CN111440610B - Multicolor fluorescent probe based on DNA nano structure and preparation method and application thereof - Google Patents
Multicolor fluorescent probe based on DNA nano structure and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a multicolor fluorescent probe based on a DNA nano structure, a preparation method and application thereof, wherein the preparation method comprises the following steps: constructing DNA tetrahedrons with different arm chain numbers; the DNA nano self-assembly structure is constructed by a strategy of layer-by-layer assembly or fractal assembly, and fluorescent molecules of different types and quantities are marked on the DNA nano self-assembly structure, so that the multicolor fluorescent probe based on the DNA nano self-assembly structure is constructed. The invention realizes the construction of the small-size multicolor fluorescent probe by a bottom-up self-assembly means, and breaks through the defects of low coding quantity and uncontrollable optical property of the traditional fluorescent label. The multicolor fluorescent probe provided by the invention can be used for single-molecule level target molecule identification and tumor cell identification, and has good biocompatibility, so that the multicolor fluorescent probe has good biomedical application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multicolor fluorescent probe based on a DNA nano structure, and a preparation method and application thereof.
Background
The simultaneous qualitative or quantitative detection of various biological small molecules, nucleic acids and proteins has important scientific significance for basic research of life science, biological medicine and the like. For example, the development of complex diseases such as malignant tumors involves a variety of biochemical reactions in the body, and diseases often have a variety of associated biomarkers. Therefore, compared with the detection of a single marker, the multi-channel detection and imaging of a plurality of markers can improve the accuracy of early detection results and reduce the detection results of false positives or false negatives. Therefore, multi-channel detection is an important means for studying physiological and pathological processes, and is extremely important for clinical medicine. Non-invasive fluorescence imaging is an important means for researchers and clinicians to conduct various biomolecular studies and further monitor physiological and pathological processes and mechanisms due to its advantages of real-time performance, high spatial and temporal resolution, and the like. In the actual fluorescence detection of various target molecules, the number of the effective fluorescent probes in a visible light region is only 4 to 5 due to the crosstalk easily generated between the fluorescent probes due to spectral overlapping and the FRET effect between adjacent spectral molecules, so that the development of a multi-channel fluorescence imaging technology is greatly limited. Therefore, the development of a method for constructing a multi-color fluorescent probe with rich codes is a urgent task for the development of multi-channel detection.
The DNA nanotechnology provides a new idea for solving the problems. Based on the self-assembly strategy of 'from bottom to bottom', DNA nano-materials with different spatial configurations are reported sequentially through DNA base complementary pairing hybridization. Compared with other nanometer materials, the DNA nanometer material has good programmability and addressability, can be used as a template, and realizes the controllable assembly of target molecules by accurately controlling the spatial position and the number of the target molecules. In addition, different from single-stranded DNA, when the DNA nano-chain is assembled into a DNA nano-structure through hybridization between sequences, the structural rigidity of the material is obviously enhanced. The rigid structure is not only beneficial to controlling the distance of target molecules, but also can enter cells automatically, and has good enzyme cutting resistance. Thus, DNA nanostructures have great potential in multichannel imaging within cells. For example, different types and quantities of fluorescent molecules are modified at different positions of the DNA origami, so that DNA fluorescent probes with abundant quantities and different fluorescent signals are constructed. However, in order to prevent FRET effect between fluorescent molecules on origami, the distance between different fluorescent molecules needs to be above 10 nm, usually several hundred nm DNA nanostructure is needed as a unit, but too large size does not utilize cellular uptake and reduces imaging resolution. While DNA nanostructures that are too small in size provide smaller modification sites and therefore cannot take advantage of spatial location to encode fluorescent molecules.
Disclosure of Invention
The invention aims to provide a multicolor fluorescent probe based on a DNA nano structure, a preparation method and application thereof, so as to solve the problems of low coding quantity, uncontrollable luminescence property (color and intensity) and the like of the fluorescent probe in the existing fluorescence imaging technology.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a method for preparing a multicolor fluorescent probe based on a DNA nanostructure, the method comprising: constructing DNA tetrahedrons with different arm chain numbers; the DNA nano self-assembly structure is constructed by a strategy of layer-by-layer assembly or fractal assembly, and fluorescent molecules of different types and quantities are marked on the DNA nano self-assembly structure, so that the multicolor fluorescent probe based on the DNA nano self-assembly structure is constructed.
