CN113980959A - Y-shaped multifunctional DNA nano assembly, preparation method and application thereof - Google Patents
Y-shaped multifunctional DNA nano assembly, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Y-shaped multifunctional DNA nano assembly, a preparation method and application thereof, wherein the multifunctional DNA nano assembly is formed by hybridizing four DNAs into a Y-shaped structure, and comprises a chain ab, a chain cb, a chain d and a chain e; and strand d is complementary to a partial sequence of strand ab, strand e is complementary to a partial sequence of strand cb, both strand ab and strand cb are modified with cell membrane anchoring groups, and strand d is provided with an o-nitrobenzyl photocleavage group. The preparation method comprises the following steps: s1, synthesizing an ab sequence; s2, synthesizing a cb sequence; s3, synthesizing a d sequence; s4, synthesizing a sequence e; s5, mixing the four DNA sequences, heating, annealing and hybridizing. The multifunctional DNA nano assembly can be quickly anchored on the surface of a cell membrane, so that the efficient light-operated regulation and control of the function of a c-Met receptor on the surface of the cell membrane are realized, and the real-time monitoring of the inhibition effect of the c-Met receptor is realized.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a cell membrane anchored Y-shaped multifunctional DNA nano assembly, a preparation method and application thereof.
Background
Multiple proteins of cells jointly construct a complex signal network to participate in various cell activities, and play an important role in maintaining normal physiological activities of cells and the occurrence and development processes of diseases. The proteins in the signal network do not exist in isolation, the functions and the expression level of the proteins are precisely regulated, wherein the function and the expression of other proteins are influenced by a signal cascade reaction caused by the function change of certain protein, and the precise regulation of cell behaviors is realized. In the process of cell signal transduction mediated by intercellular epithelial transformation factor (c-Met), the c-Met receptor can sense and integrate the stimulation signals of external environment, and the receptor dimerization induced by the binding of Hepatocyte Growth Factor (HGF) with the c-Met receptor is generally considered as the first step of the c-Met signal transduction process. The activated c-Met receptor transmits signals into cells, and promotes various cell behaviors such as proliferation, migration, invasion and angiogenesis of the cells by selectively activating or inhibiting specific signal molecules. For example, activation of the c-Met signaling pathway can increase the level of Vascular Endothelial Growth Factor (VEGF) secretion, thereby promoting the angiogenic process. Due to the important role of the c-Met signaling pathway in physiological function and disease progression, the regulation of cellular function by on-demand modulation of c-Met receptor function has attracted extensive attention. Given the close relationship between the relevant signaling molecules, changes in a particular signaling molecule can serve as feedback to evaluate the effect of upstream regulation. Therefore, on the premise of keeping the structure and the state of the protein unaffected, accurate on-demand regulation and real-time monitoring of the protein in the signal network is helpful for systematically clarifying the biological function and action mechanism of the protein, and promoting the development of intelligent treatment drugs, and has important significance for the development of precise medical treatment.
At present, traditional strategies such as immunoblotting and enzyme-linked immunosorbent assay are widely applied to detection means for verifying signal molecule changes (expression level or post-translational modification and the like). However, these methods require cumbersome experimental procedures prior to analysis and do not allow real-time monitoring of living cells. Although fluorescent biosensors based on gene editing strategies can be used for monitoring of cell signaling molecules, the introduction of new genes into cells requires complex and time-consuming steps and still faces the risk of interfering with the endogenous signaling pathway of the cell due to overexpression of recombinant components, which is not practical. In addition, due to the lack of a strategy capable of realizing multitasking, the existing research method cannot simultaneously realize the regulation and control of protein functions and the real-time monitoring of regulation and control results.
The functional nucleic acid has outstanding programmability and diversity, and has outstanding potential in the construction of multifunctional nano-assemblies. Among them, aptamers have been designed for the accurate detection of signal molecules in living cells as a functional nucleic acid capable of binding to a specific target molecule. Some aptamers are reported to interfere with the activation process of cellular signaling pathways by blocking protein-protein interactions. However, to date, the multifunctional use of functional nucleic acid-based nanoassemblies in the regulation and monitoring of protein function in living cells remains challenging. In the first aspect, since the aptamer itself has a small size, it is easily dissociated after binding to a target protein, and is greatly influenced by the environment. In a second aspect, endocytosis of living cells also makes the regulation and detection efficiency of single free aptamers still unsatisfactory, especially for cell surface receptors and secreted signaling proteins. In a third aspect, the currently constructed probe usually only contains a single functional module, can only execute a single operation of regulation or monitoring, cannot meet the requirement of realizing multifunctional response on a plurality of proteins in a cell signal network, and cannot monitor a regulation result in real time. In order to solve the above technical problems, there is a need to develop a stable and efficient multifunctional DNA nano-assembly for precise regulation of protein function and real-time monitoring of signal transduction key molecules.
