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] This invention belongs to the fields of biotechnology and medicine, and more specifically relates to mRNA encoding red fluorescent protein and its applications. Background Technology

[0002] Firefly luciferase is a novel fluorescent protein gene isolated from sea anemones. It emits red fluorescence under ultraviolet light excitation and is currently the naturally luminescent fluorescent protein with the longest excitation and emission wavelengths. Furthermore, it has relatively low background interference and is now 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 demonstrated unprecedented potential in the biomedical field, particularly in vaccine development and the treatment of genetic diseases. During the research and functional exploration of carrier materials such as lipid nanoparticles, in vivo tracking experiments are frequently conducted to characterize their delivery efficiency, distribution, and metabolism. Common practices include encapsulating mRNA containing a red fluorescent protein reporter gene into lipid nanoparticles (LNPs), injecting them into animals, and then using flow cytometry or immunoimaging techniques to detect the fluorescence intensity to assess delivery efficiency, distribution, and metabolism. In experimental design, selecting the lowest possible LNP-mRNA dose is crucial to ensuring experimental efficacy while minimizing material costs and meeting animal ethics requirements. Simultaneously, ensuring the high safety of the mRNA sequence encapsulated in the injected lipid nanoparticle (LNP) carrier is essential for guaranteeing therapeutic efficacy and patient safety. This relates to reducing potential immune responses and side effects, and ensuring the long-term stability and biocompatibility of the drug delivery system in vivo. Therefore, accurately assessing and designing the safety of mRNA sequences is not only a necessary step in drug development but also key to improving efficacy and reducing treatment risks. Based on this, developing a red fluorescent protein mRNA sequence that can produce higher protein yields will greatly enhance the economic benefits and scientific value of the experiment. Summary of the Invention

[0004] This invention provides a novel mRNA encoding red fluorescent protein. By rationally optimizing the nucleic acid sequence of the functional region, this mRNA encoding red fluorescent protein exhibits high translation efficiency, thereby increasing the production of red fluorescent protein and making it a promising candidate for gene therapy. Therefore, this invention proposes the following technical solution.

[0005] The present invention provides an mRNA encoding a red fluorescent protein, comprising an ORF sequence encoding the red fluorescent protein, the nucleotide sequence of which 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 NO: 5 to 10; preferably, the nucleotide sequence of the ORF sequence is any one of the nucleotide sequences shown in SEQ ID NO: 5 to 10.

[0006] Furthermore, 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 a complementary sequence thereof. 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 a complementary sequence thereof. 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 a 5' to 3' order: the 5' UTR shown in SEQ ID NO:1, the ORF sequence shown in any one of SEQ ID NO:5-10, and the 3' UTR shown in SEQ ID NO:2-3; preferably, the mRNA comprises, in a 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 the order of 5' to 3', the 5' UTR shown in SEQ ID NO:1, the ORF sequence shown in SEQ ID NO:5, 6, 8, or 10, and the 3' UTR shown in SEQ ID NO:4.

[0008] More preferably, the mRNA further comprises a 5' cap structure and / or a polyA sequence and / or at least one chemical modification; the 5' cap structure is selected from Cap0 cap structure, Cap1 cap structure, or Cap2 cap structure; the polyA 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, etc. At least one of the following: 1-methyl-1-deaza-pseuuridine, 2-thio-T-methyl-pseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methyl-pseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, or 5-methoxyuridine and 2'-O-methyluridine.

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

[0010] The present invention provides a carrier comprising the aforementioned DNA molecule.

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

[0012] The present invention also provides a method for preparing red fluorescent protein, which involves culturing the cells to obtain red fluorescent protein; Alternatively, the mRNA, DNA molecule, or vector described herein may be prepared into a linearized plasmid and expressed via in vitro transcription to obtain red fluorescent protein.

