Engineered vesicle and application of engineered vesicle in improvement of RNA (Ribonucleic Acid) molecular load

Abstract

The invention belongs to the field of taking an extracellular vesicle as a nucleic acid drug delivery carrier system, and particularly relates to an engineered vesicle and an application of the engineered vesicle in improvement of RNA (Ribonucleic Acid) molecular load. On the basis of fusion expression of ribosomal protein L7Ae and exosome membrane protein CD9, the L7Ae is specifically combined with a target RNA nucleic acid molecule containing a Box C / D sequence, the L7Ae is connected in series with a section of RNA binding domain, a droplet-shaped phase separation region is formed through multivalent interaction, and the target RNA nucleic acid molecule is further enriched into extracellular vesicles. The phase separation mechanism significantly improves the local concentration of the target RNA and the encapsulation efficiency in extracellular vesicles, and enhances the stability and delivery effect of the RNA. The invention is widely applicable to gene therapy, tumor immunotherapy and mRNA vaccine delivery, and has huge clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the field of extracellular vesicles as nucleic acid drug delivery carrier systems, and in particular relates to an engineered vesicle and its application in increasing RNA molecule loading. Background Art

[0002] Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles released from almost all prokaryotic and eukaryotic cells that carry proteins, lipids, RNA, and DNA. They can participate in processes such as cell communication, cell migration, angiogenesis, and tumor cell growth. They are widely present in various body fluids and cell supernatants and stably carry some important signaling molecules. Extracellular vesicles are potential RNA drug carriers based on their biocompatibility, low immunogenicity, and penetration of the blood-brain barrier. However, their mediated therapeutic delivery is limited by rough packaging methods.

[0003] There are many important applications of mRNA delivery, such as mRNA vaccines, cell reprogramming, gene therapy, and genome editing. The translation of mRNA is rapidly initiated after entering the cytoplasm, and the risk of genomic integration is close to zero when using mRNA as the delivery nucleic acid. Therefore, there is an unmet need for an effective delivery tool to introduce messenger mRNA into different cell lines. Several platforms have been reported, such as liposome transfection, lipid nanoparticles, and electroporation. However, these nucleic acid delivery methods have strong toxic side effects.

[0004] At present, the loading strategies of exosomes mainly include electroporation and transfection reagents, which have some problems, including low loading efficiency (loading efficiency 10-40%), damage to the structure and activity of exosomes, and influence on drug activity. Recently, there are also engineering designs for exosomes, which improve the controllability and loading efficiency of exosomes to a certain extent. However, further improving the loading efficiency of exosomes within the limited vesicle space is still an urgent problem to be solved. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides an engineered vesicle to solve the problem of low RNA molecule loading capacity of extracellular vesicles in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] An engineered vesicle carries a scaffold protein expression plasmid containing an RNA binding domain for loading nucleic acid into an extracellular vesicle and an expression plasmid of a nucleic acid sequence to be loaded.

[0008] Preferably, the cells from which the engineered vesicles are extracted include HEK-293T cells.

[0009] Preferably, the scaffold protein comprises exosome membrane protein, RNA binding domain (RBD, Receptor Binding Domain), and ribosomal protein L7Ae expressed in series.

[0010] Preferably, the exosomal membrane protein includes CD9.

[0011] Preferably, the nucleic acid sequence to be loaded expression plasmid comprises the nucleic acid molecule to be loaded and Box C / D sequence connected to each other.

[0012] Preferably, the RNA binding domain includes the RG-rich region of G3BP1 (Ras-GTPase activating protein SH3 domain binding protein 1) or the RRM1-2 tandem region of HnRNPA1 (heterogeneous nuclear protein A1).

[0013] The present invention also discloses a method for increasing RNA molecule loading using engineered vesicles, comprising the following steps:

[0014] Step (1), extracting RNA from the engineered vesicles;

[0015] Step (2), removing DNA and synthesizing cDNA (complementary DNA) chain through reverse transcription reaction;

[0016] Step (3), perform qPCR reaction.

[0017] Preferably, the RNA includes any RNA nucleic acid molecule carrying Box C / D.

[0018] Preferably, the qPCR reaction uses GAPDH as an internal reference and EGFP as a fluorescent protein;

[0019] The upstream primer sequence of GAPDH is shown in SEQ ID NO.24:

[0020] AGCCACATCGCTCAGACAC;

[0021] The downstream primer sequence of GAPDH is shown in SEQ ID NO.25:

[0022] GCCCAATACGATCGATCC;

[0023] The upstream primer sequence of EGFP is shown in SEQ ID NO.26:

[0024] CCTGAAGTTCATCTGCACCA;

[0025] The downstream primer sequence of EGFP is shown in SEQ ID NO.27:

[0026] GGTCAGGGTGGTCACGAG.

[0027] The theoretical basis of the engineered extracellular vesicle principle proposed in the present invention is as follows:

[0028] 1. L7AE-CD9 system in engineered vesicles. L7Ae protein is one of the large subunit proteins of archaeal ribosomes. In addition to playing an important role in ribosome structure, it is also involved in ribosomal RNA splicing, post-transcriptional modification and the stability of archaeal RNaseP complex. L7Ae can bind to RNA secondary structure, called kink turn motif. The characteristic of kink is the formation of an axially bent RNA structure, which allows interaction with L7Ae protein. There are various kink turn motifs, including BoxC / D and H / ACA motifs on snoRNA, Kt motifs on ribosomal RNA, and riboswitches. Through the specific binding of L7AE to C / D box and the targeting effect of CD9, the target RNA can be enriched into extracellular vesicles to enhance the affinity of vesicles to specific cells.