The invention content is as follows:
the invention aims to provide a Y-shaped multifunctional DNA nano assembly, a preparation method and application thereof, and aims to solve the technical problems of the Y-shaped multifunctional DNA nano assembly, including but not limited to any one of the following technical problems: on the first hand, how to construct a DNA nano assembly with multitasking capability, and on the second hand, how to realize controllable and efficient regulation and control of c-Met receptor protein function and signal transduction process; in the third aspect, how to realize real-time monitoring on the regulation and control effect of the c-Met receptor protein function. In order to achieve the purpose, the invention adopts the following technical scheme:
a cell membrane anchored 'Y-shaped' multifunctional DNA nano-assembly is formed by four DNAs in a hybridization way to form a 'Y-shaped' structure, and specifically comprises a chain ab with a sequence shown as SEQ ID NO.1, a chain cb with a sequence shown as SEQ ID NO.2, a chain d with a sequence shown as SEQ ID NO.3 and a chain e with a sequence shown as SEQ ID NO. 4; and the strand d is complementary with a partial sequence of the strand ab, the strand e is complementary with a partial sequence of the strand cb, the strand ab and the strand cb are both modified with the cell membrane anchoring group, the strand cb carries a Cy3 fluorescent group, the strand d carries an o-nitrobenzyl Photocleavable group (PC-linker), and the strand e carries a BHQ-2 fluorescence quenching group. In the scheme, the four DNAs hybridized to form the Y-shaped structure form an anchoring module, a regulation module and a monitoring module, the anchoring module is double-chain DNA modified by a cell membrane anchoring group, the regulation module comprises a nucleic acid aptamer for recognizing c-Met receptor protein and a blocking probe modified by a photocleavage group, and the monitoring module comprises a nucleic acid aptamer for recognizing VEGF protein and a blocking probe.
In a further development of the above embodiment, the chain ab comprises a nucleic acid aptamer sequence targeted to recognize the c-Met receptor protein and the chain cb comprises a nucleic acid aptamer sequence targeted to recognize the VEGF protein.
On the basis of the scheme, in another improved scheme, the chain ab is provided with a Cy5 fluorescent group, and the chain d is provided with a BHQ-2 fluorescence quenching group
On the basis of the above scheme, in another improved scheme, the cell membrane anchoring group is one of hydrophobic molecules, and the hydrophobic molecules comprise cholesterol molecules, tocopherol molecules and diacyl liposomes.
On the basis of the above scheme, in another improved scheme, the four DNAs are artificially synthesized or any other nucleic acid sequences of sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
The invention also provides a preparation method of the cell membrane anchored Y-shaped multifunctional DNA nano assembly, which comprises the following steps:
s1, synthesizing the ab sequence with a sequence shown as SEQ ID NO. 1;
s2, synthesizing the cb sequence with a sequence shown as SEQ ID NO. 2;
s3, synthesizing the d sequence with a sequence shown as SEQ ID NO. 3;
s4, synthesizing the e sequence with the sequence shown as SEQ ID NO. 4;
s5, mixing the four DNA sequences in the steps S1 to S4 according to the molar concentration of 1:1:1:1, heating at 95 ℃ for 5 minutes after mixing, carrying out annealing and mutual hybridization, and slowly cooling to room temperature.
On the basis of the above-mentioned protocol, in another modified protocol, the four DNA sequences in steps S1 to S4 are prepared as solutions with a concentration of 10. mu.M, respectively, and then mixed in proportion.
The invention also provides application of the cell membrane anchored Y-shaped multifunctional DNA nano assembly in a nucleic acid aptamer inhibitor drug.
The invention also provides application of the cell membrane anchored Y-shaped multifunctional DNA nano assembly in the research related to the function regulation effect of the real-time monitoring cell membrane surface receptor.
The invention also provides application of the cell membrane anchored Y-shaped multifunctional DNA nano assembly in related researches of protein function regulation and cell signal transduction.