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

[0014] A further provision provides a lipid nanoparticle comprising the mRNA, the DNA molecule, or the carrier. Attached Figure Description

[0015] Figure 1 This is an example of capillary electrophoresis detection results for red fluorescent protein mRNA molecules.

[0016] Figure 2 It represents the fluorescence intensity of the candidate mRNA molecule expressing red fluorescent protein after 24 hours.

[0017] Figure 3 It is the detection of the proportion of candidate red fluorescent protein mRNA aggregates. Detailed Implementation

[0018] I. Definitions and Explanations In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields.

[0019] As used herein, the terms “comprising,” “including,” “containing,” and “having,” and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0020] The term "and / or" describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship.

[0021] The terms “nucleotide,” “nucleotide molecule,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and refer to single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “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, C, or T, “I” for inosine, and “N” for any nucleotide. Although nucleotide sequences may be represented as DNA sequences (containing T) herein, when referring to RNA, those 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 ribonucleic acid molecule that carries genetic information and can guide protein synthesis within cells.

[0023] The term "protein" refers to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term "protein" may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0024] The term "open reading frame" (ORF) refers to the normal nucleotide sequence of a structural gene, possessing the potential to encode a protein or polypeptide. It begins with a start codon and ends with a stop codon, without any stop codons that would interrupt translation. On an mRNA strand, ribosomes begin translation from the start codon, synthesizing a polypeptide chain along the RNA sequence and continuously elongating it. The elongation of the polypeptide chain terminates when a stop codon is encountered. The terms "ORF" or "ORF sequence" are used interchangeably.

[0025] The term "identity" refers to the ratio, expressed as a percentage, of mismatched nucleotides in a homologous region to the total number of nucleotides. 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 forming a hydrogen bond with another nucleic acid sequence through a traditional Watson-Crick bond or other non-traditional type.

[0027] The term "5'UTR" refers to the sequence from the 5' end of an mRNA molecule to the translation initiation codon, which recruits the ribosome complex and initiates mRNA translation. The 5'UTR includes the 5'UTR region structure on the mRNA or its corresponding coding sequence on the DNA template. The 5'UTR regulates post-transcriptional modifications, translation initiation complex formation, and stability by interacting with transcription factors, ribosomes, and other transcriptional 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. The 3'UTR can regulate mRNA translation through interactions with mRNA-binding proteins, miRNAs, and other organisms. The 3'UTR includes the 3'UTR region structure on mRNA or the structure corresponding to the coding sequence on the DNA template. It is closely related to post-transcriptional modifications and mRNA stability. The sequence and structural features of the 3'UTR can affect mRNA stability, ribosome scanning, and the formation of translation termination complexes, thereby influencing protein expression levels.

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

[0030] The term "5' cap structure" encompasses 5' cap structures present on native mRNA and their analogues. A 5' cap structure on native mRNA refers to a methylated guanosine monophosphate nucleotide linked to the 5' terminal nucleotide of the RNA via pyrophosphate, forming a 5',5'-triphosphate linkage. There are generally three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as Cap0, Cap1, and Cap2, respectively. Cap0 indicates that the ribose of the terminal nucleotide is unmethylated, Cap1 indicates that the ribose of the terminal nucleotide is methylated, and Cap2 indicates that the ribose of both terminal nucleotides is methylated. Methods for capping mRNA molecules are known in the art. The aforementioned 5' cap structure of mRNA molecules can be added via an enzymatic reaction after obtaining mRNA molecules through chemical synthesis or in vitro transcription (e.g., using commercially available kits containing a vaccinia capping enzyme and an mRNA cap structure 2'-O-methyltransferase). However, capped mRNA can also be produced by directly incorporating a capped nucleotide analog as the first nucleotide into the transcript during in vitro transcription.

[0031] The terms “chemically modified” or “chemically modified” refer to modifications of one or more of the positions, patterns, percentages, or groups of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides or deoxyribonucleosides. In this document, these terms are not intended to refer to modifications of ribonucleotides in naturally occurring 5' cap structures.