[0029] 2. Phase separation of RNA binding domains in RNA binding proteins: Eukaryotic cells contain non-membrane-bound organelles. Many of these structures are rich in RNA and proteins and play a role in regulating RNA-protein complexes. Such organelles are generally called ribonucleoprotein (RNP) particles, including nucleoli, Cajal bodies, stress granules, and P bodies. RNP particles have a higher protein concentration than the surrounding cytoplasm or nucleoplasm. Fluorescence recovery after photobleaching (FRAP) analysis shows that proteins can associate, dissociate, and move within RNP particles on a time scale of seconds to minutes. Multiple interactions are important for the formation and recruitment of RNP particles. Granule assembly usually requires RNA molecules to bind to the RNA binding domains of many granule proteins. RNP granule proteins usually contain arginine / glycine-rich (RGG / RG) domains and RNA recognition motifs (RRMs).

[0030] The scaffold protein expression plasmid for loading nucleic acid into extracellular vesicles and the expression plasmid of the nucleic acid sequence to be loaded are co-transfected into HEK-293T cells, and the exosome membrane protein is fused with RBD (RG-rich region of G3BP1 or RRM1-2 tandem region of HnRNPA1) and an L7Ae protein to form a fusion protein (i.e., scaffold protein), in which the C / D box autonomously docks with L7Ae, so that the nucleic acid molecules to be loaded are loaded into the extracellular vesicles through the fusion protein, and the extracellular vesicles are secreted outside the cells while carrying specific nucleic acid molecules outside the cells.

[0031] Furthermore, the efficiency of this system was tested by detecting the target RNA content in the engineered vesicles;

[0032] The specific research method is as follows: Detect the content of target RNA nucleic acid molecules in engineered vesicles: express the scaffold protein plasmid of exosome membrane protein in series with RBD and an L7Ae, as well as the EGFP-Box C / D expression plasmid in HEK-293T cells, collect a sufficient amount of culture supernatant, extract vesicles, and use qPCR to detect the target RNA enrichment efficiency.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides an engineered extracellular vesicle, which can smoothly enrich target RNA molecules into the extracellular vesicle, and provides a new method for delivering RNA nucleic acid molecules;

[0035] Compared with vesicles carrying only CD9 protein, the engineered vesicles of the present invention can efficiently enrich the target RNA in the vesicles;

[0036] The present invention can effectively increase the loading capacity of extracellular vesicles for specific RNA nucleic acid molecules to be loaded, and provides an important theoretical and technical basis for the development of drugs using extracellular vesicles as nucleic acid drug delivery systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are images of living cells overexpressing CD9, CD9-L7Ae, CD9-HnRNPA1 (RRM1-2)-L7Ae, and pCMV-CD9-G3BP1 (RG region)-L7Ae plasmids taken by confocal microscopy in the present invention;

[0038] Figure 2 Western-blot experiments were performed on the engineered vesicles secreted after overexpressing CD9, CD9-L7Ae, CD9-HnRNPA1 (RRM1-2)-L7Ae, and pCMV-CD9-G3BP1 (RG region)-L7Ae plasmids in HEK-293T cells in the present invention, and the expression level of each protein in each group of engineered vesicles was detected;

[0039] Figure 3 The relative expression levels of mRNA in the engineered vesicles CD9, CD9-L7Ae, CD9-HnRNPA1 (RRM1-2)-L7Ae, and pCMV-CD9-G3BP1 (RG region)-L7Ae measured after qPCR reaction in the present invention;

[0040] Figure 4: In vitro purification of HnRNPA1(RRM1-2)-L7Ae and CD9-G3BP1(RG region)-L7Ae proteins, and the phase separation phenomenon observed by adding total RNA extracted from HEK-293T cells expressing EGFP-Box C / D plasmid. DETAILED DESCRIPTION

[0041] Example 1

[0042] This example discloses a method for extracting engineered vesicles. The experimental materials used are as follows: HEK-293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (SCSP-502); fetal bovine serum was purchased from Biological Industries (C04001-500); DMEM culture medium was purchased from Biological Industries (C11995500); transfection reagent Lipo-3000 was purchased from ThermoFisher (L3000015); poly-lysine was purchased from Biyuntian (P6407); ultrafiltration tube was purchased from MilliporeSigma (UFC9100); DNA polymerase was purchased from Vazyme (P505-d1); restriction endonucleases were purchased from Thermo Fisher (ER1461 and FD0694 for pCMV-CD9 plasmid, respectively);

[0043] The method for extracting the engineered vesicles comprises the following steps:

[0044] Step (1), construct the target plasmid

[0045] Select plasmid vectors: pCMV-CD9 plasmid, pCMV-CD9-L7Ae plasmid, pCMV-CD9-HnRNPA1(RRM1-2)-L7Ae plasmid (HnRNPA1(RRM1-2) fragment is constructed into the stable overexpression vector pCMV-CD9-L7Ae), pCMV-CD9-G3BP1(RG region)-L7Ae plasmid (G3BP1(RG region) is constructed into the overexpression vector pCMV-CD9-L7Ae);

[0046] The mCherry fragments were respectively constructed into the above four plasmid vectors to obtain pCMV-CD9-mCherry plasmid, pCMV-CD9-L7Ae-mCherry plasmid, pCMV-CD9-HnRNPA1 (RRM1-2)-L7Ae-mCherry plasmid, and pCMV-CD9-G3BP1 (RG region)-L7Ae-mCherry plasmid;

[0047] pET28-HnRNPA1(RRM1-2)-L7Ae plasmid (HnRNPA1(RRM1-2)-L7Ae fragment was constructed into the stable overexpression vector pET28) and pET28-G3BP1(RG region)-L7Ae plasmid (G3BP1(RG region)-L7Ae was constructed into the overexpression vector pET28) were obtained by PCR;