The technical scheme of the invention at least has the following beneficial effects: the invention provides a Y-shaped multifunctional DNA nano assembly for regulating and controlling and monitoring the signal transduction process of living cells in real time. The present invention is illustrated with the mesenchymal epithelial transition (c-Met) signal pathway as a model. On the first hand, the multifunctional DNA nano assembly can be anchored on the surface of a cell membrane rapidly, the anchoring function is utilized to improve the combination stability of the aptamer and the c-Met receptor, and further the regulation effect of the aptamer on the c-Met receptor is improved. Furthermore, by utilizing the advantages of remote control and lossless and rapid regulation and control of light control, the multifunctional DNA nano assembly can realize the function regulation and control of the c-Met receptor protein with space-time resolution. Furthermore, the multifunctional DNA nano-assembly can monitor the secretion change of key signal molecules VEGF caused by the change of the function of the c-Met receptor protein in real time, thereby realizing the real-time monitoring of the regulation and control effect of the c-Met receptor. Therefore, the multifunctional DNA nano-assembly with the modular design can simultaneously realize the high-efficiency and controllable real-time monitoring of key molecules in the protein function regulation and signal transduction processes, and is converted from the independent research of a single signal molecule to the associated research of a plurality of signal molecules in signal transduction, thereby being beneficial to the deep research of the physiological function and the action mechanism of the living cell protein in a complex signal network and playing an important role in the research of early diagnosis, treatment, curative effect evaluation and the like of tumors.
Drawings
FIG. 1 is a schematic structural diagram of a "Y-shaped" multifunctional DNA nano-assembly in an embodiment of the present invention;
FIG. 2 is a schematic diagram of the principle of the Y-type multifunctional DNA nano-assembly for protein function regulation and signal transduction key molecule monitoring in the embodiment of the present invention;
FIG. 3 is a graph of the optical response of the DNA nano-assembly 2CH-ab: b: d analyzed by (a) 12% native polyacrylamide gel electrophoresis and (b) confocal imaging in Experimental example 1 of the present invention, wherein the scale: 20 μm;
FIG. 4 is a graph showing the effect of 2CH-b on the function of c-Met receptor analyzed by immunoblotting in Experimental example 2 of the present invention.
FIG. 5 shows the results of immunoblot analysis of the effect of DNA nano-assembly 2CH-ab: d on the photocontrol of c-Met receptor in Experimental example 3 of the present invention, (b) immunoblot analysis of the effect of DNA nano-assembly 2CH-ab: b: d or ab: b: d on the photocontrol of c-Met receptor after incubation with DU145 cells for 24 hours;
FIG. 6 shows fluorescence spectra of (a) VEGF at various concentrations in the experimental example 3 of the present invention reacted with a 200nM2 CH-cb: b: e probe for 30 minutes, and (b) 200nM of a 5. mu.g/mL protein with a 2 CH-cb: b: e probe for 30 minutes;
FIG. 7 is a graph of (a) ELISA assay of VEGF secretion promotion mediated by c-Met receptor activation in DU145 cells, (b) ELISA assay and (c) confocal imaging analysis of 2CH-ab: b effects on VEGF secretion mediated by c-Met receptor activation in DU145 cells in Experimental example 4 of the present invention, scale: 20 μm;
FIG. 8 is a gel electrophoresis diagram of the "Y-type" multifunctional DNA nano-assembly 2CH-ab: b: d: e in Experimental example 5 of the present invention.
Fig. 9 is a confocal image of the "Y-type" multifunctional DNA nano-assembly 2CH-ab: b: d: e in experimental example 6 of the present invention for realizing the function of the light-controlled c-Met receptor on DU145 cells and monitoring VEGF secretion in real time, and the scale: 20 μm.
Detailed Description
The following preferred embodiments of the present invention are provided to aid in a further understanding of the invention. It should be understood by those skilled in the art that the description of the embodiments of the present invention is by way of example only, and not by way of limitation.