The DNA nano self-assembly structure is a two-dimensional plane or three-dimensional DNA nano structure formed by hybridizing a plurality of DNA tetrahedral monomers through arm chains.
The DNA nano self-assembly structure comprises a DNA tetrahedron, a DNA cube, a DNA triangular bipyramid, a DNA octahedron, a DNA icosahedron and DNA origami.
Preferably, the positions of the arm chains can be designed at other sites of the DNA tetrahedron, and the positions of the arm chains comprise 5 'ends, 3' ends or any positions in the middle.
The labeling method of the fluorescent molecule on the DNA nano self-assembly structure preferably comprises any one or more of covalence, intercalation and coordination.
The labeled fluorescent molecules include Aleax488, ROX, and Cy5. It should be understood that other fluorescent molecules can be used in the system.
The buffer system for preparing DNA tetrahedron comprises magnesium chloride and Tris-HCl, and the reaction system is maintained at pH 8.0 to ensure the formation of DNA tetrahedron.
Preferably, the DNA nano self-assembly mode is that DNA tetrahedrons with different arm chain numbers are incubated and assembled in a shaking table according to a certain metering ratio in a layer-by-layer assembly or fractal assembly mode.
According to a preferred embodiment of the present invention, there is provided a method for preparing a multicolor fluorescent probe based on a DNA nano self-assembled structure, the method comprising the steps of: mixing 4 DNA single chains, and obtaining DNA tetrahedrons which contain different numbers of arm chains and have the side length of 20 bp in a high-temperature annealing mode; and (3) obtaining a high-order DNA nano self-assembly structure by assembling the DNA tetrahedrons with different arm chain numbers layer by layer or fractal assembly, and marking different types and numbers of fluorescent molecules on the high-order DNA nano self-assembly structure so as to construct the multicolor fluorescent probe based on the DNA nano self-assembly structure. The high-purity high-order DNA nano self-assembly structure can be obtained by HPLC purification and separation.
Preferably, the DNA tetrahedron has an edge length of 20 bp, but it is understood that other DNA tetrahedrons having lengths greater than 20 bp can be used in the present invention.
The assembly mode of the DNA tetrahedron monomer comprises modes such as double-strand hybridization, covalent connection and the like, as long as the assembly of the DNA tetrahedron can be ensured.
The modified aptamers include AS1411, SYL3C, sgc8, or other aptamers or antibodies.
According to a second aspect of the present invention, there is provided a multicolor fluorescent probe based on a DNA nano self-assembled structure prepared according to the above preparation method.
According to a third aspect of the invention, the application of the multicolor fluorescent probe based on the DNA nano self-assembly structure in ordered recognition of single-molecule level target molecules is provided. According to the application provided by the invention, the multicolor fluorescent probe based on the DNA nano self-assembly structure is modified on a glass surface dish by adopting a biotin and avidin combination method, so that the research of the fluorescent probe and a target sequence on the aspect of ordered molecular recognition can be observed in a total internal reflection fluorescent microscope.
The invention also provides an application of the multicolor fluorescent probe based on the DNA nano self-assembly structure in multicolor imaging of tumor cells. According to the application provided by the invention, the imaging condition of the fluorescent probe in different tumor cells can be observed in a total internal reflection fluorescence microscope by self-assembling the high-order DNA tetrahedron multicolor fluorescent probe with MCF7, hela and Hek293 cells. The multicolor fluorescent probe is preferably incubated in an amount of 0.5 to 5 nM, for example: 0.5 nM, 1nM, 2 nM,3 nM,4 nM,5 nM, more preferably 1 nM. The tumor cells comprise one or more of MCF7, hela and Hek293 cells. Wherein the total internal reflection fluorescence microscope has a laser wavelength of 488, 561 and 647 nm and an exposure time of 100 ms.
The invention mainly discloses a multicolor fluorescent probe based on a DNA nano structure, a preparation method and application thereof, and creativity of the multicolor fluorescent probe is that the multicolor fluorescent probe is constructed by accurately controlling the quantity and the type of fluorescent molecules on a high-order DNA tetrahedron self-assembly structure by utilizing addressability and programmability of the DNA nano structure. The invention provides a simple, quick and effective construction strategy for constructing an intensity-quantifiable multicolor fluorescent label. Therefore, the invention realizes the construction of the small-size multicolor fluorescent probe by a bottom-up self-assembly means, and breaks through the defects of low coding quantity and uncontrollable optical property of the traditional fluorescent label. The multicolor fluorescent probe prepared according to the invention can be used for single-molecule level target molecule identification and tumor cell identification, and the multicolor fluorescent probe has good biocompatibility, so the multicolor fluorescent probe has good biomedical application prospect.