[0048] The EGFP-Box C / D fragment was constructed into the pCMV plasmid;

[0049] Step (2): Obtaining the target gene

[0050] According to the requirements of the constructed plasmid and the primer design principles, the corresponding primers were designed. The DNA sequences of the plasmid primers and the amino acid sequences of CD9 protein, L7Ae protein, HnRNPA1 (RRM1-2 protein), G3BP1 (RG region), and EGFP protein are as follows:

[0051] The upstream primer sequence of pCMV-CD9 plasmid is shown in SEQ ID NO.1:

[0052] CAGATCCGCTAGCGCTACCGGTCGCCACCCTCGAGATGCCGGTCAAAG GAGGCACCAAGTGCA;

[0053] The downstream primer sequence of pCMV-CD9 plasmid is shown in SEQ ID NO.2:

[0054] CCTCCACCACCGCTACCTCCGAATTCGACCATCTCGCGGTTCCTGCGG ATAGCACAGCACAAGATC;

[0055] The upstream primer sequence of pCMV-CD-L7Ae plasmid is shown in SEQ ID NO.3:

[0056] TGGTGGAGGATCAGGTGGTAAGCTTATGGACTTGCGCTTCGAGGCT;

[0057] The downstream primer sequence of pCMV-CD9-L7Ae plasmid is shown in SEQ ID NO.4:

[0058] TCAGTTATCTAGATCCGGTGGATCCTCACTTCGCAGGCCCTGTTATTTC CGATT;

[0059] The upstream primer sequence of pCMV-CD9-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.5:

[0060] ATCAGGTGGTAAGCTTAGGAAGCTCTTCATTGGAGGGTT;

[0061] The downstream primer sequence of pCMV-CD9-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.6:

[0062] GAAGCGCACGTACATGTCGACTCCGGAGCCTCTCTTGCTTTGACAGGGC TTTTCTAACTTC;

[0063] The upstream primer sequence of pCMV-CD9-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.7:

[0064] ATCAGGTGGTAAGCTTGTCGAAGAGAAGAAGACTCGAGC;

[0065] The downstream primer sequence of pCMV-CD9-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.8:

[0066] GACTCCGGAGCCTCCCTGCCGTGGCGCAAGC;

[0067] The upstream primer sequence of pCMV-CD9-mCherry plasmid is shown in SEQ ID NO.9:

[0068] GGATCAGGTGGTAAGCTTATGGTGAGCAAGGGCGAGGAG;

[0069] The downstream primer sequence of pCMV-CD9-mCherry plasmid is shown in SEQ ID NO.10:

[0070] TTATCTAGATCCGGTGGATCCTTGTACAGCTCGTCCATGCC;

[0071] The upstream primer sequences of pCMV-CD9-L7Ae-mCherry plasmid, pCMV-CD9-HnRNPA1 (RRM1-2)-L7Ae-mCherry plasmid and pCMV-CD9-G3BP1 (RG region)-L7Ae-mCherry plasmid are shown in SEQ ID NO.11:

[0072] ATCAAGGGCCTGVAGAAGATGGTGAGCAAGGGCGAGG;

[0073] The downstream primer sequences of pCMV-CD9-L7Ae-mCherry plasmid, pCMV-CD9-HnRNPA1 (RRM1-2)-L7Ae-mCherry plasmid and pCMV-CD9-G3BP1 (RG region)-L7Ae-mCherry plasmid are shown in SEQ ID NO.12:

[0074] TATCTAGATCCGGTGGATCCTTGTACAGCTCGTCCATGC;

[0075] The upstream primer sequence of pET28-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.13:

[0076] GGGACACCATCACCATCACCATATGAGGAAGCTCTTCATTGGAGGGTT GA;

[0077] The downstream primer sequence of pET28-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.14:

[0078] GGTGGTGGTGGTGGTGCTCGAGTCACTTCTGCAGGCCCTTGATCTTT; the upstream primer sequence of pET28-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.15:

[0079] GGGACACCATCACCATCACCATATGGTCGAAGAGAAGAAGA;

[0080] The downstream primer sequence of pET28-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.16:

[0081] GGTGGTGGTGGTGGTGGTGCTCGAGTCACTTCTGCAGGCCCTTGATCTTT; the upstream primer sequence of pCMV-EGFP-Box C / D plasmid is shown in SEQ ID NO.17:

[0082] TTAGTGAACCGTCAGATCCCGCTAGCgccaccATGGTGAGCAAGGGCGAG GAG;

[0083] The upstream primer sequence of pCMV-EGFP-Box C / D plasmid is shown in SEQ ID NO.18:

[0084] ATTCGAAGCTTGAGCTCGAGATCTGAGGGTCACCTTTCGGATCACGCCCGTACTTGTACAGCTCGTCCATGCCG;

[0085] The amino acid sequence of CD9 is shown in SEQ ID NO.19:

[0086] MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGVYILIGAGALMmLVGFLGCCGAVQESQCmLGLFFGFLLVIFAIEIAAAIWGYSHK DEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV;

[0087] The amino acid sequence of L7Ae is shown in SEQ ID NO.20:

[0088] MYVRFEVPEDMQNEALSLLEKVRESGKVKKGTNETTKAVERGLAKLVYIAEDVDPPEIVAHLPLLCEEKNVPYIYVKSKNDLGRAVGIEVPCASAAIINEGELRKELGSLVEKIKGLQKGGSGRHPDSHQLFIGNLPHEVDKSELKDFFQSYGNVVELRINSGGKLPNFGFVVFDDSEPVQKVLSNRPIMFRGEVRLNVEEKKTRAAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGFGVGRGLAPRQGGGGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK;

[0089] The amino acid sequence of HnRNPA1 (RRM1-2) is shown in SEQ ID NO.21:

[0090] RKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDAAMNARPHKVDGRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQ;

[0091] The amino acid sequence of G3BP1 (RG region) is shown in SEQ ID NO.22:

[0092] VEEKKTRAAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGF GVGRGLAPRQ;

[0093] The amino acid sequence of EGFP is shown in SEQ ID NO.23:

[0094] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

[0095] The primer length was kept at 18-30 bp, and the GC content was kept at 40-60% during design. The components of the PCR reaction system are shown in Table 1:

[0096] Table 1

[0097]

[0098] The whole process was carried out on ice, and the mixture was gently pipetted and mixed before centrifugation.