Referring to the schematic of FIG. 1, in this example, we use the unique molecular recognition mechanism of functional nucleic acids and the precise self-assembly property of nucleic acid structures to construct "Y-type" DNA nano-assemblies of multifunctional modules. The Y-type multifunctional DNA nano assembly in the embodiment of the invention is formed by hybridizing four DNAs into a Y-type structure, and specifically comprises a chain ab with a sequence shown in SEQ ID NO.1, a chain cb with a sequence shown in SEQ ID NO.2, a chain d with a sequence shown in SEQ ID NO.3 and a chain e with a sequence shown in SEQ ID NO. 4; and the chain d is complementary with a partial sequence of the chain ab, the chain e is complementary with a partial sequence of the chain cb, the chain ab and the chain cb are both modified with cell membrane anchoring groups, the chain cb is provided with Cy3 fluorescent groups, the chain d is provided with o-nitrobenzyl Photocleavable groups (Photocleavable linker, PC-linker), and the chain e is provided with BHQ-2 fluorescence quenching groups. Wherein chain ab comprises a nucleic acid aptamer sequence targeted to recognize the c-Met receptor protein and chain cb comprises a nucleic acid aptamer sequence targeted to recognize the VEGF protein; the chain ab is provided with a Cy5 fluorescent group, and the chain d is provided with a BHQ-2 fluorescence quenching group. The technology how to modify the above groups on four DNA chains belongs to the routine operation in the field, and does not belong to the improvement point of the application, and is not described in detail in the specification.
The cell membrane anchoring group in this example is a cholesterol molecule; in another improved embodiment, the cell membrane anchoring group is one of other hydrophobic molecules, such as a tocopherol molecule or a diacyl liposome.
The principle of the "Y-type" multifunctional DNA nano-assembly in this embodiment for realizing protein function regulation and signal transduction key molecule monitoring can be seen in the schematic illustration of fig. 2. The feasibility of the strategy is verified by taking HGF/c-Met signal transduction related c-Met and VEGF as experimental objects. When c-Met is activated by HGF, the initiated signaling cascade is shown to promote VEGF secretion, thereby playing an important role in the angiogenesis process. In this embodiment, the "Y-type" multifunctional DNA nano-assembly 2CH-ab: cb: d: e is divided into 3 functional modules, and is assembled by DNA sequences with different functions (see FIG. 1). Wherein the region 2CH-b: b plays an anchoring function to anchor the multifunctional DNA nano-assembly on the surface of a cell membrane. The regions a and d constitute the function of regulating the activity of the receptor protein to exert photoresponse. Region a is a nucleic acid aptamer recognizing the c-Met receptor, which is capable of specifically binding to and inhibiting the functional activity of the c-Met receptor. The probe d is designed to contain a sequence of an o-nitrobenzyl photocleaving group (PC-linker) and a BHQ-2 quenching group, can be completely hybridized with a partial sequence of the region a, enables Cy5 fluorescence modified at the end of the region a to be quenched, and prevents the region a from playing a regulation function when the probe is not driven by illumination. When the multifunctional DNA nano assembly is triggered by illumination, the breakage of the probe d enables the area a to recover to a three-dimensional space configuration capable of being combined with a c-Met receptor, so that the protein function is inhibited, and the fluorescence of Cy5 is recovered. The areas c and e are used to implement the real-time monitoring function. And the region c is the sequence of the aptamer of VEGF, the probe e for modifying the BHQ-2 quenching group is completely complementary with part of the sequence of the region c, and the Cy3 fluorescence modified by the region c is quenched. When VEGF exists, the configuration of the area c is changed due to the combination of VEGF and aptamer, so that the area c is dissociated from the probe e, and the Cy3 fluorescence signal at the end of the area c is recovered. Therefore, the multifunctional DNA nano-assembly can realize complex functional operation, realize efficient and controllable function regulation of the c-Met receptor of the living cell, and simultaneously monitor the change of key signal molecule VEGF secretion caused by the function change of the c-Met receptor.
In this example, chain ab comprises a nucleic acid sequence recognizing the c-Met receptor protein (part of the regulatory module) and an anchor sequence with a modification of the cell membrane anchor group (part of the anchor module), and chain cb comprises a nucleic acid sequence recognizing the VEGF protein (part of the monitoring module) and an anchor sequence with a modification of the cell membrane anchor group (part of the anchor module), chain d is a photoactivation sequence modified with an ortho-nitrobenzyl photocleavage group (part of the regulatory module), and chain e is a blocking probe sequence (part of the monitoring module).
The four DNAs in this example were artificially synthesized, and in other examples could be nucleic acid sequences of the sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 from any other source.
