CN116606811B

CN116606811B - Method for programming extracellular vesicles and cell interactions based on receptor ligand encoded by DNA

Info

Publication number: CN116606811B
Application number: CN202310415585.2A
Authority: CN
Inventors: 王凯喆; 魏雨涵; 闫庆龙; 李江; 王丽华; 樊春海
Original assignee: Xiangfu Laboratory
Current assignee: Xiangfu Laboratory
Priority date: 2023-04-18
Filing date: 2023-04-18
Publication date: 2024-12-20
Anticipated expiration: 2043-04-18
Also published as: CN116606811A

Abstract

The invention provides a method for programming extracellular vesicles and cell interactions based on a receptor ligand coded by DNA, which comprises the steps of inserting cholesterol modified DNA single chains into phospholipid bilayer of extracellular vesicles by shaking with the extracellular vesicles as the ligand, mixing and annealing three cholesterol modified DNA single chains with a DNA containing a complementary pairing sequence with the ligand to form a receptor with a DNA tetrahedron structure, inserting the receptor into a cell membrane through cholesterol at three vertexes, and identifying the extracellular vesicles functionalized by the DNA ligand, so that the extracellular vesicles from different sources can be efficiently and specifically ingested by the cell. According to the invention, a programmable, general, efficient and specific control method for interaction between extracellular vesicles and cells is provided, and a novel tool is provided for applications such as living cell delivery and cell communication regulation.

Description

Method for programming extracellular vesicles and cell interactions based on receptor ligand encoded by DNA

Technical Field

The present invention relates to the field of cell engineering, and more particularly to a method for programming extracellular vesicles and cellular interactions based on DNA-encoded receptor ligands.

Background

Extracellular vesicles are a highly heterogeneous class of natural biological vesicles secreted by cells with a phospholipid bilayer structure. Extracellular vesicles contain bioactive substances such as proteins, lipids, nucleic acids, etc., which can be secreted by donor cells, transferred to recipient cells, and alter the phenotype and function of the recipient cells. The "extracellular vesicle-cell" interactions are involved in a variety of normal physiological responses, as well as in pathological processes. Extracellular vesicles have the unique advantages of wide sources, high biocompatibility, abundant contents and the like, and are widely focused in the fields of drug delivery, regenerative medicine, tissue repair, synthetic biology and the like. Thus, the interaction of the reprogrammed extracellular vesicles with the cells is not only critical to elucidating the relevant biological processes, but also helps to advance the construction of extracellular vesicles as information transfer units in synthetic biology. In recent years, research has been greatly advanced to manually intervene in extracellular vesicles and cell interactions. For example, expression of VSV-G protein (vesicular stomatitis virus G protein) on the surface of extracellular vesicles by genetic engineering has been used to promote uptake into target cells, modification of c (RGDyK) polypeptide on the surface of extracellular vesicles by copper-free click chemistry has been used to promote aggregation into cerebrovascular endothelial cells, and functionalization of macrophage-derived extracellular vesicles by AS1411 aptamer has been used to enhance targeting to glioma cells. However, the specificity, programmability and versatility of these methods remain limited.

Disclosure of Invention

The invention aims to provide a method for programming interaction between extracellular vesicles and cells based on a receptor ligand coded by DNA, so as to solve the problems of limited specificity, programmability and universality existing in the existing interaction technology of the reprogrammed extracellular vesicles and the cells.

In order to solve the problems, the invention adopts the following technical scheme:

A method for programming extracellular vesicles and cell interactions based on a receptor ligand encoded by DNA is provided, which comprises inserting cholesterol modified DNA single strands as ligands into phospholipid bilayer of extracellular vesicles by shaking with the extracellular vesicles, mixing three cholesterol modified DNA single strands with a DNA containing complementary pairing sequences with the ligands, annealing to form a receptor with a DNA tetrahedron structure, inserting cholesterol at three vertices into cell membranes, and recognizing extracellular vesicles functionalized by the DNA ligands, thereby realizing efficient and specific uptake of extracellular vesicles of different sources by cells.