[0099] Among them, the PCR instrument reaction program is shown in Table 2:

[0100] Table 2

[0101]

[0102] In order to obtain the sticky ends of the plasmid vector, a double enzyme digestion reaction is required. The specific components of the double enzyme digestion reaction system are shown in Table 3:

[0103] Table 3

[0104]

[0105] Step (3), fragment recovery

[0106] 1) Glue preparation: Prepare a nucleic acid gel with a mass-to-volume ratio of 1% by weighing 0.2 g agarose powder on a weighing balance and placing it in 20 mL 1×TAE (nucleic acid electrophoresis buffer), boiling it in a microwave oven, adding 1.3 μL of Gelred (non-toxic nucleic acid dye) after cooling, mixing well and pouring it into a gel-making plate containing a comb to solidify for 30 minutes, removing the comb and placing it in an electrophoresis tank containing electrophoresis solution;

[0107] 2) Sample preparation: Add DNA Loading Buffer to the PCR reaction product and the linearized enzyme digestion vector, mix well with a pipette tip, and then add to the sample loading well. The electrophoresis conditions are 85V for 35min.

[0108] 3) Gel cutting: After electrophoresis, take out the nucleic acid gel, photograph the gel in a gel imager, cut out the nucleic acid gel band that matches the expected band size, carefully put it into a 1.5 mL EP tube, weigh the nucleic acid gel on a weighing balance, add 100 μL Buffer GDP (DNA binding solution) for every 100 mg of nucleic acid gel according to the weight of the nucleic acid gel, and completely dissolve the nucleic acid gel in a 55°C oven, turning it upside down to mix;

[0109] 4) Transfer solution: transfer the completely dissolved nucleic acid gel solution to the adsorption column and centrifuge at 12000 rpm for 30 seconds;

[0110] 5) Discard the waste liquid in the collection tube, add 300 μL Buffer GDP (DNA binding solution) to the adsorption column, let it stand for 1 min, and centrifuge at 12000 rpm for 30 s;

[0111] 6) Discard the waste liquid in the collection tube, add 700 μL Buffer GW (rinsing solution) to the adsorption column, and centrifuge at 12000 rpm for 30 seconds;

[0112] 7) Repeat the above steps;

[0113] 8) Discard the waste liquid in the collection tube, centrifuge at 12000 rpm for 1 min, and leave it open for 2 min. Place the adsorption column in a new 1.5 mL EP tube, add 20 μL of preheated Elution Buffer, let it stand for 2 min, centrifuge at 12000 rpm for 1 min, and measure the concentration using a micro-spectrophotometer Nano-500;

[0114] Step (4), recombination reaction

[0115] The recovered product was subjected to a recombination reaction (recombination conditions were 37° C., 30 min). The components of the recombination reaction system are shown in Table 4:

[0116] Table 4

[0117]

[0118] Step (5), conversion of reaction products

[0119] 1) Take out the DH5α competent cells in advance at -80°C, add 50 μL of DH5α competent cells to the recombination reaction system for transformation, and keep on ice for 30 minutes;

[0120] 2) Heat shock the cells in a 42°C water bath for 60 seconds, place on ice for 2 minutes, add 1 mL of LB medium, and shake the cells in a 37°C shaker at 250 rpm for 45 minutes;

[0121] Step (6): coating the board

[0122] 1) Prepare a sterile glass rod, pass it through an alcohol lamp for 10 seconds, and then irradiate it with ultraviolet light for 30 minutes. Take 80 μL of the shaken bacterial solution and apply it to the solid plate of the corresponding resistance, and place it in a 37°C incubator for overnight culture for 15 hours;

[0123] 2) After the culture is completed, remove the solid culture medium, add 5 mL of culture medium to a 15 mL centrifuge tube, pick a single clone with a 10 μL pipette tip and place it on the 5 mL culture medium, and culture it at 37°C at 250 rpm for 12 h;

[0124] Step (7), extracting plasmid

[0125] 1) Take out the overnight culture solution and centrifuge it at 3500rpm for 10min, then discard the supernatant;

[0126] 2) Add 250 μL of Buffer P1 (bacterial suspension) to the obtained precipitate and mix well by pipetting with a pipette;

[0127] 3) Take 250 μL of Buffer P2 (bacterial lysis solution) and add it to the EP tube. Invert and mix well for 5 minutes.

[0128] 4) Take 350 μL of Buffer P3 (neutralizing solution) and add it to the EP tube. Invert and mix to terminate the reaction. Centrifuge at 12000 rpm for 10 min. Take the supernatant after centrifugation and add it to the adsorption column. Centrifuge at 12000 rpm for 30 s.

[0129] 5) Take 700 μL of Buffer PW2 (to remove residual salt ions in the plasmid) into the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat the above steps;

[0130] 6) Place the EP tube in a centrifuge, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and leave it open for 2 min;

[0131] 7) Take out the adsorption column and place it in a new 1.5 mL EP tube, and add 50 μL of Elution Buffer (elution solution) preheated in the oven to the adsorption column. After standing for 2 minutes, centrifuge at 12000 rpm for 1 minute. After the end, use a micro-spectrophotometer Nano-500 to measure its concentration, and store the obtained plasmid in a -20°C refrigerator;

[0132] Step (8), cell transfection

[0133] 1) Cell plating: HEK-293T cells with good growth conditions were seeded in a 100 mm culture dish (the cells were cultured in DMEM medium and 10% fetal bovine serum) with a cell density of 50%. The dish was shaken in an "8" pattern until the cell density reached 70% before transfection.

