candidate red fluorescent protein mRNA is much higher than that in the control red fluorescent protein mRNA, indicating that the candidate red fluorescent protein mRNA has higher bioavailability and safety.

Claims

1. An mRNA encoding a red fluorescent protein, characterized in that, It comprises an ORF sequence encoding a red fluorescent protein, the nucleotide sequence of the ORF sequence is the nucleotide sequence shown in SEQ ID NO:

10.

2. The mRNA of claim 1, wherein The mRNA further comprises a 5' UTR and / or a 3' UTR.

3. The mRNA of claim 2, wherein 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 the nucleotide sequence shown in SEQ ID NO:

2.

4. The mRNA of claim 3, wherein, The mRNA comprises, in the order from 5' to 3', a 5' UTR shown in SEQ ID NO: 1, an ORF sequence shown in SEQ ID NO: 10, and a 3' UTR shown in SEQ ID NO:

3.

5. The mRNA according to any one of claims 1 to 4, 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 at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 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.

6. A DNA molecule encoding the mRNA of any one of claims 1-5.

7. A vector comprising the DNA molecule of claim 6.

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

9. A method of producing a red fluorescent protein, characterized by, It comprises culturing the cell of claim 8 to obtain firefly luciferase; or preparing the mRNA of any one of claims 1-5, the DNA molecule of claim 6, or the vector of claim 7 into a linearized plasmid to obtain a red fluorescent protein by in vitro transcription and expression.

10. Use of the mRNA of any one of claims 1-5 or the DNA molecule of claim 6 or the vector of claim 7 as a reporter gene.

11. A lipid nanoparticle comprising the mRNA of any one of claims 1-5, the DNA molecule of claim 6, or the vector of claim 7.

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