According to a preferred scheme of the invention, the method comprises the following steps of S1, respectively preparing a DNA-encoded artificial receptor and a ligand, S2, adopting the DNA-encoded artificial receptor modified cells prepared in the step S1, S3, separating and extracting extracellular vesicles, adopting the DNA-encoded artificial ligand modified extracellular vesicles prepared in the step S1, and S4, adding the DNA-encoded artificial ligand modified extracellular vesicles into the DNA-encoded artificial receptor modified cells, incubating for 2-3 hours at 37 ℃, and observing DNA receptor ligand mediated extracellular vesicle uptake by a confocal laser microscope or a flow cytometer.

According to a preferred embodiment of the invention, step S1 comprises the following sub-steps:

(1) Dissolving magnesium chloride hexahydrate in phosphate buffer solution to make the final concentration of magnesium ions be 1-5mM;

(2) Respectively dissolving four DNA tetrahedral short chains in the solution prepared in the step (1), and uniformly mixing in a PCR tube, wherein the final concentration range of each short chain is 0.5-2 mu M;

(3) Placing the PCR tube in the step (2) in a PCR instrument, heating at 95 ℃ for 10 minutes, rapidly cooling to 4 ℃ to obtain a DNA coded artificial receptor, and storing at 4 ℃ for later use;

(4) And (3) dissolving ligand DNA-chol short chain in the solution prepared in the step (1) to make the concentration 80-120 mu M, and preparing the DNA coded artificial ligand, and storing at 4 ℃ for later use.

According to a preferred embodiment of the invention, step S2 comprises the following sub-steps:

(5) Inoculating cells into a cell culture dish with a glass bottom, removing a culture medium, and adding PBS for cleaning;

(6) Adding the DNA tetrahedron solution obtained in the step (2) into a serum-free culture medium;

(7) Adding the solution in the step (6) into the cells in the step (5), and incubating for 8-12 minutes at room temperature to obtain DNA-encoded artificial receptor modified cells;

(8) Step (7) cells were washed three times with PBS and serum-free medium was added.

5. The method according to claim 4, characterized in that step S3 comprises the sub-steps of:

(9) Inoculating and culturing the cells of the extracellular vesicles to be extracted in a plurality of T75 cell culture flasks, when the cell growth area reaches about 80%, washing three times by using 10mL of PBS, finally adding 10mL of serum-free culture medium respectively, culturing for 24 hours to obtain about 100mL of cell culture supernatant, centrifuging 300g for 5-10 minutes to remove dead cells, collecting supernatant, centrifuging 3000g for 10-20 minutes to remove cell fragments, collecting supernatant, centrifuging 10000g for 20-30 minutes to remove organelles and large cell vesicles, collecting supernatant, filtering by using a 0.22 mu m filter, collecting lower-layer solution, centrifuging 100000g for 80-90 minutes, gently removing upper-layer liquid, re-suspending the bottom by using 500 mu L of PBS, and storing at-80 ℃ for later use.

According to a preferred embodiment of the invention, step S3 further comprises the sub-steps of:

(10) Taking 100 mu L of extracellular vesicles for quick dissolution, adding 1 mu L of Deep-red extracellular vesicle staining reagent, uniformly mixing, incubating at 37 ℃ for 30-40 minutes in a dark place, transferring all the solutions into a filter tube, centrifuging at 3000g for 3-5 minutes at room temperature, adding 100 mu L of PBS into the filter tube, centrifuging at 3000g for 3-5 minutes, repeating once, centrifuging at 3000g for 3-5 minutes at room temperature to remove the residual solution, sucking 50 mu L of PBS, adding the solution into the tube, lightly blowing, and resuspending extracellular vesicles;

(11) Taking 50 mu L of the solution in the step (10), and adding 5.6 mu L of ligand DNA-chol short chain with the concentration of 80-120 mu M;

(12) Placing the solution in the step (11) on a vortex oscillator, and oscillating for 3-5 minutes at 600 rpm/min;

(13) Adding the solution obtained in the step (12) into a sleeve of a ultrafilter tube, adding 350 mu L of PBS solution, centrifuging for 3-5 minutes at 5000g, and removing the bottom solution;

(14) 400 mu L of PBS solution is added into the ultrafiltration tube sleeve, 5000g is centrifuged for 5 minutes, and the washing is repeated for 2 times;

(15) The residual solution in the tube of the ultrafiltration tube in step (14) was aspirated to about 20. Mu.L, added to a 500. Mu.L centrifuge tube, and placed at 4℃for further use.