The preparation method of the cell membrane anchored 'Y-shaped' multifunctional DNA nano-assembly in the embodiment comprises the following steps:
s1, synthesizing the ab sequence with a sequence shown as SEQ ID NO. 1;
s2, synthesizing the cb sequence with a sequence shown as SEQ ID NO. 2;
s3, synthesizing the d sequence with a sequence shown as SEQ ID NO. 3;
s4, synthesizing the e sequence with the sequence shown as SEQ ID NO. 4;
s5, mixing the four DNA sequences in the steps S1 to S4 according to the molar concentration of 1:1:1:1, heating at 95 ℃ for 5 minutes after mixing, carrying out annealing and mutual hybridization, and slowly cooling to room temperature.
On the basis of the above embodiment, in another modified embodiment, the four DNA sequences in steps S1 to S4 are prepared as solutions with a concentration of 10. mu.M, respectively, and then mixed in proportion.
The invention also provides application of the cell membrane anchored Y-shaped multifunctional DNA nano assembly in a nucleic acid aptamer inhibitor drug.
The invention also provides application of the cell membrane anchored Y-shaped multifunctional DNA nano assembly in the research related to the function regulation effect of the real-time monitoring cell membrane surface receptor.
The invention also provides application of the cell membrane anchored Y-shaped multifunctional DNA nano assembly in related researches of protein function regulation and cell signal transduction.
The main instruments used in experimental examples 1 to 6 were:
SH-1000UV-Vis Spectrophotometer (Corona Electric Co., Japan); a1 confocal laser scanning microscope (nikon, japan); CHB 202 constant temperature metal bath (hangzhou bori technologies, china); cary Eclipse fluorescence spectrometer (Agilent Technologies, USA); Milli-Q Integarl pure water/ultrapure water integrated system (Millipore Corp., USA); a small vertical electrophoresis cell (Bio-Rad, USA); ChemiDocTM Touch gel imaging system (Bio-Rad, USA); chromatography experiment refrigerator (Beijing Detianyou science and technology company, China).
The main reagents used in experimental examples 1 to 6 were:
the DNA sequences (Table 1) used in Experimental examples 1 to 6 were synthesized by Shanghai Bioengineering technology, Inc. of China and purified by HPLC. ELISA detection kit for VEGF was purchased from Abcam, USA. Recombinant human HGF was purchased from PeproTech, usa. Recombinant human VEGF and recombinant human PDGF-BB were purchased from China near-shore protein technology, Inc. The antibodies used in this experiment were purchased from Cell Signaling Technology, usa. MEM medium, RPMI1640 medium, BSA, PBS, and FBS, etc., were purchased from Gibco, USA.
TABLE 1 oligonucleotide sequences used in the present invention
In Table 1 above, the chain CH-ab comprises a nucleic acid aptamer sequence of c-Met; chain CH-cb nucleic acid aptamer sequences comprising VEGF; "-CH-" represents a cholesterol molecule and "//" represents a photocleavable group PC-linker.
Experimental example 1
The experiment example 1 verifies the light control response performance of the DNA nano assembly 2CH-ab: b: d consisting of the anchoring module and the regulating module in the solution and on the surface of the living cell. As can be seen from the non-denaturing polyacrylamide gel electrophoresis analysis of FIG. 3a, a higher molecular weight band 2CH-ab: b: d (lane 2) appears when d is successfully assembled with 2CH-ab: b. After 5 minutes of illumination with an ultraviolet flashlight, region a regains its aptamer conformation due to cleavage by PC-linker, with a cleaved d band (lane 3). We examined this photoresponse process on the surface of HeLa cells by assembling Cy3 fluorophore-modified 2CH-ab: b with BHQ-2 quencher-modified d to form 2CH-ab: b: d, and incubating the 2CH-ab: b: d with HeLa cells. As shown in the confocal image of FIG. 3b, only under the condition of light triggering, a circle of obvious Cy3 fluorescence appeared on the HeLa cell membrane, while the non-illuminated group had no obvious fluorescence signal, indicating the light-responsive performance of 2CH-ab: b: d.