[0032] The term "vector" refers to a segment of DNA extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment can be inserted or has already been inserted for cloning and / or expression purposes. In some embodiments, the vector can be stably maintained in the organism. A vector may contain, for example, an origin of replication, a selection 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, linear plasmid fragments), plasmid vectors, viral vectors, granules, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc.

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

[0034] The term "lipid nanoparticles" refers to particles containing lipid components and having a nanoscale size.

[0035] The term "ionizable cationic lipid" refers to lipid molecules that can carry a positive charge under physiological pH conditions. For example, ionizable cationic lipids are amino lipids.

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

[0037] The term "structural lipids" 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 both a polymer portion and a lipid portion. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid.

[0039] II. Detailed Implementation Plan This invention provides an mRNA encoding a red fluorescent protein, comprising an ORF sequence encoding the 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 NO: 5 to 10. Preferably, the nucleotide sequence of the ORF sequence is any one of the nucleotide sequences shown in SEQ ID NO: 5 to 10.

[0040] In some embodiments, the mRNA further 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 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: the 5' UTR shown in SEQ ID NO:1, the ORF sequence shown in any one of SEQ ID NO:5-10, and the 3' UTR shown in SEQ ID NO: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: the 5' UTR shown in SEQ ID NO:1, the ORF sequence shown in SEQ ID NO:5, 6, 8, or 10, and the 3' UTR shown in SEQ ID NO:3.

[0042] In some embodiments, the mRNA further comprises a 5' cap structure and / or a polyA sequence and / or at least one chemical modification. The 5' cap structure is selected from Cap0, Cap1, or Cap2 cap structures; preferably, the 5' cap structure is selected from Cap1 cap structures. The polyA sequence comprises 20-500 adenine nucleotides; preferably, the polyA 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 polyA sequence comprises 70 adenine nucleotides. The chemical modification is selected from at least one of pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseuuridine, 2-thio-T-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, or 5-methoxyuridine and 2'-O-methyluridine.

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

[0044] The present invention provides a carrier comprising the aforementioned DNA molecule.

[0045] In some implementations, 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, granules, bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs). Preferably, the vector is a plasmid vector or a viral vector.

[0047] In some embodiments, the vector comprises an RNA polymerase promoter sequence, an RNA coding sequence, and / or a restriction endonuclease site and a selection marker. The RNA polymerase promoter sequence is operatively linked to the RNA coding sequence, which allows RNA molecules to be transcribed in vivo and / or in vitro. The RNA polymerase promoter can be of various types, including but not limited to the T7 RNA polymerase promoter, the T6 viral RNA polymerase promoter, the SP6 viral RNA polymerase promoter, the T3 viral RNA polymerase promoter, or the T4 viral RNA polymerase promoter.

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

[0049] Methods for obtaining RNA molecules from nucleic acid vectors through in vitro transcription are known in the art, for example, in vitro transcription can be performed using commercially available kits.

[0050] The present invention provides a cell comprising the mRNA, the DNA molecule, or the vector described herein.

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

[0052] This invention provides a method for preparing red fluorescent protein, which involves culturing the cells to obtain red luciferase; Alternatively, the mRNA, nucleotide molecules, or vector contained herein can be prepared into a linearized plasmid and expressed via in vitro transcription to obtain red fluorescent protein.

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

[0054] In some embodiments, the mRNA, DNA molecule, or vector described above 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 present invention provides a lipid nanoparticle comprising the mRNA, the DNA molecule, or the carrier described herein.

[0056] In some preferred embodiments, the lipid nanoparticles further include one or more lipid portions selected from the group consisting of ionizable cationic lipids, structural lipids, neutral lipids, or polymeric lipids.

[0057] The present invention will be described below through specific embodiments to better understand the present invention, but this does not constitute a limitation on the present invention.