According to a preferred embodiment of the invention, step S4 comprises the following sub-steps:

(16) Adding 20 mu L of the DNA-chol short-chain modified extracellular vesicles prepared in the step (15) into the DNA tetrahedron modified cell serum-free medium prepared in the step (8);

(17) Placing the cells in a cell culture medium, and incubating for 2-3 hours at 37 ℃;

(18) Removing the cell culture medium in the step (17), adding 1mL of a culture medium containing 5 mu L PlasMem Bright Green cell membrane dye and 10 mu L of Hoechst 33342 (100×) nuclear dye, and incubating at 37 ℃ for 20 minutes;

(19) Removing the medium containing the dye in the step (18), washing with PBS for 2 times, and re-adding the cell culture medium;

(20) DNA receptor ligand mediated extracellular vesicle uptake was detected by confocal laser microscopy or flow cytometry analysis.

Preferably, the extracellular vesicles of different sources include STO cells, watermelon, and extracellular vesicles of E.coli origin.

Preferably, the cells include cells of human origin, murine origin, etc., and may be selected from MDA-MB-231 cells, heLa cells, etc.

According to a preferred embodiment of the present invention, the extracellular vesicle uptake capacity of a cell can be regulated by changing the length of the complementary strand of DNA, and the extracellular vesicle uptake capacity increases with the number of complementary paired bases of DNA.

The invention relates to a novel method for controlling interaction between an extracellular vesicle and a cell, in particular to a method for controlling interaction between the extracellular vesicle and the cell, which is characterized in that a DNA single chain is inserted into the surface of the extracellular vesicle through cholesterol to serve as a ligand, a complementary strand is extended from vertexes of a DNA tetrahedron to serve as a receptor, and the other three vertexes are inserted into the surface of the cell membrane through cholesterol, so that the extracellular vesicle can be efficiently and specifically ingested by the cell, and the extracellular vesicle and the cell can be controlled. A schematic representation of such a DNA-encoded receptor ligand programmed "extracellular vesicle-cell" interaction provided in accordance with the present invention is shown in fig. 1.

It should be appreciated that the prior art never discloses the interaction technique of extracellular vesicles and cells through DNA encoding receptor ligands, and the invention solves the problem of reprogramming and controllable interaction of extracellular vesicles and cells of different genus sources by adopting such a technical scheme.

Compared with the prior art, the invention has the following remarkable advantages:

1) The receptor and the ligand synthesized artificially are DNA codes, and the receptor ligand coded by the DNA codes has high specificity and predictable orthogonal reaction effect by changing the DNA tetrahedron on extension DNA single strand on cells and the DNA single strand base sequence on extracellular vesicles due to the complementary pairing of DNA specific bases and the abundant programmable sequences;

2) Because the extracellular vesicles from different sources and the cells of different types are formed by phospholipid bilayer to form a basic membrane structure, and the receptor ligand coded by DNA is inserted into the phospholipid bilayer of the biological membrane by utilizing cholesterol hydrophobic effect, the operation is simple and convenient without complex covalent modification and genetic engineering, so the programming of the extracellular vesicle-cell interaction by the receptor ligand coded by DNA has wide universality.

3) The DNA tetrahedral structure provides a basis for the stable presence of DNA receptors on cells, which are not rapidly endocytosed by the cell.

4) The invention provides a versatile platform for cell engineering to tailor the exchange of intercellular substances and signals across species.

In summary, according to the present invention, a programmable, universal, efficient, and specific method for manipulating extracellular vesicles and cell interactions is provided, which provides a new tool for applications such as living cell delivery, cell communication regulation, and the like.

Drawings

FIG. 1 is a schematic representation of the programmed "extracellular vesicle-cell" interactions of a DNA-encoded receptor ligand designed according to the present invention;

FIG. 2 shows TEM image (a) and particle size analysis (b), dynamic light scattering results (c), and Chol-DNA ligand map (d) after extracellular vesicle extraction in example 1;

FIG. 3 is a graph of polyacrylamide gel electrophoresis (a) of DNA tetrahedron receptor synthesis of example 1, modified by laser confocal microscopy of MDA-MB-231 cell membranes (b) and a statistical graph of fluorescence over time (c);

FIG. 4 is a statistical plot of DNA encoding extracellular vesicle uptake laser confocal microscopy results (a) and fluorescence over time (b) of example 2;