[0134] 2) Transfection: prepare two sterile EP tubes A and B, add 1000 μL Opti-MEM to each EP tube, add 10 μL liposome transfection reagent to tube A, add 10 μg plasmid obtained in step (7) to tube B, mix well by pipetting in each tube, add the liquid in tube A to tube B with a pipette, mix well by pipetting, incubate at room temperature for 20 min, remove the old complete medium in the culture dish, and add fresh complete medium;

[0135] 3) After the incubation, add the liposome transfection mixture to the culture medium, shake it gently, and place it in a cell culture incubator. After 8 hours of transfection, remove the culture medium containing the transfection reagent and replace it with fresh serum-free culture medium;

[0136] Step (9): Observation of scaffold protein expression using confocal microscopy

[0137] 1) First add poly-lysine to the confocal glass bottom dish to wet it, and then suck it off; then add deionized water to wet it, and then suck it off; then HEK-293T cells are seeded in a 35mm confocal glass bottom dish, and each group of HEK-293T cells is transfected with 2μg of the corresponding plasmid, and the images are taken after 24 hours;

[0138] 2) Turn on the 37°C constant temperature function of the microscope imaging system, use a 100x microscope to photograph the cells (when using a 100x microscope, drop specific immersion oil on the lens), add 2 μL of hoechest dye for 10 minutes, and then use the 405 channel and 594 channel to photograph the cells. The results are as follows: Figure 1 As shown, CD9, CD9-L7Ae, CD9-HnRNPA1(RRM1-2)-L7Ae, and pCMV-CD9-G3BP1(RG region)-L7Ae proteins were correctly expressed and localized in HEK-293T cells;

[0139] Step (10), extracellular vesicle extraction

[0140] 1) On the third day after the medium change, collect the supernatant from each dish into a 50 mL sterile centrifuge tube;

[0141] 2) Centrifuge at 300 rpm for 10 min to remove cells, discard the precipitate and collect the supernatant;

[0142] 3) Centrifuge at 2000 rpm for 10 min to remove apoptotic bodies. After centrifugation, discard the precipitate and collect the supernatant.

[0143] 4) The harvested supernatant was added to a 100 kDa sterile ultrafiltration tube in batches, with the volume added each time not exceeding 15 mL, followed by centrifugation at 5000 rpm. When the liquid in the concentrated collection tube was ultrafiltered to about 1000 μL, the liquid filtered out of the collection tube was discarded, and the supernatant was added to the concentrated collection tube, followed by centrifugation at 5000 rpm again until all the supernatant was concentrated to 500 μL and then the centrifugation was stopped;

[0144] 5) Add 9.5 mL of PBS, then place the two tubes at 4°C and centrifuge at 5000 rpm. Stop centrifugation when the supernatant in the concentrated collection tube is concentrated to 500 μL;

[0145] 6) Carefully aspirate the supernatant in the concentrated collection tube with a pipette and add it to a 1.5 mL EP tube. If it is used in the short term, store it in a 4°C refrigerator. If it needs to be stored for a long time, store it in a -80°C refrigerator.

[0146] Further, the extracted extracellular vesicles (engineered vesicles) are subjected to protein identification, comprising the following steps:

[0147] 1) Obtaining samples: Perform BCA protein assay on the collected vesicles, add appropriate loading buffer and cook the samples in a 100°C water bath for 10 min (open the tube cover at the 5th minute);

[0148] 2) Prepare protein gel: Place the washed and dried glass plate on the gel rack, add the prepared separation gel first, and flatten it with 75% anhydrous ethanol. After about 30 minutes, pour out the anhydrous ethanol, invert it to allow the ethanol to flow out, add the prepared concentrated gel, and insert a 15-hole comb for about 1 hour to solidify it;

[0149] 3) Sample loading: The target protein loading volume per well should reach 10 μL, and the maker loading volume is 3 μL. After loading, use 1x Loading Buffer to fill the volume of each well to the same level;

[0150] 4) Electrophoresis: Run at 80V for 30 minutes to separate the maker, then adjust the voltage to 120V. End the electrophoresis when the Loading Buffer reaches the bottom of the gel.

[0151] 5) Transfer: Tightly clamp the negative electrode surface, sponge, filter paper, gel, PVDF membrane activated with anhydrous ethanol, filter paper, sponge, and positive electrode surface in the order of the negative electrode surface, and pay attention to avoid the generation of bubbles. Use 0.22 μm PVDF membrane and 300 mA transfer for 80 min.

[0152] 6) Blocking: Carefully remove the transferred membrane, soak it in TBST buffer containing 5% skim milk powder, and place it on a shaker at room temperature for 2 hours;

[0153] 7) Incubation with primary antibody: After blocking, the membrane was slowly rinsed with TBST buffer for 3 times, 10 min each time, and then immersed in TBST primary antibody dilution containing 5% BSA and incubated on a shaker at 4°C overnight;

[0154] 8) Incubation with secondary antibody: After the primary antibody incubation is completed, the membrane is slowly washed with TBST buffer for 3 times, 10 minutes each time, and then the membrane is immersed in TBST secondary antibody diluent containing 5% skim milk powder and incubated on a shaker at room temperature for 1 hour;

[0155] 9) Development: After the secondary antibody incubation, the membrane was slowly washed with TBST for 3 times, 5 minutes each time, and then placed in a developer for development. The results are as follows: Figure 2 As shown, the prepared engineered vesicles successfully contained the above-mentioned proteins.