In conclusion, according to the multicolor fluorescence probe based on the DNA nanostructure, the preparation method and the application thereof provided by the invention, the problems that the existing multicolor fluorescence label is low in coding quantity and the fluorescence probe strength is difficult to accurately control are solved, the high-order DNA tetrahedral multicolor fluorescence probe prepared according to the invention has the advantages of small size, large cell uptake, no obvious toxicity in cells, high biocompatibility and the like, has obvious advantages in cell imaging, and opens up a new path for the development of medical imaging technology.
Drawings
FIG. 1 is a schematic structural diagram of the present invention for assembling DNA tetrahedron by layer-by-layer assembly or fractal assembly;
FIG. 2 shows the result of electrophoretic characterization of DNA tetrahedral polyacrylamide with arm chain;
FIG. 3 AFM representation of DNA tetrahedrons;
FIG. 4 is a diagram of agarose electrophoresis characterization of a high-order DNA tetrahedral self-assembly structure;
FIG. 5 is a diagram of agarose electrophoresis characterization of the yield of high-order DNA tetrahedral self-assembly structure prepared by layer-by-layer assembly and fractal assembly;
FIG. 6 is an AFM representation of the high order DNA tetrahedral self-assembly structure;
FIG. 7 is an experimental result demonstration of a chain high-order DNA nanostructure composed of different DNA tetrahedra, wherein a is an AFM characterization of the chain high-order DNA nanostructure, and b is a size analysis result of the chain high-order DNA nanostructure;
FIG. 8 shows STROM characterization results of dendritic high-order DNA nanostructures;
FIG. 9 shows the optical property characterization experiment results of the high-order DNA tetrahedral self-assembly multicolor fluorescent probe, wherein a is the labeling diagram of fluorescent molecules on nanometers, b is the statistical result of the spacing between adjacent tetrahedrons, c is the corresponding relationship between the fluorescence intensity of the fluorescent probe labeled with Alexa488 and the number of fluorescent molecules, d is the corresponding relationship between the fluorescence intensity of the fluorescent probe labeled with ROX and Cy5 and the number of fluorescent molecules, and e is the confocal imaging characterization result of the multicolor fluorescent probe;
FIG. 10 shows fluorescence emission spectrum characterization results of different fluorescent probes;
FIG. 11 shows the result of single-molecule identification total internal reflection fluorescence imaging with Target1 added first and Target2 added later;
FIG. 12 shows the result of single-molecule recognition total internal reflection fluorescence imaging by adding Target2 first and then adding Target 1;
FIG. 13 shows the results of stability analysis of fluorescent probes under physiological conditions;
FIG. 14 shows the results of cytotoxicity analysis of three fluorescent probes;
FIG. 15 shows the results of fluorescent analysis of three fluorescent probes for tumor cell identification.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
According to a preferred embodiment of the present invention, a method for preparing a high-order DNA tetrahedron self-assembly structure is provided, it should be understood that, by way of example only and not limitation, in this embodiment, 20 bp DNA tetrahedrons with different numbers of arm chains are selected as monomers, then the DNA tetrahedrons are assembled by layer-by-layer assembly or fractal assembly at room temperature (as shown in fig. 1), and multicolor DNA nanostructure fluorescent probes are obtained by labeling different kinds and numbers of Alexa488, ROX and Cy5 fluorescent molecules on the DNA tetrahedron assemblies; then, modifying the obtained multicolor DNA nano-structure fluorescent probe in a glass surface dish, and observing the identification behavior with a target sequence under a total internal reflection fluorescent microscope; finally, the multicolor DNA nanostructure fluorescent probes respectively marked with the AS1411, SYL3C and sgc8 aptamers are incubated with MCF7, hela and Hek293 cells, and the cell imaging conditions of the three fluorescent probes are observed in a total internal reflection fluorescence microscope imaging system, and the following examples specifically illustrate the implementation effect of the invention.
