Mitochondrial nucleic acid delivery system and application thereof
By constructing a mitochondrial nucleic acid delivery system, mitochondria targeting and nucleic acid delivery are achieved using components such as stem-loop sequence MDS and 3’UTR sequence, the disease problems caused by mitochondrial DNA mutations are solved, and efficient mitochondrial gene editing and disease treatment are achieved.
Patent Information
- Application Number
- CN202510182596.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art is difficult to effectively solve the diseases caused by mitochondrial DNA mutations, especially due to the multi-copy characteristics of the mitochondrial genome and the bilayer membrane shielding of organelles, gene editing technology cannot effectively edit or correct mutations in mtDNA.
A mitochondrial nucleic acid delivery system is proposed, including stem-loop sequence MDS, 3'UTR sequence, target RNA sequence and regulatory sequence. The target gene sequence is amplified by PCR method and the stem-loop sequence MDS is introduced, the plasmid is constructed and the expression position of the target RNA sequence is judged by fluorescence microscopy to achieve effective targeting and nucleic acid delivery of mitochondria.
It has achieved efficient targeting and nucleic acid delivery of mitochondria, and can degrade mutant RNA or DNA through the mitochondrial pathway, solves the disease problems caused by various mtDNA mutations, and overcomes the difficulty of guide RNA in the CRISPR system that cannot be introduced into mitochondria.
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Figure CN119876210A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a mitochondrial nucleic acid delivery system and application thereof. Background Art
[0002] Mitochondria are important hubs for aerobic energy metabolism in cells. They have independent genomes that encode specific proteins. Mitochondria play a vital role in maintaining cellular balance and are associated with programmed cell death and the metabolism of lipids, amino acids, and iron. Therefore, if mitochondrial DNA (mtDNA) mutates and accumulates, and there are not enough wild-type copies to compensate, it will lead to mitochondrial dysfunction and eventually mitochondrial disease. Mutations in the human mitochondrial genome are associated with neuromuscular diseases, metabolic defects, and aging, such as Leber's hereditary optic neuropathy (LHON) and neuropathy ataxia and retinitis pigmentosa (NARP). However, because the mitochondrial genome is difficult to manipulate, mtDNA mutations cannot be corrected by gene editing for mitochondrial diseases; in basic research, the establishment of mtDNA mutation models is also difficult, making scientific research difficult to carry out.
[0003] The application of gene editing technology in the nuclear genome has become mature, but precise editing cannot be achieved in the mitochondrial genome. The reason is that, on the one hand, mtDNA has a multi-copy characteristic, which makes it difficult to edit completely, and on the other hand, it is shielded by the double membrane of the organelle, which prevents the entry of nucleic acids, making gene editing technologies such as CRISPR unable to be applied to mitochondria. Therefore, existing methods for treating mitochondrial diseases aim to reduce the level of mutant mtDNA by degrading mutant mtDNA molecules, but current tools are still unable to induce or edit other types of mutations in mtDNA, such as insertions, deletions, and transitions, which makes the operation of gene editing technology on mtDNA very limited.
[0004] In addition to direct manipulation of the mitochondrial genome, allotopic expression is also a potential method for treating mitochondrial genetic diseases. However, there are still many obstacles to be overcome for this method to be widely and effectively applied. First, the efficiency of allotopic expression needs to be improved. According to the hydrophobicity hypothesis, mitochondrial genes were not completely transferred to the nucleus during evolution. This is because the high hydrophobicity of a subset of mitochondrial-encoded proteins interferes with their transport from the cytoplasm to the mitochondria. Secondly, the imported mitochondrial proteins cannot be properly assembled in the respiratory complex, and it is not clear whether the presence of related polypeptides in the cytoplasm will affect cell function. At the same time, this strategy cannot correct mutations in non-coding RNAs such as tRNA in mitochondria.
[0005] If the mutant RNA or DNA can be corrected or the wild type can be complemented directly at the nucleic acid level, the impact of the mutant gene can be better eliminated. However, due to the difficulty of delivering nucleic acids to mitochondria, there are still few studies on the correction of these RNA and DNA. At present, the research on nucleic acid delivery mainly focuses on some vector systems, but the vector systems have potential immunogenicity and have certain challenges in preparation conditions and cell uptake efficiency.