The specific operation process is as follows: (a) 200nM Cy3-CH-ab, BHQ-2-d, and CH-b were self-assembled at a molar ratio of 1:1:1, and 2CH-ab: b: d was constructed for gel electrophoresis experiments. The experiments were divided into an illuminated group and an unilluminated group, wherein the solution in the illuminated group was under a 365nm UV lamp (5 mW/cm)2) After 5 minutes of irradiation, the reaction was carried out for 10 minutes. 2CH-ab: b was used as a positive control. (b) The assembled 200nM2CH-ab: b: d cells were incubated with HeLa cells for 15 min and the non-anchored probes were washed with PBS. The experiments were divided into light-controlled and non-light-controlled groups, wherein the condition of the light-controlled group is illuminationAfter 5 minutes, the cells were incubated for another 10 minutes and confocal imaging was performed.
Experimental example 2
In order to further examine the capability of the light response of the DNA nano assembly 2CH-ab: b: d for regulating the function of the c-Met protein, the expression level of the p-Met protein of the cell under different experimental conditions is examined by using a classical immunoblotting experiment in the experimental example 2, and the expression level of the p-Met is the most direct means for verifying whether the c-Met is activated. As shown in fig. 4, incubation of DU145 cells with the "anchor module" 2CH-b alone did not affect the protein phosphorylation process caused by binding of 20ng/mL HGF to the c-Met receptor. As shown in fig. 5a, 2CH-ab: b: d, after incubation with DU145 cells, showed similar levels of p-Met expression in the absence of light as in the positive control with HGF alone. Meanwhile, the binding of HGF to the c-Met protein was not affected by mere light treatment of the cells. When the light triggers the regulation and control performance of the 'regulation and control module', almost no protein band of p-Met is detected, which shows that the DNA nano assembly can efficiently realize the function of optically controlling the c-Met receptor. As shown in fig. 5b, this experimental example 2 also demonstrated the light response modulating performance of 2CH-ab: b: d after a long incubation time. After incubating 2CH-ab: b: d with DU145 cells for 24 hours, light was controlled. The light triggering 2CH-ab: b: d still can show obvious effect of inhibiting p-Met protein expression, and the light without the light has no obvious effect on the p-Met protein expression induced by HGF stimulation. However, ab: b: d without anchoring function failed to inhibit c-Met receptor phosphorylation mediated by HGF after 24 hours of incubation with cells, regardless of light. The experimental result shows that 2CH-ab: b: d can still respond to illumination to trigger the regulation function after long-time incubation, and the regulation of the photoresponse function of the c-Met receptor is realized.
The specific operation process is as follows: DU145 cells (3X 10 per well)5) After culturing for 24 hours in a 6-well plate, the culture medium was starved for 24 hours by replacing the culture medium with a MEM medium containing 0.5% BSA, and experiments were performed.
(1) Different concentrations of 2CH-b: b were incubated with DU145 cells for 15 min, followed by addition of 20ng/mL HGF and incubation with the cells for 30 min. After washing the cells 3 times with PBS, the cells were lysed to extract the protein for immunoblotting.
(2) The experiments for optically controlling and regulating the activity of the c-Met protein are divided into an illumination group and a non-illumination group for carrying out experiments. DU145 cells were incubated with 30nM 2CH-ab: b: d for 15 min, with the light group at 5 min and 10 min after incubation. Then 20ng/mL HGF was added and incubated with the cells for 30 minutes. After washing the cells 3 times with PBS, the cells were lysed to extract the protein for immunoblotting.
(3) To examine the ability of the "regulatory module" to respond to photocontrolled regulation of c-Met protein activity after prolonged incubation, 250nM 2CH-ab: bd or ab: b: d was added to DU145 cells, and the well plates were incubated in a cell culture chamber for 24 hours and then exposed to light. Then 20ng/mL HGF was added and incubated with the cells for 30 minutes before protein extraction.
Experimental example 3
This experimental example 3 examined the performance of DNA nano-assemblies 2CH-cb b: e consisting of an "anchor module" and a "monitor module" in detecting VEGF in solution. As shown in fig. 6a, the fluorescence signal of 2CH-cb b: e increased with increasing VEGF concentration, indicating the feasibility of the DNA nano-assembly to detect VEGF. In addition, no significant fluorescent signal response was observed in the presence of other proteins, including BSA, HGF and PDGF-BB, demonstrating the selectivity of 2CH-cb b: e (FIG. 6 b).
The specific operation process is as follows: 200nM Cy3-CH-cb, BHQ2-e, and CH-b were self-assembled at a molar ratio of 1:1:1, and 2 CH-cb: b: e was constructed for fluorescence detection experiments. Different concentrations of recombinant human VEGF (0-6000ng/mL) were added for 30 min of reaction. Spectral data for each set was recorded using a fluorescence spectrometer. To verify the selectivity of 2CH-cb b: e, 5000ng/mL VEGF, BSA, HGF and PDGF-BB were reacted with 200nM 2CH-cb b: e for 30 min, respectively. Spectral data for each set was recorded using a fluorescence spectrometer.