Wherein the functionalized DNA is purchased from a living organism (Shanghai); human cervical cancer cell Hela, breast cancer cell MCF-7 and human embryonic kidney cell HEK293 were purchased from Shanghai Life sciences research institute, culture medium 1640 and DMEM used for culturing the cells were purchased from Ensifier Weijie (China), and the rest of the reagents were purchased from Sigma Aldrich China.
The DNA sequence is as follows:
example 1
The invention provides a multicolor fluorescent probe based on a DNA nano structure and a preparation method and application thereof, and firstly, the preparation of a DNA tetrahedron comprises the following steps:
all DNA was dissolved in Q water, and the absorbance of single strands at 260 nm was measured by UV spectrophotometer to quantify all DNA single strand concentrations to 100. Mu.M. Four single-stranded DNAs for preparing DNA tetrahedrons were mixed in equal amounts in TM buffer (10 mM Tris-HCl,5 mM MgCl) 2 pH 8.0) was added to the reaction solution, and the final concentration of DNA single strand was 1. Mu.M. Keeping the temperature in a PCR instrument at 95 ℃ for 10 min, then quickly cooling to 4 ℃ and keeping the temperature in the PCR instrument at 4 ℃ for 10 min to prepare a DNA tetrahedron. And then, characterizing the DNA tetrahedron by 8% polyacrylamide electrophoresis and AFM, wherein the electrophoresis operation condition is 120V for 120 min. The morphology of the DNA tetrahedron was characterized by AFM.
As a result: polyacrylamide electrophoresis showed that as the number of arm strands on the DNA tetrahedron increased, the rate of DNA tetrahedron migration slowed and the DNA tetrahedron was successfully prepared (fig. 2). The AFM results clearly show DNA tetrahedrons, with the tetrahedrons having an edge length of about 12 nm and a height of 2.6 nm (FIG. 3).
Example 2
The preparation of the high-order DNA tetrahedron self-assembly structure comprises the following steps:
and mixing the DNA tetrahedrons modified with the complementary arm chains according to the assembly molar ratio of 1, and incubating for 2h at 37 ℃ to obtain a first-generation high-order DNA tetrahedron self-assembly structure (G1). And then incubating for 2h at 37 ℃ in a layer-by-layer assembly or fractal assembly mode to synthesize second generation (G2), third generation (G3) and fourth generation (G4) high-order DNA tetrahedron self-assembly structures. The assembly rule of the high-order DNA tetrahedron is as follows, and each additional layer from the central tetrahedron a-T0 as an assembly center represents the generation of a new generation of high-order structure. The n-1 th generation tetrahedron high-order structure a-Gn-1 with arm chains has the arm chain number of 3 x 2 (n-1), and is hybridized with tetrahedrons with complementary hybrid arm chains to obtain the n-th generation high-order DNA tetrahedron assembly structure. When the assembly order is adjusted, the same structure is also obtained by fractal assembly.
Purifying the DNA tetrahedron self-assembly structure by adopting high performance liquid chromatography, wherein a separation column is SEC-4000, the flow rate of a mobile phase is 1 ml/min, collecting target peak components, and then concentrating by using a 30 kDa ultrafiltration tube, the ultrafiltration condition is 3000 g and 10 min, and ultrafiltration is carried out for 3 times. The mobile phase was 25 mM Tris-HCl,450 mM NaCl, pH 7.2.
Atomic force microscopy characterization: treating the newly uncovered mica with 0.5% (v/v) APTES solution for 1 min, washing with a large amount of Q water to remove unadsorbed APTES, and blow-drying the mica surface with ear washing balls for later use. And (3) dripping 10 mu L of 1nM sample on the surface of mica, adsorbing for 5 min, and scanning and imaging under an atomic force microscope, wherein the imaging probe is SCANASYST-FLUID +, and the imaging mode is PeakForce QNM in FLUID.
Agarose gel electrophoresis characterization: characterization was performed using 1% agarose gel electrophoresis, run at 100V for 1h.
STORM imaging: two-color fluorescence labeled 4-G4 self-assemblies were used in STORM imaging, with the central tetrahedron labeled Cy3 and the outermost tetrahedron labeled Alexa647. Firstly, washing glass sheet with surfactant for 2min, washing with Q, treating in Q water, acetone and anhydrous ethanol for 15 min, and treating with N 2 And drying for later use. The glass sheet was then left to stand at 110 ℃ for 45 min. 1 uL of 10 pM G4 sample was dropped onto a slide for 10 min for modification, and 1. Mu.L of fluorescent microspheres and 70. Mu.L of an imaging buffer (purchased from Nikon) were added thereto, and after standing for 10 min, STORM imaging was performed.