[0058] Example 1: Construction of a vector containing a red fluorescent protein mRNA sequence To improve the yield of red fluorescent protein, a UTR combination that effectively enhances 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 with the amino acid sequence (SEQ ID NO: 11), conserved codon sequences were screened through sequence alignment. Furthermore, techniques from the field of natural language processing and RNA secondary structure prediction models (such as CDSfold and Ribotree) were used to assign weights and identify stable mRNA sequences. By balancing the CAI and MFE parameters, a batch of mRNA sequences with the potential to achieve high protein yields were screened. The ORF sequence of red fluorescent protein from the cloning vector pGBT-R14 was used as a control sequence. These sequences were paired with two types of UTR pairs and named: RFP-ctrl 1 (5' UTR sequence SEQ ID NO:1, ORF sequence SEQ ID NO:4, 3' UTR sequence SEQ ID NO:2) and RFP-ctrl 2 (5' UTR sequence SEQ ID NO:1, ORF sequence SEQ ID NO:4, 3' UTR sequence SEQ ID NO:3). These mRNA sequences underwent gene synthesis, in vitro transcription template preparation, plasmid linearization, IVT mRNA synthesis, purification, and quality control to obtain mRNA molecules for in vitro cell experiments. Finally, 10 candidate superior red fluorescent protein mRNA sequences 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, nucleic acid fragments containing a T7 promoter, a 5'UTR, an ORF sequence encoding red fluorescent protein (Table 2), a 3'UTR, a poly(A) sequence containing 70 A nucleotide residues, and IIS type restriction endonuclease cleavage sites (corresponding to RFP-1 to RFP-6) were synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgeneplasmid#101362).

[0062] Example 2: Detection of mRNA molecule length and integrity The vector obtained in Example 1 was linearized and in vitro transcribed using T7-RNA polymerase to produce mRNA molecules, with a 5' cap structure added simultaneously. The 5' cap structure, through co-transcriptional capping, incorporates a cap analog as the first nucleotide into the transcript during in vitro transcription, directly producing mRNA molecules with the Cap1 structure. The resulting mRNA molecules were purified and resuspended in water. Quality control of the mRNA molecules was performed using an Agilent 5200 fragment analyzer to detect mRNA molecule length and integrity (values ​​derived from the area under the curve of the expected length fragment). The mRNA molecules met the requirements (see [link to Agilent Fragment Analyzer]). Figure 1 This information can then be used for subsequent testing of different candidate sequences.

[0063] Example 3: Validation of the in vitro expression level of red fluorescent protein mRNA sequence Whether the candidate red fluorescent protein mRNA sequences generated by the algorithm for in vitro experimental testing have higher protein yield is determined by the following method: Mammalian 293T cells were co-transfected with equal amounts of the aforementioned candidate mRNA molecules and green fluorescent protein (GFP) mRNA molecules. 24 hours post-transfection, the fluorescence value of red fluorescent protein (RFP) was measured, representing its protein expression level; the fluorescence value of GFP represented the transfection efficiency of the group. The relative fluorescence ratio of the group was obtained by dividing the fluorescence value of red RFP by the fluorescence value of GFP. The relative fluorescence value obtained for each candidate mRNA molecule in this experiment represents the protein expression level of the candidate red RFP mRNA molecule.

[0064] The specific experimental procedure for determining the fluorescence value of the red fluorescent protein used to determine translation efficiency is as follows: 293T human embryonic kidney cells were used at 5×10 4Cells were seeded at a density of 100 cells / well in 48-well plates. The following day, cells were washed in Opti-MEM and then co-transfected with a mixture of 2 μl of Lipofectamine 2000, 200 ng / well of candidate mRNA encoding red fluorescent protein, and 100 ng / well of green fluorescent protein mRNA. Cells without any added RNA were used as the background group. Six hours after transfection, the mixed medium was aspirated and replaced with complete medium.