FIG. 5 is a flow cytometry fluorescence of extracellular vesicles taken up by cells with and without paired DNA in example 2;

FIG. 6 is a laser confocal fluorescence analysis of extracellular vesicles and endocytic localization in example 2;

FIG. 7 is a fluorescence image of extracellular vesicle uptake flow cell encoded by DNA of different base lengths in example 2;

FIG. 8 is a graph showing the uptake analysis of DNA-encoded mouse STO cells, watermelon, E.coli-derived extracellular vesicles in MDA-MB-231 cells in example 3;

FIG. 9 is a graph showing the analysis of the selective uptake of DNA encoding HeLa cells and watermelon-derived extracellular vesicles in HeLa cells in example 4.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1 preparation of DNA-encoded artificial receptors and ligands

In this example 1, the preparation of DNA-encoded artificial receptors and ligands comprises the following steps:

(1) Magnesium chloride hexahydrate (MgCl ₂·6H₂ O,10.16 mg) was dissolved in phosphate buffered saline (PBS, 10 mL).

(2) Four DNA tetrahedral short chains (40. Mu.M, 2.5. Mu.L) were dissolved in the solution (90. Mu.L) of (1) and mixed well in a PCR tube, the sequences of which are shown in Table 1.

(3) And (3) placing the PCR tube in the step (2) in a PCR instrument, heating for 10 minutes at 95 ℃, rapidly cooling to 4 ℃, and assembling four DNA single strands to form a DNA tetrahedron structure. Stored at 4 ℃ prior to use.

(4) Ligand cholesterol modified DNA short chain a' -chol (L1) (100 μm) was stored at 4 ℃ prior to use.

(5) DNA tetrahedral gel running was performed using a 6% polyacrylamide gel in 1 XTAE-Mg ²⁺(40mM Tris,2mM EDTA-NH₂,12.5mM MgAc·4H₂ O, and 20mM HAc, pH 8.0) buffer at 100V for 1h. The Gel was stained with Gel red. And (5) carrying out imaging analysis on the DNA tetrahedron synthesis effect by a gel imager.

In this embodiment, the method for modifying cells using the artificial receptor encoded by the above DNA comprises the steps of:

(1) MDA-MB-231 cells were seeded in glass-bottomed cell culture dishes, the medium was removed, and washed once with 2mL PBS.

(2) Mu.L of DNA tetrahedron solution (1. Mu.M) was added to 300. Mu.L of serum-free medium.

(3) The solution of (2) was added to the cells of (1) and incubated at room temperature for 10 minutes.

(4) The cells in (3) were washed three times with 2mL PBS and finally added to serum-free medium. As shown in FIG. 1, cholesterol-modified DNA (Chol-DNA) is inserted into the phospholipid bilayer of extracellular vesicles as a ligand by shaking with the extracellular vesicles, and three cholesterol-modified DNA single strands and one DNA containing a complementary pairing sequence with the ligand are mixed and annealed to form a receptor having a DNA tetrahedron structure, and the DNA receptor is inserted into the cell membrane through cholesterol at three vertices to recognize extracellular vesicles functionalized with the DNA ligand.

The DNA sequences used in this example 1 are shown in Table 1 below.

TABLE 1 DNA sequence

As shown in FIG. 2, the extracellular vesicles derived from MDA-MB-231 cells are obtained by an ultracentrifugation method, the extracellular vesicles are subjected to negative staining by uranyl acetate, TEM results (a) show that the extracellular vesicles extracted from MDA-MB-231 cells are in a typical spherical cup-shaped structure, the diameter of the extracellular vesicles is mainly 50-150nm through nanoparticle tracking analysis (b), dynamic light scattering results (c) show that the hydration particle size of the extracellular vesicles is enlarged after DNA modification, ligand Chol-DNA modifies FAM fluorescent groups, and enzyme-labeled instrument detects (d) that the extracellular vesicles have obvious fluorescent signal increase, DNaseI digestion and fluorescent signal disappearance, so that ligand Chol-DNA is proved to be successfully inserted into the extracellular vesicle membrane.

As shown in FIG. 3, polyacrylamide gel electrophoresis (a) showed successful assembly of the DNA tetrahedral receptor and complementary binding to the ligand DNA, and laser confocal microscopy results (b, c) showed that three DNA tetrahedral receptors with apex modified cholesterol were successfully inserted into the cell membrane, and after 2h, there was a 55% fluorescent signal on the cell membrane.