As a result: from 1% agarose gel electrophoresis, it can be seen that the structure size and molecular weight increase and the migration speed of the band becomes slower as the number of generations of the assembly increases. The chain assembly F was analyzed by ImageJ software 2,0 、F 2,1 ，F 2,2 And F 2,3 The productivity of the layer-by-layer assembly is 63%, 60%, 40% and 30%, respectively, and the dendritic self-assembly F 3,0 、F 3,1 ，F 3,2 And F and 3,3 the yield of the layer-by-layer assembly is 89%, 88%, 67% and 78%, respectively, and the dendritic self-assembly F 4,0 、F 4,1 ，F 4,2 And F and 4,3 the yields of layer-by-layer assembly were 81%, 72%, 32% and 57%, respectively (fig. 4). But also for F 3,3 Structurally, the yield of fractal assembly under the same conditions is 84% much higher than the layer-by-layer assembly efficiency, because all intermediate structure yields can be purified during the fractal assembly process, which is beneficial for accurately controlling the assembly ratio, and thus the yield is high (fig. 5). F is clearly seen in the AFM 2,0 、F 2,1 ，F 2,2 ，F 2,3 ，F 3,0 ，F 3,1 ，F 3,2 ，F 3,3 ，F 4,0 、F 4,1 ，F 4,2 And F and 4,3 higher order DNA tetrahedra assembled structures with yields of 95%,64%,246%,96%,66%,41%,64%,39% and 42%, respectively (FIG. 6).
In addition, AFM characterization analysis shows that the included angles between three adjacent tetrahedra in the chain-shaped assembly are distributed within 50-180 degrees, which shows that the chain-shaped self-assembly has certain structural flexibility under the premise of ensuring rigidity (figure 7)
The stram results show that Cy3 is surrounded by Alexa fluorescence, the location of Cy3 is offset from the geometric center of the structure, and the central spot is offset from the center by 79 ± 36 nm, indicating that the structure has certain flexibility (fig. 8).
Example 3
By utilizing the structural characteristics of the high-order DNA tetrahedron self-assembly structure, the construction of the multicolor fluorescent probe without adjacent molecular crosstalk and with quantifiable fluorescence intensity by marking of fluorescent molecules is further researched. Each DNA tetrahedron in the high-order DNA tetrahedron self-assembly structure has a site capable of labeling a fluorescent molecule, so that each tetrahedron can label 4 fluorescent molecules. Three fluorescent molecules Alexa488, ROX and Cy5 are selected to construct a multicolor fluorescent probe. For the F3,2 structure 28 fluorescent molecules can be labeled.
And (3) adopting an Edinburgh FS920 fluorescence spectrophotometer to carry out fluorescence spectrum collection on the constructed fluorescence probe. All samples tested were at a concentration of 10 nM. The excitation wavelength of Alexa488 is 488 nm, and the spectrum acceptance range is 495-590 nm; the ROX excitation wavelength is 588 nm, and the spectral receiving range is 595-640 nm; the Cy5 excitation wavelength is 650 nm, and the spectral acceptance range is 655-720 nm.
As a result: the results in FIG. 9 b show that the average spacing between adjacent tetrahedra is 18.5. + -. 3.8 nm, which is effective for avoiding energy transfer between fluorescent molecules. As shown in fig. 9 c and d, the number of fluorescent molecules is linearly related to the fluorescence intensity of the probe, and the number of fluorescent molecules in the probe has a good linear relationship with the fluorescence intensity of the probe (R2 > 0.99). In addition, when the fluorescent probe is modified with Alexa488, ROX and Cy5 simultaneously, fluorescence crosstalk and FRET effect do not exist among fluorescent molecules (as shown in FIG. 10), and the high-order DNA tetrahedron is suitable for being used as a structural element for amplifying fluorescence brightness and can be used for constructing the fluorescent probe with quantifiable intensity.
In a chain shape F 2,3 For structural motifs, by controlling the number and location of Alexa488, ROX and Cy5, we constructed 36 fluorescent probes, named in the manner of Cy5-ROX-Alexa488, such as 111 labeled with 1 Cy5,1 ROX and 1 Alexa488.