[0006] In addition, existing studies have revealed that a small amount of nuclear-encoded RNA can enter mitochondria, including small non-coding RNA, miRNA (MicroRNA), tRNA, and viral RNA. The functional research of these RNAs imported into mitochondria is not limited to the analysis of mechanism and function, but also has potential therapeutic applications. For example, by modifying the tRNA that is naturally taken up by mitochondria from the cytoplasm, the wild-type mitochondrial tRNA can be replenished. Karicheva et al. studied the tRNA that spontaneously enters mitochondria in yeast. Lys (cuu) The anticodon and aminoacylation recognition site of tRNA were mutated to enable it to recognize and carry leucine. The introduction of this recombinant tRNA into human cells can rescue the tRNA in human cells to a certain extent. Leu(UUR) However, this method is inefficient and limited to tRNA mutations.
[0007] In summary, there is still a lack of an effective and simple mechanism to neutralize harmful mtDNA mutations. Summary of the invention
[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a mitochondrial nucleic acid delivery system and its application.
[0009] According to one aspect of the present invention, a mitochondrial nucleic acid delivery system is proposed, which comprises a stem-loop sequence MDS, a 3'UTR sequence, a target RNA sequence and a regulatory sequence, wherein both the stem-loop sequence MDS and the 3'UTR sequence target mitochondria.
[0010] Furthermore, the stem-loop sequence MDS is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in the sequence listing, or a combination thereof.
[0011] Furthermore, the target RNA sequence is a sequence encoded by the mitochondrial genetic code.
[0012] Furthermore, the regulatory sequence assists the target RNA sequence to be translated in the mitochondrial environment and degraded through the mitochondrial pathway.
[0013] According to a second aspect of the present invention, a method for constructing a mitochondrial nucleic acid delivery system is provided, comprising the following steps:
[0014] S1, amplifying the target gene sequence by PCR, and introducing the stem-loop sequence MDS by primers, connecting the target gene sequence and the stem-loop sequence MDS after gel recovery to obtain a connection product, and the connection product is transformed and verified to complete the plasmid construction of the delivery system;
[0015] S2. The plasmid described in step S1 is transformed into a reporter strain, induced, and a fluorescence microscope is used to determine whether the expression position of the target RNA sequence is consistent with the expression position of the red fluorescent protein Redstar.
[0016] Furthermore, the target gene sequence in step S1 is a target RNA sequence, a 3'UTR sequence and a regulatory sequence.
[0017] Furthermore, the expression of the target RNA sequence in step S2 is based on the construction of a reporter strain.
[0018] Furthermore, the reporter strain described in step S2 carries a red fluorescent protein Redstar gene fused with a mitochondrial membrane protein.
[0019] Furthermore, the reporter strain described in step S2 is induced by overnight activation in a liquid culture medium with glucose as the carbon source, and then transferred to a liquid culture medium with raffinose as the carbon source for 4-6 hours. After the glucose is consumed, it is induced in a liquid culture medium with galactose as the carbon source for 18 hours.
[0020] According to the third aspect of the present invention, an application of the delivery system in mitochondrial genetic diseases and mitochondrial gene editing is proposed.
[0021] Beneficial effects of the present invention:
[0022] The present invention achieves effective targeting and exerting effects on mitochondria by rationally engineering the target RNA. The mRNA fused with the RNA targeting sequence can be imported into the mitochondria after transcription in the cell nucleus, and the translation can be restored while being processed, and can be degraded through normal pathways in the mitochondria. The mitochondrial nucleic acid delivery system proposed in the present invention is highly efficient, and is expected to solve diseases caused by various mtDNA mutations, facilitate the establishment of mitochondrial disease models, and overcome the difficulty that the guideRNA in the CRISPR system cannot be introduced into the mitochondria. The mitochondrial nucleic acid delivery system of the present invention not only brings new hope for the treatment of mitochondrial genetic diseases, but also brings new inspiration and possibilities for basic research on mitochondria and RNA and mitochondrial genome editing. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 A schematic diagram of the working principle of the mitochondrial nucleic acid delivery system provided by the present invention;
[0024] Figure 2 This is the plasmid map of pRS403-pGAL1-COX25'UTR-sfGFPm-COX23'UTR-MDS-ATM13'UTR of a specific embodiment of the present invention;
[0025] Figure 3 This is a schematic diagram of the strain induction process of a specific embodiment of the present invention;
[0026] Figure 4 The expression and localization map of sfGFPm in a specific embodiment of the present invention. DETAILED DESCRIPTION
[0027] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.
[0028] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.
[0029] The first object of the present invention is to propose a mitochondrial nucleic acid delivery system, which provides a stable and precise tool for delivering targeted mitochondrial nucleic acids.
[0030] The second purpose of the present invention is to propose a method for constructing a mitochondrial nucleic acid delivery system to achieve effective targeting and exerting effects on mitochondria.