EXAMPLE 2DNA encoded receptor ligand Programming MDA-MB-231 cell "extracellular vesicle-cell" interactions

In this example 2, the method of DNA-encoded receptor ligand programming the MDA-MB-231 cell "extracellular vesicle-cell" interaction comprises the steps of:

(1) MDA-MB-231 cells were cultured in 10T 75 cell culture flasks, and when the cells were grown to 80% -90% of the area of the dish, washed three times with PBS, and replaced with serum-free medium for 24 hours. The supernatant was collected, and extracellular vesicles were isolated from the medium by ultracentrifugation and resuspended in 500. Mu.L of PBS.

(2) And (3) staining and marking the extracellular vesicles, namely taking 100 mu L of the extracellular vesicles in the step (1), adding 1 mu L of Deep-red extracellular vesicle staining reagent, and uniformly mixing. Incubate at 37 ℃ for 30 minutes in the dark. The whole solution was transferred to a filter tube and centrifuged at 3000g for 5 minutes at room temperature. 100. Mu.L of PBS was added to the filter tube, and the mixture was centrifuged at 3000g for 5 minutes and repeated once. Centrifuge at 3000g for 5min at room temperature to remove the remaining solution. mu.L of PBS was pipetted into the tube, gently blown, and the extracellular vesicles resuspended.

(3) Extracellular vesicle-modified DNA was aliquoted in two aliquots of 60. Mu.L of the solution from step (2), each at 30. Mu.L. One group (DNA modified group) was added with 10. Mu.L of ligand DNA-chol short chain (40. Mu.M) at 30. Mu.L, placed on a vortex shaker, shaken at 600 rpm/min for 5min, the reacted solution was added to a tube-in-tube (100 k MWCO) of a ultrafilter, and the other was added with 350. Mu.L of PBS solution, and centrifuged at 5000g for 5 min. The bottom solution was removed and the wash repeated 2 times. The other group (control group) repeats the above operation. Finally, about 20. Mu.L of the same residual solution was obtained for each group and placed at 4℃for further use. Thus, DNA ligand-modified extracellular vesicles were obtained.

(4) Cell modification DNA tetrahedra MDA-MB-231 cells were seeded in 2 glass-bottomed cell culture dishes, the medium was removed, and washed once with 2mL PBS. One group of cells was incubated at room temperature for 10 minutes by adding 100. Mu.L of DNA tetrahedron solution (1. Mu.M) to 300. Mu.L of serum-free medium, washing the cells three times with 2mL of PBS, and finally adding the serum-free medium to obtain DNA tetrahedron receptor-modified cells.

(5) DNA receptor ligand encoding extracellular vesicles interaction with cells two groups of extracellular vesicles from step (3) were added to two groups of cells from step (4), incubated at 37℃for 2h without serum, medium removed, 1mL of medium containing 5. Mu. L PlasMem Bright Green cell membrane dye and 10. Mu.L of Hoechst 33342 (100×) nuclear dye was added and incubated at 37℃for 20 min. The medium containing the dye was removed and washed 2 times with PBS. The cell culture medium was re-added.

(6) DNA receptor ligand mediated extracellular vesicle uptake was observed with confocal laser microscopy or flow cytometry.

The DNA sequences used in this example 2 are shown in Table 2 below.

TABLE 2 DNA sequence

TABLE 3 DNA sequences of different pairing lengths

As shown in fig. 4, the laser confocal microscopy results (a, b) showed that the extracellular vesicle uptake of the DNA encoding group was significantly increased, and the fluorescence intensity was increased by about 6.5 times, compared to the extracellular vesicle uptake in the natural state after co-incubation for 2 hours.

As shown in FIG. 5, the fluorescence intensity measurement results by the flow cytometer show that the cell can efficiently take up the extracellular vesicles only when the DNA receptor ligand bases are complementary and the extracellular vesicles cannot be taken up when the receptor ligand bases are not matched.

As shown in fig. 6, cells trace the endocytic process through ECGreen dye, and confocal laser microscopy imaging shows that after co-incubation for 2 hours, the extracellular vesicles in the natural state (control group) are mainly co-localized with endocytic vesicles, and the extracellular vesicles encoded by the DNA receptor ligand are distributed on the cell membrane, in the cytoplasm and in the endocytic vesicles.