36 fluorescent probes to be detected are spotted on a clean glass slide according to a certain sequence by adopting a SpotBot 2 microarray spotting instrument, the spotting distance is 400 mu m, and a Leica TCS SP8 laser confocal microscope is adopted for imaging after 1h. The imaging condition is 633 nm excitation, the receiving range is 650 nm-720 nm,561 nm laser, the receiving window is set to 575-620 nm;488 nm laser, and the receiving window is set to be 495-550 nm.
As a result: as shown in e in FIG. 9, each fluorescent probe presents different colors, which proves that the construction of the multicolor fluorescent probe is successful, and the high-order DNA tetrahedron self-assembly structure is a powerful tool for constructing the multicolor fluorescent probe.
Example 4
The application of the high-order DNA tetrahedral self-assembly structure in single molecule recognition comprises the following steps:
a131 high-order DNA tetrahedron self-assembly body is used as a research object, and a 20 pM fluorescent probe modified with avidin is firstly modified on a biotin-modified glass sheet, and the adsorption time is 5 min. The 131 self-assemblies not adsorbed on the petri dish were then removed by washing with 1 × PBS buffer. 100 nM Target1 single strand was added to the sample tank, incubated at 37 ℃ for 30 min, then washed three times with PBS and the fluorescent signal of the fluorescent probe was collected in the 488, 561 and 647 nM channels. Then, 100 nM Target2 single strand was added to the sample well, incubated at 37 ℃ for 30 min, and then washed three times with PBS, and the fluorescent signals of the fluorescent probes in the 488, 561 and 647 nM channels were collected for 100 ms exposure.
As a result: when the Target sequence is not added, the three channels of 488, 561 and 647 nm have signals, when Target1 is added firstly, the Cy5 signal disappears, only ROX and Alexa488 signals can be collected, and the number of steps in the ROX and Alexa488 bleaching curves is 1 and 3 respectively. With the addition of Target2, both Cy5 and Cy3 signals disappeared, only Alexa488 signal was collected, and the number of steps in the bleaching curve was 1 (fig. 11). When Target2 is added first, the Cy5 and Cy3 signals disappear simultaneously, and only Alexa488 signals exist; when Target1 is added, only Alexa488 signal is still available, so that the multicolor fluorescent probe can realize ordered recognition of Target molecules (FIG. 12).
Example 5
The application of the high-order DNA tetrahedron self-assembly structure in multiple cell imaging and tumor cell classification and identification comprises the following steps:
in the structural stability study of the fluorescent probe, 10. Mu.L of 10 nM fluorescent probe was added to 100. Mu.L of 1640 cell culture medium containing 10% (v/v) fetal bovine serum and incubated at 37 ℃ for 2h, 8h, 12h and 24 h, respectively. And respectively taking 30 mu L of samples to be detected, characterizing by 1% agarose gel electrophoresis, running for 1h under the condition of 100V, and determining the structural stability of the fluorescent probe according to the brightness of an electrophoresis strip.
As a result: the tree-structure fluorescent probe has good structural stability in serum within 12h, can effectively inhibit the degradation of enzyme to DNA, and is suitable for cell imaging analysis (figure 13).
In fluorescent probe cytotoxicity studies, 1X 10 5 The HeLa cells and MCF-7 cells were individually placed in a 96-well plate and cultured overnight at 37 ℃. Then, a fluorescent probe was added to the medium at a final concentration of 20 nM for 6 h, 12h and 24 h, respectively. After the incubation was completed, the cells were washed 3 times with 1 × PBS buffer. Then, 100. Mu.L of 1.5 mM MTT solution was added to each well. After 4 hours, the MTT solution was aspirated, and 100 μ L DMSO was added to each well. The absorbance at 570 nm of each well was then read using a Bio Tek synergy MX H1 microplate reader. Repeat 5 groups for each cell sample.
As a result: after the cells were incubated with the probe, the cell survival rate was greater than 90%, indicating that the fluorescent probe was not cytotoxic and had good biocompatibility (fig. 14).
We further investigated the use of multicolor fluorescent probes for multiplex imaging studies of cells, and we chose 410, 041 and 104 fluorescent probes for cell imaging. Firstly, respectively marking AS1411, SYL3C and sgc8 on fluorescent probes to construct AS1411-410 fluorescent probes, SYL3C-041 and sgc8-104 fluorescent probes. 1nM of mixed fluorescent probes AS1411-410, SYL3C-041 and sgc8-104 were added to 5X 10, respectively 5 The HeLa cells, MCF7 cells and HEK293 cells were incubated at 37 ℃ for 1h. Cells were washed 3 times with 1 × PBS prior to imaging, and then fluorescence signals were collected from cells in the 488, 561 and 647 nm channels.