[0156] Example 2

[0157] This embodiment discloses a method for increasing the RNA molecule loading capacity by using engineered vesicles, comprising the following steps:

[0158] Step (1), extracting total RNA

[0159] 1) Sample treatment: Add 200 μL of extracted extracellular vesicles (EVs) to 1 mL of trizol, mix well by pipetting, and place at room temperature for 5 minutes;

[0160] 2) Add chloroform: add 0.2 mL of chloroform to every 1 mL of trizol, invert and place at room temperature for 3 minutes, then centrifuge at 4°C, 12,000 rpm for 15 minutes. Centrifuge to separate the sample into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase and an intermediate layer. RNA is mainly in the aqueous phase.

[0161] 3) RNA precipitation: transfer the aqueous phase to a new tube, add isopropanol to precipitate RNA, leave at room temperature for 10 minutes, and then centrifuge at 4°C, 12,000 rpm for 10 minutes to collect the RNA precipitate;

[0162] 4) Washing and re-dissolving: Add 1 mL of 75% ethanol to wash the RNA precipitate, then place it at room temperature to dry for 5 minutes, and add 20 μL of RNase-free (enzyme-free and sterile) water to dissolve the RNA;

[0163] Step (2), reverse transcription

[0164] 1) Prepare the reaction system

[0165] 1.1 Prepare the DNA removal reaction system. The components of the reaction system are shown in Table 5:

[0166] Table 5

[0167]

[0168] After the above system is prepared, mix it evenly and place it at 42°C for 2 minutes;

[0169] 1.2 Prepare the cDNA chain synthesis reaction system. The components of the reaction system are shown in Table 6:

[0170] Table 6

[0171]

[0172]

[0173] After the above system is prepared, mix well, place at 50℃ for 15min, and then place at 85℃ for 2min;

[0174] Step (3), qPCR reaction

[0175] After reverse transcription, 80 μL RNase-free deionized water was added to the cDNA solution for dilution, and then the qPCR reaction system was prepared. The qPCR reaction used GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal reference, and the upstream primer sequence of GAPDH was shown in SEQ ID NO.24:

[0176] AGCCACATCGCTCAGACAC;

[0177] The downstream primer sequence of GAPDH is shown in SEQ ID NO.25:

[0178] GCCCAATACGATCGATCC;

[0179] The upstream primer sequence of EGFP (enhanced green fluorescent protein) is shown in SEQ ID NO.26:

[0180] CCTGAAGTTCATCTGCACCA;

[0181] The downstream primer sequence of EGFP is shown in SEQ ID NO.27:

[0182] GGTCAGGGTGGTCACGAG;

[0183] The components of the qPCR reaction system are shown in Table 7:

[0184] Table 7

[0185]

[0186] After the reaction system is prepared, a fluorescent quantitative PCR instrument is used for the reaction. The qPCR reaction program is shown in Table 8:

[0187] Table 8

[0188]

[0189]

[0190] After the reaction, data processing was performed and relative quantification was performed using 2-ΔΔ Ct Method, GAPDH was selected as the internal reference gene, and the mRNA expression level was calculated. The results are as follows Figure 3 As shown in the figure, after L7Ae was connected to CD9 in series, and Box C / D was connected to EGFP in series, the EGFP mRNA content in the vesicles was significantly higher than that in the group expressing only the CD9 scaffold protein. When HnRNPA1 (RRM1-2) and G3BP1 (RG region) were connected to CD9-L7Ae in series, and Box C / D was connected to EGFP in series, the EGFP mRNA content in the vesicles was further increased compared with the group expressing the CD9-L7Ae scaffold protein.

[0191] Example 3

[0192] This embodiment discloses a method for observing the interaction between protein and RNA in vitro, comprising the following steps:

[0193] (1) Protein purification

[0194] 1) Transformation and expansion: transform the plasmid into BL21 competent cells and plate; clone into 5 mL LB (add the corresponding kanamycin at a ratio of 1:1000 according to the vector resistance), shake at 37°C overnight; expand 10 mL of bacterial solution of each protein into 400 mL LB (add the corresponding kanamycin at a ratio of 1:1000 according to the vector resistance), shake at 37°C until OD600 = 1.0 for 3 h;

[0195] 2) Induction: 400 mL bacterial solution + 200 μL 1 M IPTG (inducer) (2000:1, final concentration 0.5 mM); induce overnight at 16°C for 18 h;

[0196] 3) Ultrasound: Before harvesting, place on ice for 30 minutes, centrifuge at 4°C, 5000 rpm, 10 minutes; resuspend the precipitate in 20 mL Bingding buffer (1M NaCl, 20 mM Tris), amplitude 6, engineering 2, 25% power, 2s on, 4s off, ultrasonic for 40 minutes; centrifuge at 4°C, 14500 rpm, 30 minutes, and take the supernatant;

[0197] 4) Purification: first add a tube of deionized water to wash the gel; wash the filler with 5 column volumes of binding buffer, repeat 3 times, and the last time the binding buffer is retained in the column for a period of time; add the supernatant after ultrasound to the balanced filler (to ensure that the protein is in full contact with Ni+); after the protein solution has flowed out, elute the impurities with wash buffer (30mM imidazole) and wash twice; add 12mL Elution buffer (500mM imidazole) and elute 6 times, 2mL each time, collect the eluate, and record the eluate as F1, F2, F3, F4, F5, and F6 respectively; use 3 column volumes of binding buffer and 5 column volumes of deionized water to balance the filler in turn, and then use 5 column volumes of 20% ethanol to balance, and finally store in an equal volume of 20% ethanol at 4°C to prevent contamination;