[0065] 24 hours after transfection, the culture medium was aspirated, and 50 μL of 0.2% trypsin was added to ensure all cells were infiltrated. Digestion was carried out at 37°C for 1 minute, and serum-containing culture medium was added to terminate the digestion. The cells were transferred to 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 by flow cytometry.

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

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

[0068] Example 4: Detection of the proportion of candidate red fluorescent protein mRNA aggregates A detailed analysis of the aggregate proportions of candidate red fluorescent protein mRNA sequences was performed using size exclusion chromatography (SEC). This data reflects the aggregation state of candidate mRNAs and also indicates the stability and usability of candidate mRNA sequences in practical applications, thereby determining their efficiency and safety in biomedical applications. Larger aggregates may affect the bioavailability and safety of mRNAs, reducing the safety and efficacy of mRNA therapies by inducing 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 assessing the distribution of monomers and aggregates in RNA samples.

[0069] First, the candidate mRNA sequences were dissolved in a suitable buffer to ensure complete dissolution and dispersion. The sample containing the candidate mRNA molecules was diluted to 100-200 ng / µl with buffer (100 mM Tris-Ac, 2.5 mM EDTA, pH 7.4) and purified by size exclusion chromatography on a Waters Arc HPLC instrument at a loading rate of 0.5 mL / min. The column was chosen based on its ability to effectively resolve different molecular sizes to ensure the differentiation between mRNA monomers and aggregates of various sizes. The column used in this study was an Agilent Bio SEC-5 2000A 7.8*300 mm, 5.0 μm. During the sample passage and elution process, the RNA uptake was monitored in real time using a UV detector. As the eluent flowed, mRNA molecules of different sizes were separated due to their varying residence times within the column. The proportions of each component (monomer mRNA and different aggregates) are analyzed and determined based on the elution time and peak area of ​​each molecule. The detection results are as follows: Figure 3 As shown in Table 3. Figure 3 Table 3 shows the percentages of monomeric mRNA, primary aggregates (aggregate component 1), secondary aggregates (aggregate component 2), and tertiary aggregates (aggregate component 3) in each sample. Monosomic mRNA also predominated in the control group. This provides a benchmark for comparison, demonstrating that the candidate mRNA sequences perform comparably to or even better than industry standards in aggregate control.

[0070] Table 3. Detection results of the proportion of candidate red fluorescent protein mRNA aggregates

[0071] The proportion of monomeric mRNA in the candidate red fluorescent protein mRNA was much higher than that in the control group, 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, 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 of SEQ ID NO: 5 to 10; preferably, the nucleotide sequence of the ORF sequence is a nucleotide sequence shown in any of SEQ ID NO: 5 to 10.

2. The mRNA as described in claim 1, characterized in that, 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 a complementary sequence thereof. 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 a complementary sequence thereof. Preferably, the nucleotide sequence of the 3'UTR is the nucleotide sequence shown in SEQ ID NO: 2 or 3.

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

3.

4. The mRNA according to any one of claims 1-3, characterized in that, The mRNA further comprises a 5' cap structure and / or a polyA sequence and / or at least one chemical modification; the 5' cap structure is selected from Cap0 cap structure, Cap1 cap structure, or Cap2 cap structure; the polyA 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-thio-1- At least one of methyl-1-deaza-pseuuridine, 2-thio-T-methyl-pseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methyl-pseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, or 5-methoxyuridine and 2'-O-methyluridine.

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

6. A vector comprising the DNA molecule of claim 5.

7. A cell comprising the mRNA as claimed in any one of claims 1-4, the DNA molecule as claimed in claim 5, or the vector as claimed in claim 6.

8. A method for preparing red fluorescent protein, characterized in that, It obtains red fluorescent protein by culturing the cells as described in claim 7; Alternatively, a linearized plasmid containing the mRNA as described in any one of claims 1-4, the DNA molecule as described in claim 5, or the vector as described in claim 6 can be prepared and expressed via in vitro transcription to obtain red fluorescent protein.

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

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

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