As a result: from the total internal reflection fluorescence image, all cells showed fluorescence spots of mixed colors, indicating that the cells can effectively take up the three fluorescent probes, mainly due to the expression of nucleolin, epCAM, PTK7 and other proteins in HeLa cells, MCF7 cells and HEK293 cells. However, there is a large difference in the mean fluorescence intensity between the three cells. The differences between individuals within the same cell line are also large, resulting in weaker differences between tumor cells. Thus, these cells are difficult to distinguish by any one aptamer. By statistically analyzing the fluorescence of the three fluorescent probes at 488, 561 and 647 nm in each cell, the intensity of different cells in the three channels is obviously different, and the three tumor cells are easily distinguished (FIG. 15). Therefore, the system can more accurately identify different tumor cells and has great potential in tumor typing.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in the conventional technical content.
SEQUENCE LISTING
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<400> 6
aaaaaaaaaa aaaaaaaaaa aaaaagctac acgattcaga cttaggaatg ttcgacatgc 60
gagggtccaa taccgacgat tacagctt 88
<210> 7
<211> 88
<212> DNA
<213> Artificial sequence
<400> 7
aaaaaaaaaa aaaaaaaaaa aaaaagtgat aaaacgtgta gcaagctgta atcgacggga 60
agagcatgcc catccactac tatggcgg 88
<210> 8
<211> 88
<212> DNA
<213> Artificial sequence
<400> 8
aaaaaaaaaa aaaaaaaaaa aaaaactcgc atgactcaac tgcctggtga tacgaggatg 60
ggcatgctct tcccgacggt attggacc 88
<210> 9
<211> 88
<212> DNA
<213> Artificial sequence
<400> 9
tttttttttt tttttttttt tttttcagtt gagacgaaca ttcctaagtc tgaaatttat 60
cacccgccat agtagacgta tcaccagg 88
<210> 10
<211> 88
<212> DNA
<213> Artificial sequence
<400> 10
aaagccggta aggtgaggaa tccgagctac acgattcaga cttaggaatg ttcgacatgc 60
gagggtccaa taccgacgat tacagctt 88
<210> 11
<211> 88
<212> DNA
<213> Artificial sequence
<400> 11
aaagccggta aggtgaggaa tccgagtgat aaaacgtgta gcaagctgta atcgacggga 60
agagcatgcc catccactac tatggcgg 88
<210> 12
<211> 88
<212> DNA
<213> Artificial sequence
<400> 12
aaagccggta aggtgaggaa tccgactcgc atgactcaac tgcctggtga tacgaggatg 60
ggcatgctct tcccgacggt attggacc 88
<210> 13
<211> 88
<212> DNA
<213> Artificial sequence
<400> 13
tcggattcct caccttaccg gctttcagtt gagacgaaca ttcctaagtc tgaaatttat 60
cacccgccat agtagacgta tcaccagg 88
<210> 14
<211> 63
<212> DNA
<213> Artificial sequence
<400> 14
gctacacgat tcagacttag gaatgttcga catgcgaggg tccaataccg acgattacag 60
ctt 63
<210> 15
<211> 63
<212> DNA
<213> Artificial sequence
<400> 15
gtgataaaac gtgtagcaag ctgtaatcga cgggaagagc atgcccatcc actactatgg 60
cgg 63
<210> 16
<211> 63
<212> DNA
<213> Artificial sequence
<400> 16
ctcgcatgac tcaactgcct ggtgatacga ggatgggcat gctcttcccg acggtattgg 60
acc 63
<210> 17
<211> 88
<212> DNA
<213> Artificial sequence
<400> 17
acctgagtga gtatgcggag tacgactcgc atgactcaac tgcctggtga tacgaggatg 60
ggcatgctct tcccgacggt attggacc 88
<210> 18
<211> 83
<212> DNA
<213> Artificial sequence
<400> 18
ctccgcatac tcactcaggt cagttgagac gaacattcct aagtctgaaa tttatcaccc 60
gccatagtag acgtatcacc agg 83
<210> 19
<211> 88
<212> DNA
<213> Artificial sequence
<400> 19
catctctgct ctagtgagtc tcgaagtgat aaaacgtgta gcaagctgta atcgacggga 60
agagcatgcc catccactac tatggcgg 88
<210> 20
<211> 83
<212> DNA
<213> Artificial sequence
<400> 20
gactcactag agcagagatg cagttgagac gaacattcct aagtctgaaa tttatcaccc 60
gccatagtag acgtatcacc agg 83
<210> 21
<211> 25
<212> DNA
<213> Artificial sequence
<400> 21
tcgtactccg catactcact caggt 25
<210> 22
<211> 25
<212> DNA
<213> Artificial sequence