As shown in FIG. 7, by changing the length of the complementary strand of DNA (DNA sequences of different lengths are shown in Table 3), the uptake capacity of extracellular vesicles by cells can be regulated, and the uptake capacity of extracellular vesicles increases with the increase in the number of complementary paired bases of DNA.

EXAMPLE 3 DNA-encoded receptor ligand Programming extracellular vesicles of different origins to interact with cells

In this example 3, the programming of extracellular vesicles of different origins by DNA-encoded receptor ligands to interact with cells comprises the following steps:

(1) Extraction of mouse STO cell-derived extracellular vesicles As shown in the MDA-MB-231 cell method of example 2, mouse STO cell-derived extracellular vesicles were obtained.

(2) Extraction of extracellular vesicles from watermelon by squeezing a piece of watermelon by physical extrusion, taking 50mL of watermelon juice, diluting with PBS according to a ratio of 1:2, and separating extracellular vesicles by differential ultracentrifugation. The specific steps are that the solution is centrifuged for 10 minutes with 1000g, the supernatant is taken, and then centrifuged for 20 minutes with 3000g, and the supernatant is taken repeatedly for 2 times. The supernatant was centrifuged at 10000g for 30 minutes, and the supernatant was collected and repeated 2 times, and filtered through a 0.22 μm filter. Extracellular vesicles were collected by centrifugation at 100000g for 90 min at 4℃using a Beckmann ultracentrifuge (Optima x-90) and a 50.2Ti rotor, carefully decanting the supernatant, and re-suspending with PBS.

(3) E.coli-derived extracellular vesicles extraction 50mL of E.coli LB (Luria-Bertani) liquid medium was centrifuged at 10000g for 30min, and the supernatant was taken and repeated twice. The solution was filtered twice through a 0.22 μm filter to remove cells and cell debris. Coli-derived extracellular vesicles were collected at 100000g ultracentrifugation for 120min at 4 ℃. Finally, the vesicles were resuspended in 500 μl PBS.

(4) The extracellular vesicles of different origins isolated in step (1-3) were subjected to labelling staining as shown in example 2, modifying the DNA ligand (A' -chol, L1). Co-incubation with MDA-MB-231 cells modified by the DNA receptor (R1) was performed, and the DNA sequences are shown in Table 4 below.

(5) DNA receptor ligand mediated uptake of extracellular vesicles of different origins was observed with confocal laser microscopy or flow cytometry.

As shown in FIG. 8, we have extracted mouse STO cells, watermelon, E.coli-derived extracellular vesicles in addition to MDA-MB-231-derived extracellular vesicles for studying the versatility of DNA-encoded receptor ligands. TEM images show that mouse STO cells, watermelon, E.coli-derived extracellular vesicles all take on cup-like, circular structures. MDA-MB-231 cells and extracellular vesicles from different sources are respectively modified by DNA receptor ligands, and then incubated for 2 hours, and a laser confocal fluorescence diagram and a flow cytometry analysis result show that the receptor ligands coded by the DNA can obviously promote the uptake of the MDA-MB-231 cells to the extracellular vesicles from different sources.

EXAMPLE 4 DNA-encoded receptor ligand Programming HeLa cells and Selective uptake of watermelon-derived extracellular vesicles in HeLa cells

In this example 4, DNA-encoded receptor ligand programming HeLa cells and selective uptake of watermelon-derived extracellular vesicles in HeLa cells comprises the steps of:

(1) HeLa cells and watermelon-derived extracellular vesicles were isolated and stained for markers by the method described in example 3, and DNA of different sequences was modified as different ligands A '-chol (L1) or B' -chol (L2), respectively, and the specific DNA sequences are shown in Table 4.

(2) According to the method of example 2, heLa cells were modified simultaneously to carry tetrahedra of different DNA receptors R1 and R2.

(3) According to the experimental design of fig. 9, heLa cells with modified or unmodified DNA ligand in step (1) and watermelon-derived extracellular vesicles were incubated with HeLa cells modified with DNA receptor in step (2) in serum-free medium, respectively.

(4) DNA-encoded receptor ligands were photographed using a confocal laser microscope to program the selective uptake of HeLa cells and watermelon-derived extracellular vesicles in HeLa cells.