[0031] The third purpose of the present invention is to propose an application of a mitochondrial nucleic acid delivery system in mitochondrial genetic diseases and mitochondrial gene editing, which brings new hope to the research of mitochondrial genetic diseases, and also brings new inspiration and possibilities to the basic research of mitochondria and RNA and mitochondrial genome editing.
[0032] In order to achieve the above object, the present invention provides a mitochondrial nucleic acid delivery system. Figure 1 The schematic diagram of the working principle of the mitochondrial nucleic acid delivery system is as follows:
[0033] S1, amplifying the target gene sequence by PCR, and introducing the stem-loop sequence MDS by primers, connecting the target gene sequence and the stem-loop sequence MDS after gel recovery to obtain a connection product, and the connection product is transformed and verified to complete the plasmid construction of the delivery system;
[0034] S2. The plasmid described in step S1 is transformed into a reporter strain, induced, and a fluorescence microscope is used to determine whether the expression position of the target RNA sequence is consistent with the expression position of the red fluorescent protein Redstar.
[0035] In an embodiment of the present invention, the yeast is Saccharomyces cerevisiae s288c.
[0036] The stem-loop sequence MDS described in the present invention is any one of the sequence listings SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 or a combination thereof. Although the specific embodiments of the present invention are described using the stem-loop sequence MDS as SEQ ID NO:1 as a selection, it should be understood that the technical problems to be solved by the present invention and the innovative solutions on which the basic technical effects rely are also applicable to these other selections listed above.
[0037] The mitochondrial nucleic acid delivery system and its application provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.
[0038] Example 1: Construction of sfGFPm-MDS-ATM1 3'UTR plasmid
[0039] (1) Primers were designed to PCR amplify the promoter sequence of GAL1, COX2 5'UTR sequence, COX2 3'UTR sequence, sfGFPm ORF sequence and ATM1 3'UTR sequence from the yeast genome and WWP001 plasmid, as shown in SEQ ID NO:7 to SEQ ID NO:11 in the sequence table, respectively. The stem-loop sequence MDS was introduced by primers, and the specific sequence is shown in SEQ ID NO:1 in the sequence table. For ease of characterization, the target RNA sequence uses sfGFPm encoded by the mitochondrial genetic code, that is, the ORF sequence of sfGFPm. The above fragments were used for the subsequent construction of pRS403-pGAL1-COX2 5'UTR-sfGFPm-MDS-COX23'UTR-ATM1 3'UTR plasmid. The plasmid map of pRS403-pGAL1-COX2 5'UTR-sfGFPm-COX23'UTR-MDS-ATM13'UTR is shown in Figure 2 shown.
[0040] (2) Fragment gel recovery: The above PCR products were run on 1.2% agarose gel electrophoresis, and the gel blocks with the correct bands were cut out in the blue light gel excision, and the DNA (full gold) was recovered through the column of the gel recovery kit.
[0041] (3) Add the fragment amplification product in step (2) (5 μL in total) and 5 μL of 2×FS PCR Master MIX (AG) in a molar ratio of 1:1 to 10 μL of the ligation system, mix well, and ligate the fragments according to the following PCR procedure.
[0042] PCR procedure:
[0043] step specific conditions first step 98℃2min Step 2 98℃10s Step 3 73℃1s Step 4 55℃15s(5% power) Step 5 72℃4min Step 6 Return to step 2 and repeat 15 times Step 7 72℃5min
[0044] (4) Product transformation: Transformation and plating of ligation products: Add the ligation products to the competent cells, place on ice for 30 min, heat shock in a 42°C water bath for 90 s, immediately place on ice for 5 min, add 800 μL of LB medium, resuscitate in a 37°C shaker for 1 h, remove from the shaker, centrifuge at 12,000 rpm for 1 min, discard the supernatant, add 200 μL of ddH20 and mix well, and spread on LB+Carb plates according to 90% and 10% of the total volume respectively. Wait until the liquid is completely dry and place the plates in a 37°C incubator overnight.
[0045] (5) Interface verification, enzyme digestion verification and sequencing: Transformants were picked from the transformation plate, and the remaining bacterial solution was used as a template after dripping on the plate to design cross-interface verification primers for PCR verification of their interfaces. Strains with correct interface verification were streaked for bacterial preservation, plasmid extraction, enzyme digestion verification and sequencing.
[0046] Example 2: Detection of sfGFPm expression and expression location
[0047] (1) Construction of reporter strain:
[0048] The Redstar gene was fused to the original COX4 position in the BY4741 (leu2Δ0ura3Δ0his3Δ1met15Δ0) genome, and the reporter strain was generated by screening with KanMX marker.