Experimental example 4
This example 4 examined the VEGF response performance of cell membrane anchored "monitor module" 2CH-cb b: e on live cells. The promotion of VEGF secretion by DU145 cells mediated by c-Met receptor protein activation was examined by enzyme-linked immunosorbent assay. As shown in fig. 7a, stimulation with HGF promoted VEGF secretion by DU145 cells compared to the blank control. As shown in FIG. 7b, the decreased VEGF secretion was shown when the c-Met receptor activity was inhibited by 2CH-ab: b followed by stimulation with HGF. The confocal image of fig. 7c also verifies this phenomenon.
The specific operation process is as follows: DU145 cells (3X 10 per well)4) Seeded in 48-well plates. To verify the ability of the HGF/c-Met pathway to promote VEGF secretion, the experiment was divided into 2 groups, the first group being a blank control group; the second group is an experimental group stimulated by 20ng/mL HGF; the plates were incubated in a 37 ℃ cell incubator for 2, 4, 8 or 12 hours. Culture supernatant of each group of cells was taken and the VEGF content was determined by ELISA assay kit. To verify the ability of 2CH-ab b to inhibit VEGF secretion, the experiments were divided into 3 groups: the first group is a blank control group; the second group was a positive control group stimulated with 20ng/mL HGF only; the third group of cells was incubated with 200nM2CH-ab b for 15 min and then stimulated with 20ng/mL HGF. The plates were incubated in a 37 ℃ cell incubator for 4 or 8 hours. Culture supernatant of each group of cells was taken and the VEGF content was determined by ELISA assay kit. To verify the VEGF-detecting performance of 2 CH-cb: b: e, DU145 cells were reacted with 200nM2CH-ab: b for 15 minutes and then reacted for 4 hours with 20ng/mL HGF. DU145 cells supplemented with 20ng/mL HGF alone were used as a positive control. Cy3-2CH-cb b: e was added and incubated with DU145 cells for 30 minutes for confocal imaging.
Experimental example 5
This example 5 examined the assembly of "Y-type" multifunctional DNA nano-assemblies 2CH-ab: cb: d: e in buffer solution. As shown in fig. 8, 2CH-ab cb x d e enables successful assembly.
The specific operation steps are as follows: the DNA dry powders of CH-ab, CH-cb, d and e were dissolved in ultrapure water to prepare 100. mu.M stock solutions, respectively, and the DNA stock solutions were diluted to 10. mu.M with PBS. The DNA solution was placed in a constant temperature metal bath, annealed by heating at 95 ℃ for 5 minutes, and slowly cooled to room temperature. Self-assembling CH-ab and d, and CH-cb and e according to a molar concentration ratio of 1:1 to respectively construct CH-ab: d and CH-cb: e; the self-assembly of CH-ab: d and CH-cb: e was carried out at a molar ratio of 1:1 to construct 2CH-ab: cb: d: e, each at a final concentration of 1. mu.M, for gel electrophoresis experiments.
Experimental example 6
This example 6 uses "Y-type" multifunctional DNA nano-assembly 2CH-ab: cb: d: e for the functional regulation of c-Met protein in photocontrol response and monitoring of VEGF secretion changes triggered by related signal transduction. The region a and the region c of the multifunctional DNA nano-assembly are labeled with Cy5 and Cy3 fluorescence, respectively, and in the initial stage, the Cy5 and Cy3 fluorescence are quenched by BHQ-2 on the probes d and e, respectively. As shown in FIG. 9, 2CH-ab: cb: d: e was incubated with DU145 cells, and Cy5 fluorescence signal appeared on the cell membrane due to illumination triggering the "regulatory module". The 'regulatory module' inhibits the function of c-Met protein, further inhibits the secretion of VEGF, and no obvious Cy3 fluorescent signal is observed after the cells are incubated for 2, 3 and 4 hours. In contrast, in the control experiment without light, there was no significant Cy5 fluorescence signal. After 2, 3, 4 hours of cell incubation, a progressively increasing Cy3 fluorescence signal was produced due to VEGF secretion. The results prove that the combination of the region a and the c-Met protein in the multifunctional DNA nano assembly 2CH-ab: cb: d: e of the illumination triggering multifunctional module can effectively inhibit the combination of HGF and the c-Met protein, realize the function of optically controlling and regulating the c-Met protein, and simultaneously, the multifunctional DNA nano assembly can realize the monitoring and analysis of VEGF secretion change caused by HGF/c-Met channel signal transduction change.