TABLE 4 DNA sequence

Thanks to the DNA specific base complementary pairing and the abundance of programmable sequences, we can construct multiple sets of specific recognition units by altering the base sequence of DNA tetrahedrally extended DNA single strands on cells and DNA single strands on extracellular vesicles. We extracted HeLa cells, watermelon-derived extracellular vesicles, and used to study the selective uptake of HeLa cells into different sources of extracellular vesicles by altering the DNA recognition sequence of the receptor ligand.

As shown in fig. 9, the HeLa cells with different fluorescent markers and the extracellular vesicles derived from watermelons respectively modify different DNA ligands L1 or L2, the HeLa cells modify corresponding receptors R1 and R2, and the co-incubation for 2h, and the confocal laser fluorescence image results show that the extracellular vesicles can be efficiently taken up by the cells only when the base sequences of the receptor ligands encoded by the DNA meet complementary pairing in a mixed system. In the control group, heLa cells and watermelon-derived extracellular vesicles were not modified at all, and HeLa cells were taken up in very little amounts. HeLa cells can take up HeLa vesicles with high efficiency when HeLa cell-derived extracellular vesicles modify the DNA ligand L1. When the extracellular vesicles derived from the watermelons modify the DNA ligand L2, heLa cells can efficiently ingest the vesicles derived from the watermelons. When the HeLa cells and watermelon-derived extracellular vesicles modify the DNA ligands L1 and L2, respectively, the HeLa cells can efficiently ingest HeLa and watermelon-derived vesicles in the bench system.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.

Claims

1. A method for programming the interaction between extracellular vesicles and cells based on DNA-encoded receptor ligands, which is not used for the diagnosis and treatment of diseases, and is characterized in that the method comprises: inserting a cholesterol-modified DNA single strand as a ligand into the phospholipid bilayer of the extracellular vesicle by oscillating with the extracellular vesicle to obtain an extracellular vesicle modified with a DNA-encoded artificial ligand; mixing three cholesterol-modified DNA single strands and one DNA single strand containing a complementary pairing sequence with the ligand, annealing, and forming a receptor with a DNA tetrahedral structure; using the receptor-modified cell to obtain a cell modified with a DNA-encoded artificial receptor; adding the extracellular vesicle modified with the DNA-encoded artificial ligand to the cell modified with the DNA-encoded artificial receptor, wherein the receptor is inserted into the cell membrane through the cholesterol at three vertices, and recognizes the extracellular vesicle functionalized with the DNA ligand, thereby realizing the efficient and specific uptake of extracellular vesicles from different sources by the cell.

2. The method according to claim 1, characterized in that the method comprises the following steps:

S1: Prepare DNA-encoded artificial receptors and ligands respectively;

S2: cells modified with the artificial receptor encoded by the DNA prepared in step S1;

S3: isolating and extracting extracellular vesicles, and modifying the extracellular vesicles with the artificial ligand encoded by the DNA prepared in step S1;

S4: Add extracellular vesicles modified with DNA-encoded artificial ligands to cells modified with DNA-encoded artificial receptors, incubate at 37°C for 2h~3h, and observe DNA receptor ligand-mediated extracellular vesicle uptake using laser confocal microscopy or flow cytometry.

3. The method according to claim 2, characterized in that step S1 comprises the following sub-steps:

(1) Dissolve magnesium chloride hexahydrate in phosphate buffer solution to make the final concentration of magnesium ions 1-5 mM;

(2) Dissolve three cholesterol-modified DNA single strands and one DNA single strand containing a sequence complementary to the ligand in the solution prepared in step (1), and mix them in a PCR tube. The final concentration of each of the four DNA single strands is in the range of 0.5-2 μM.

(3) Place the PCR tube in step (2) in a PCR instrument, heat at 95°C for 10 minutes, and quickly cool to 4°C to obtain a DNA tetrahedral structure, i.e., the DNA-encoded artificial receptor, which is stored at 4°C for future use;

(4) Dissolve a cholesterol-modified DNA single strand in the solution prepared in step (1) to a concentration of 80-120 μM to prepare a DNA-encoded artificial ligand, which is then stored at 4°C for future use.