[0049] (2) Plasmid transformation-LiOAC transformation method
[0050] A. Activation of strains: Pick a fresh single clone and place it in a shaking tube containing 3 mL of YPD liquid medium, and culture it overnight in a constant temperature shaker at 30°C;
[0051] B. Determination of bacterial solution density: Measure the OD600 value of the bacterial solution, the measured value should be between 0.2-0.8
[0052] C. Transfer: Pipette an appropriate volume of bacterial solution and dilute it into a shaking tube containing 3 mL of the corresponding liquid culture medium, so that the initial OD is 0.1, and culture it in a constant temperature shaker at 30°C for 4-6 hours;
[0053] D. Collect bacteria: Take 1 mL of bacterial solution and measure OD600, the value should be around 0.6. Centrifuge the remaining 2 mL of bacterial solution at 4200 rpm for 1 min, discard the supernatant, and collect the bacteria into a 2 mL EP tube with an appropriate label;
[0054] E. Washing: resuspend each EP tube with 700 μL 0.1 M LiAc / TE buffer, centrifuge at 4200 rpm for 1 min, and discard the supernatant;
[0055] F. Resuspension: Add an appropriate volume of 0.1 M LiAc / TE to resuspend the cells to a final concentration of 50 OD / mL.
[0056] G. Conversion MIX preparation:
[0057] MIX System μL / each 50%(W / V)PEG3350 62.4 1MLiOAc 8.22 ssDNA (Salmon sperm, 2mg / ml) 5
[0058] H. Transformation system preparation:
[0059] Transformation system μL / each MIX 75.62 Yeast competent cells 20 Plasmid to be transformed 20
[0060] I. Incubation: After the transformation system is fully mixed, it is placed in a 30°C incubator for 35 min. The transformation system is heat-shocked in a 42°C water bath for 15 min.
[0061] J. Plate coating: Plate the transformation system on SC-His plate to select transformants.
[0062] (3) Figure 3 The schematic diagram of the process of strain induction is as follows Figure 3 The bacteria were induced for 18 h and then the expression of sfGFPm was determined by fluorescence microscopy.
[0063] (4) Observe the expression and localization of sfGFPm using a fluorescence microscope. Figure 4 As shown, it was determined that the expression position of sfGFPm was consistent with the expression position of Redstar.
[0064] The above describes the specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.
Claims
1. A mitochondrial nucleic acid delivery system, characterized in that: The delivery system comprises a stem-loop sequence MDS, a 3'UTR sequence, a target RNA sequence and a regulatory sequence, wherein both the stem-loop sequence MDS and the 3'UTR sequence are targeted to mitochondria.
2. The delivery system according to claim 1, characterized in that The stem-loop sequence MDS is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in the sequence list or a combination thereof.
3. The delivery system according to claim 1, characterized in that The target RNA sequence is a sequence encoded by the mitochondrial genetic code.
4. The delivery system according to claim 1, characterized in that The regulatory sequence assists the target RNA sequence to be translated within the mitochondrial environment and degraded through the mitochondrial pathway.
5. A method for constructing a delivery system according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, amplifying the target gene sequence by PCR, and introducing the stem-loop sequence MDS by primers, connecting the target gene sequence and the stem-loop sequence MDS after gel recovery to obtain a connection product, and the connection product is transformed and verified to complete the plasmid construction of the delivery system; S2. The plasmid described in step S1 is transformed into a reporter strain, induced, and a fluorescence microscope is used to determine whether the expression position of the target RNA sequence is consistent with the expression position of the red fluorescent protein Redstar.
6. The method for constructing a delivery system according to claim 5, characterized in that: The target gene sequence in step S1 is the target RNA sequence, 3'UTR sequence and regulatory sequence.
7. The method for constructing a delivery system according to claim 5, characterized in that: The expression of the target RNA sequence in step S2 is based on the construction of a reporter strain.
8. The method for constructing a delivery system according to claim 5, characterized in that: The reporter strain described in step S2 carries the red fluorescent protein Redstar gene fused with the mitochondrial membrane protein.
9. The method for constructing a delivery system according to claim 5, characterized in that: The reporter strain described in step S2 is induced by overnight activation in a liquid culture medium with glucose as the carbon source, then transferred to a liquid culture medium with raffinose as the carbon source for 4-6 hours, and then induced with a liquid culture medium with galactose as the carbon source for 18 hours after the glucose is consumed.
10. Use of the delivery system according to any one of claims 1-4 in mitochondrial genetic diseases and mitochondrial gene editing.