[0198] 5) Dialysis: Immerse the used dialysis bag in an appropriate amount of dialysis bag treatment solution (2% (mass percentage) NaHCO 3 , 1mM EDTA, Ph=8.0), boiling water bath for 10min; fully wash the dialysis bag after boiling water bath with distilled water, clamp the dialysis bag with a dialysis clamp at about 1.5cm of the opening at one end, fill it with water and apply appropriate pressure by hand to ensure that there is no obvious leakage; carefully add the protein eluent into the dialysis bag from the other end, reserve 20% of the top space, and clamp the other port with a dialysis clamp; transfer the sample to 2L stock buffer (without glycerol and DTT) for overnight dialysis, and continue dialysis for 4h after changing the solution the next day;

[0199] 6) Ultrafiltration: Pour the sample carefully from the dialysis bag into a 50mL centrifuge tube, centrifuge at 4°C, 14500rpm for 20min, remove the precipitate, and take the supernatant; add the supernatant to the upper layer of a 50kDa ultrafiltration tube, centrifuge at 4°C, 8000rpm for 20min; when the upper layer liquid is less than 500μL, add 15mL stock buffer, centrifuge at 4°C, 8000rpm for 25min, until the upper layer liquid is less than 250μL, carefully blow and mix the upper layer liquid with a yellow gun tip, then aspirate it, measure the concentration with a nano, and dispense it into PCR tubes and store at -80°C;

[0200] (2) Photography of extracorporeal phenomena

[0201] 1) Shooting was performed at 30 μM protein concentration, 50 ng / μL total RNA, and 150 μM NaCl;

[0202] 2) Use high vacuum silicone grease to draw a rectangle on the slide that fits the size of the cover glass, drop the protein sample to be observed in the middle, cover it with the cover glass, and observe it with the cover glass side facing down using a confocal microscope. Figure 4As shown, HnRNPA1 (RRM1-2) and G3BP1 (RG region) are connected in series with L7Ae. After the addition of RNA, phase separation occurs, but without RNA, phase separation does not occur.

[0203] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An engineered vesicle, characterized in that: It also carries a scaffold protein expression plasmid containing an RNA binding domain for loading nucleic acids into extracellular vesicles and an expression plasmid for the nucleic acid sequence to be loaded.

2. An engineered vesicle according to claim 1, characterized in that The cells for extracting engineered vesicles include HEK-293T cells.

3. An engineered vesicle according to claim 1, characterized in that The scaffold protein includes exosome membrane protein, RNA binding domain and ribosomal protein L7Ae which are fused and expressed in series.

4. An engineered vesicle according to claim 3, characterized in that The exosomal membrane protein includes exosomal membrane protein CD9.

5. An engineered vesicle according to claim 1, characterized in that The nucleic acid sequence expression plasmid to be loaded includes the nucleic acid molecule to be loaded and the Box C / D sequence connected to each other.

6. An engineered vesicle according to claim 1, characterized in that The RNA binding domain includes the RG-rich region of G3BP1 or the RRM1-2 tandem region of HnRNPA1.