The specific operation steps are as follows: 200nM multifunctional DNA nano-assembly 2CH-ab: cb: d: e was incubated with DU145 cells for 15 min, anchoring it to the cell membrane surface of DU 145. Wherein, the cell reaction condition of the light group is that the cells are firstly illuminated for 5 minutes and then incubated for 10 minutes. Confocal imaging was performed after 2, 3 and 4 hours incubation with 20ng/mL HGF.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present application and not for limiting the protection scope thereof, and although the present application is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: numerous variations, modifications, and equivalents will occur to those skilled in the art upon reading the present application and are within the scope of the claims as issued or as granted.
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Claims (10)
1. A cell membrane anchored 'Y-shaped' multifunctional DNA nano-assembly is characterized in that four DNAs are hybridized to form a 'Y-shaped' structure, and specifically comprises a chain ab with a sequence shown as SEQ ID NO.1, a chain cb with a sequence shown as SEQ ID NO.2, a chain d with a sequence shown as SEQ ID NO.3 and a chain e with a sequence shown as SEQ ID NO. 4; and the chain d is complementary with a partial sequence of the chain ab, the chain e is complementary with a partial sequence of the chain cb, the chain ab and the chain cb are both modified with cell membrane anchoring groups, the chain cb is provided with a Cy3 fluorescent group, the chain d is provided with an o-nitrobenzyl photocleavage group, and the chain e is provided with a BHQ-2 fluorescence quenching group.
2. The cell membrane-anchored "Y-type" multifunctional DNA nano-assembly of claim 1, wherein said chain ab comprises a nucleic acid aptamer sequence targeted to recognize the c-Met receptor protein and said chain cb comprises a nucleic acid aptamer sequence targeted to recognize the VEGF protein.
3. The cell membrane-anchored "Y-type" multifunctional DNA nano-assembly of claim 2, the chain ab carrying a Cy5 fluorophore and the chain d carrying a BHQ-2 fluorescence quenching group.
4. The cell membrane-anchored "Y-type" multifunctional DNA nano-assembly of claim 3, wherein the cell membrane anchoring group is one of hydrophobic molecules, including cholesterol molecules, tocopherol molecules, and diacyl liposomes.
5. The cell membrane anchored "Y-shaped" multifunctional DNA nano-assembly according to any of claims 1 to 4, characterized in that said four DNAs are artificially synthesized, or any other source of nucleic acid sequences of the sequences shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
6. A method for preparing the cell membrane anchored "Y-type" multifunctional DNA nano-assembly according to any one of claims 2 to 5, comprising the following steps:
s1, synthesizing the ab sequence with a sequence shown as SEQ ID NO. 1;
s2, synthesizing the cb sequence with a sequence shown as SEQ ID NO. 2;
s3, synthesizing the d sequence with a sequence shown as SEQ ID NO. 3;
s4, synthesizing the e sequence with the sequence shown as SEQ ID NO. 4;
s5, mixing the four DNA sequences in the steps S1 to S4 according to the molar concentration of 1:1:1:1, heating at 95 ℃ for 5 minutes after mixing, carrying out annealing and mutual hybridization, and slowly cooling to room temperature.
7. The method for preparing a cell membrane anchored "Y-type" multifunctional DNA nano-assembly of claim 6, wherein the four DNA sequences of steps S1 to S4 are prepared as solutions with a concentration of 10 μ M, respectively, and then mixed in proportion.
8. The use of the cell membrane anchored "Y-shaped" multifunctional DNA nano-assembly according to any one of claims 2 to 5 in the preparation of a medicament for aptamer inhibitors.
9. The use of the cell membrane anchored "Y-shaped" multifunctional DNA nano-assembly according to any one of claims 2-5 in research related to real-time monitoring of cell membrane surface receptor function modulating effects.
10. Use of the cell membrane anchored "Y-shaped" multifunctional DNA nano-assembly according to any one of claims 2 to 5 for protein function regulation and cell signaling related studies.
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