4. The method according to claim 2 or 3, characterized in that step S2 comprises the following sub-steps:

(5) Seed the cells in a glass-bottomed cell culture dish, remove the culture medium, and add PBS to wash;

(6) adding the DNA-encoded artificial receptor to serum-free culture medium;

(7) adding the solution containing the DNA-encoded artificial receptor in step (6) to the cells in step (5), and incubating at room temperature for 8 to 12 minutes to obtain cells modified with the DNA-encoded artificial receptor;

(8) Wash the cells in step (7) with PBS and add serum-free culture medium.

5. The method according to claim 2, characterized in that step S3 comprises the following sub-steps:

(9) Inoculate the cells from which extracellular vesicles are to be extracted into several T75 cell culture flasks. When the cell growth area reaches 80%, wash them three times with 10 mL PBS. Finally, add 10 mL of serum-free medium to each flask. After culturing for 24 h, obtain 100 mL of cell culture supernatant; centrifuge at 300 g for 5-10 minutes to remove dead cells and take the supernatant; centrifuge at 3000 g for 10-20 minutes to remove cell debris and take the supernatant; centrifuge at 10000 g for 20-30 minutes to remove organelles and large cell vesicles and take the supernatant; filter with a 0.22 μm filter and take the lower layer of solution; centrifuge at 100000 g for 80-90 minutes, gently remove the upper layer of liquid, resuspend the bottom with 500 μL PBS, which is the extracellular vesicles, and store at -80 °C for later use.

6. The method according to claim 5, characterized in that step S3 further comprises the following sub-steps:

(10) Take 100 μL of extracellular vesicles for rapid dissolution, add 1 μL of Deep-red extracellular vesicle staining reagent, and mix well; incubate at 37 °C in the dark for 30 to 40 minutes; transfer all the solution to a filter tube and centrifuge at 3000 g for 3 to 5 minutes at room temperature; add 100 μL of PBS to the filter tube and centrifuge at 3000 g for 3 to 5 minutes, repeat once; centrifuge at 3000 g for 3 to 5 minutes at room temperature to remove the remaining solution; aspirate 50 μL of PBS into the tube and gently blow to resuspend the extracellular vesicles;

(11) Take 50 μL of the extracellular vesicle solution in step (10) and add 5.6 μL of cholesterol-modified DNA single-stranded ligand with a concentration of 80-120 μM;

(12) Place the solution in step (11) on a vortex shaker and shake at 600 rpm/min for 3 to 5 minutes;

(13) Add the solution in step (12) to the ultrafiltration tube sleeve, add 350 μL of PBS solution, centrifuge at 5000 g for 3-5 minutes, and remove the bottom solution;

(14) Add another 400 μL of PBS solution to the ultrafiltration tube sleeve, centrifuge at 5000 g for 5 min, and repeat the wash;

(15) Pipette 20 μL of the residual solution in the ultrafiltration tube sleeve in step (14) and add it to a 500 μL centrifuge tube to obtain DNA-encoded artificial ligand-modified extracellular vesicles, which are then placed at 4°C for later use.

7. The method according to claim 2, characterized in that step S4 comprises the following sub-steps:

(16) Take 20 μL of extracellular vesicles modified with DNA-encoded artificial ligands and add them to the serum-free culture medium of cells modified with DNA-encoded artificial receptors;

(17) Place the cells in cell culture medium and incubate at 37 °C for 2–3 h.

(18) Remove the cell culture medium in step (17), add 1 mL of culture medium containing 5 μL of PlasMem Bright Green cell membrane dye and 10 μL of 100× Hoechst 33342 cell nuclear dye, and incubate at 37 °C for 20 min;

(19) Remove the culture medium containing the dye in step (18), wash twice with PBS, and add cell culture medium again;

(20) Detect DNA receptor ligand-mediated extracellular vesicle uptake using confocal laser scanning microscopy or flow cytometry.

8. The method according to claim 1, characterized in that the extracellular vesicles from different sources are selected from: extracellular vesicles from STO cells, watermelon, and Escherichia coli.

9. The method according to claim 1, characterized in that the cells are selected from human and mouse cells.

10. The method according to claim 9, characterized in that the cells are selected from: MDA-MB-231 cells and HeLa cells.

11. The method according to claim 1 is characterized in that the length of the complementary DNA pairing chain is changed to adjust the cell's ability to uptake extracellular vesicles, and the extracellular vesicle uptake ability increases with the increase in the number of complementary DNA pairing bases.