7. An engineered vesicle according to claim 1, characterized in that The extraction process comprises the following steps: Step (1), constructing a target plasmid; Select plasmid vectors: pCMV-CD9 plasmid, pCMV-CD9-L7Ae plasmid, pCMV-CD9-HnRNPA1(RRM1-2)-L7Ae plasmid (HnRNPA1(RRM1-2) fragment is constructed into the stable overexpression vector pCMV-CD9-L7Ae), pCMV-CD9-G3BP1(RG region)-L7Ae plasmid (G3BP1(RG region) is constructed into the overexpression vector pCMV-CD9-L7Ae); The mCherry fragments were respectively constructed into the above four plasmid vectors to obtain pCMV-CD9-mCherry plasmid, pCMV-CD9-L7Ae-mCherry plasmid, pCMV-CD9-HnRNPA1 (RRM1-2)-L7Ae-mCherry plasmid, and pCMV-CD9-G3BP1 (RG region)-L7Ae-mCherry plasmid; pET28-HnRNPA1(RRM1-2)-L7Ae plasmid (HnRNPA1(RRM1-2)-L7Ae fragment was constructed into the stable overexpression vector pET28) and pET28-G3BP1(RG region)-L7Ae plasmid (G3BP1(RG region)-L7Ae was constructed into the overexpression vector pET28) were obtained by PCR; The EGFP-Box C / D fragment was constructed into the pCMV plasmid; Step (2), obtaining the target gene; According to the requirements of the constructed plasmid and the primer design principles, the corresponding primers were designed. The DNA sequences of the plasmid primers and the amino acid sequences of CD9 protein, L7Ae protein, HnRNPA1 (RRM1-2 protein), G3BP1 (RG region), and EGFP protein are as follows: The upstream primer sequence of pCMV-CD9 plasmid is shown in SEQ ID NO.1: CAGATCCGCTAGCGCTACCGGTCGCCACCCTCGAGATGCCGGTCAAAG GAGGCACCAAGTGCA; The downstream primer sequence of pCMV-CD9 plasmid is shown in SEQ ID NO.2: CCTCCACCACCGCTACCTCCGAATTCGACCATCTCGCGGTTCCTGCGG ATAGCACAGCACAAGATC; The upstream primer sequence of pCMV-CD-L7Ae plasmid is shown in SEQ ID NO.3: TGGTGGAGGATCAGGTGGTAAGCTTATGGACTTGCGCTTCGAGGCT; The downstream primer sequence of pCMV-CD9-L7Ae plasmid is shown in SEQ ID NO.4: TCAGTTATCTAGATCCGGTGGATCCTCACTTCGCAGGCCCTGTTATTTC CGATT; The upstream primer sequence of pCMV-CD9-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.5: ATCAGGTGGTAAGCTTAGGAAGCTCTTCATTGGAGGGTT; The downstream primer sequence of pCMV-CD9-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.6: GAAGCGCACGTACATGTCGACTCCGGAGCCTCTCTTGCTTTGACAGGGC TTTTCTAACTTC; The upstream primer sequence of pCMV-CD9-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.7: ATCAGGTGGTAAGCTTGTCGAAGAGAAGAAGACTCGAGC; The downstream primer sequence of pCMV-CD9-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.8: GACTCCGGAGCCTCCCTGCCGTGGCGCAAGC; The upstream primer sequence of pCMV-CD9-mCherry plasmid is shown in SEQ ID NO.9: GGATCAGGTGGTAAGCTTATGGTGAGCAAGGGCGAGGAG; The downstream primer sequence of pCMV-CD9-mCherry plasmid is shown in SEQ ID NO.10: TTATCTAGATCCGGTGGATCCTTGTACAGCTCGTCCATGCC; The upstream primer sequences of pCMV-CD9-L7Ae-mCherry plasmid, pCMV-CD9-HnRNPA1 (RRM1-2)-L7Ae-mCherry plasmid and pCMV-CD9-G3BP1 (RG region)-L7Ae-mCherry plasmid are shown in SEQ ID NO.11: ATCAAGGGCCTGVAGAAGATGGTGAGCAAGGGCGAGG; The downstream primer sequences of pCMV-CD9-L7Ae-mCherry plasmid, pCMV-CD9-HnRNPA1 (RRM1-2)-L7Ae-mCherry plasmid and pCMV-CD9-G3BP1 (RG region)-L7Ae-mCherry plasmid are shown in SEQ ID NO.12: TATCTAGATCCGGTGGATCCTTGTACAGCTCGTCCATGC; The upstream primer sequence of pET28-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.13: GGGACACCATCACCATCACCATATGAGGAAGCTCTTCATTGGAGGGTT GA; The downstream primer sequence of pET28-HnRNPA1(RRM1-2)-L7Ae plasmid is shown in SEQ ID NO.14: GGTGGTGGTGGTGGTGCTCGAGTCACTTCTGCAGGCCCTTGATCTTT; The upstream primer sequence of pET28-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.15: GGGACACCATCACCATCACCATATGGTCGAAGAGAAGAAGA; The downstream primer sequence of pET28-G3BP1 (RG region)-L7Ae plasmid is shown in SEQ ID NO.16: GGTGGTGGTGGTGGTGCTCGAGTCACTTCTGCAGGCCCTTGATCTTT; The upstream primer sequence of pCMV-EGFP-Box C / D plasmid is shown in SEQ ID NO.17: TTAGTGAACCGTCAGATCCCGCTAGCgccaccATGGTGAGCAAGGGCGAG GAG; The downstream primer sequence of pCMV-EGFP-Box C / D plasmid is shown in SEQ ID NO.18: ATTCGAAGCTTGAGCTCGAGATCTGAGGGTCACCTTTCGGATCACGCCCGTACTTGTACAGCTCGTCCATGCCG; The amino acid sequence of CD9 is shown in SEQ ID NO.19: MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGVYILIGAGALMmLVGFLGCCGAVQESQCmLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRNREMV; The amino acid sequence of L7Ae is shown in SEQ ID NO.20: MYVRFEVPEDMQNEALSLLEKVRESGKVKKGTNETTKAVERGLAKLVYIAEDVDPPEIVAHLPLLCEEKNVPYIYVKSKNDLGRAVGIEVPCASAAIINEGELRKELGSLVEKIKGLQKGGSGRHPDSHQLFIGNLPHEVDKSELKDFFQSYGNVVELRINSGGKLPNFGFVVFDDSEPVQKVLSNRPIMFRGEVRLNVEEKKTRAAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGFGVGRGLAPRQGGGGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK; The amino acid sequence of HnRNPA1 (RRM1-2) is shown in SEQ ID NO.21: RKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDAAMNARPHKVDGRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQ; The amino acid sequence of G3BP1 (RG region) is shown in SEQ ID NO.22: VEEKKTRAAREGDRRDNRLRGPGGPRGGLGGGMRGPPRGGMVQKPGF GVGRGLAPRQ; The amino acid sequence of EGFP is shown in SEQ ID NO.23: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK; Step (3), fragment recovery; Step (4), recombination reaction; Step (5), conversion of reaction products; Step (6), coating the plate; Step (7), extracting the plasmid; Step (8), cell transfection; Step (9), protein purification; Step (10), extracellular vesicle extraction.

8. A method for increasing RNA molecular loading using engineered vesicles, characterized in that: The following steps are involved: Step (1), extracting RNA from the engineered vesicles; Step (2), removing DNA and synthesizing cDNA chain through reverse transcription reaction; Step (3), perform qPCR reaction.

9. A method for increasing RNA molecule loading using engineered vesicles according to claim 8, characterized in that: The RNA includes any RNA nucleic acid molecule carrying Box C / D.

10. A method for increasing RNA molecule loading using engineered vesicles according to claim 8, characterized in that: The qPCR reaction uses GAPDH as an internal reference and EGFP as a fluorescent protein; The upstream primer sequence of GAPDH is shown in SEQ ID NO.24: AGCCACATCGCTCAGACAC; The downstream primer sequence of GAPDH is shown in SEQ ID NO.25: GCCCAATACGATCGATCC; The upstream primer sequence of EGFP is shown in SEQ ID NO.26: CCTGAAGTTCATCTGCACCA; The downstream primer sequence of EGFP is shown in SEQ ID NO.27: GGTCAGGGTGGTCACGAG.

Images