<400> 22
ttcgagactc actagagcag agatg 25
<210> 23
<211> 109
<212> DNA
<213> Artificial sequence
<400> 23
atctaactgc tgcgccgccg ggaaaatact gtacggttag atttttgcta cacgattcag 60
acttaggaat gttcgacatg cgagggtcca ataccgacga ttacagctt 109
<210> 24
<211> 96
<212> DNA
<213> Artificial sequence
<400> 24
ttggtggtgg tggttgtggt ggtggtggaa aaagctacac gattcagact taggaatgtt 60
cgacatgcga gggtccaata ccgacgatta cagctt 96
<210> 25
<211> 116
<212> DNA
<213> Artificial sequence
<400> 25
cactacagag gttgcgtctg tcccacgttg tcatgggggg ttggcctgtt tttgctacac 60
gattcagact taggaatgtt cgacatgcga gggtccaata ccgacgatta cagctt 116
Claims (5)
1. Use of a multicolor fluorescent probe based on DNA nanostructures for ordered recognition of single-molecule level targets and multicolor imaging of tumor cells, wherein the use comprises:
constructing DNA tetrahedrons with different arm chain numbers; constructing a DNA nano self-assembly structure by a strategy of layer-by-layer assembly or fractal assembly, wherein the DNA nano self-assembly structure is a two-dimensional plane or three-dimensional DNA nano structure formed by hybridizing a plurality of DNA tetrahedrons through arm chains, the DNA nano self-assembly structure comprises a DNA tetrahedron, a DNA cube, a DNA triangular bipyramid, a DNA octahedron, a DNA icosahedron and a DNA origami, and different types and quantities of fluorescent molecules are marked on the DNA nano self-assembly structure and comprise Alexa488, ROX and Cy5, so that a multicolor fluorescent probe based on the DNA nano self-assembly structure is constructed;
the multicolor fluorescent probe based on the DNA nano self-assembly structure is modified on a glass surface dish by adopting a biotin and avidin combination method, so that the ordered molecular recognition of the fluorescent probe and a target sequence can be observed in a total internal reflection fluorescent microscope; and
the multicolor fluorescent probe based on the DNA nano self-assembly structure is respectively marked with AS1411, SYL3C and sgc8 aptamers, and then the multicolor fluorescent probe respectively marked with the aptamers is incubated with three different tumor cells, namely MCF7, hela and Hek293, so that the imaging conditions of the multicolor fluorescent probe in the different tumor cells can be observed in a total internal reflection fluorescent microscope, and the different tumor cells can be identified.
2. The use according to claim 1, wherein the positions of the arm chains comprise 5 'end, 3' end or any position in the middle when constructing DNA tetrahedrons with different numbers of arm chains.
3. The use according to claim 1, wherein the labeling method of the fluorescent molecule comprises any one or more of covalent, intercalation, and coordination.
4. The use according to claim 1, wherein the buffer system used for the preparation of DNA tetrahedra comprises magnesium chloride and Tris-HCl.
5. The use of claim 1, wherein the layer-by-layer assembly or fractal assembly is performed by incubating and assembling DNA tetrahedrons with different arm chain numbers in a shaking table according to a certain metering ratio.
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| CN112326762B (en) * | 2020-10-29 | 2023-03-21 | 常州大学 | Method for detecting DNA tetrahedron by capillary electrophoresis |
| CN113189066A (en) * | 2021-04-26 | 2021-07-30 | 东北大学 | Bionic nano probe and preparation method and application thereof |
| CN114084871B (en) * | 2021-11-18 | 2023-05-23 | 电子科技大学长三角研究院(湖州) | Method for assembling nano pattern based on DNA molecule |
| CN114438079B (en) * | 2021-12-31 | 2023-08-08 | 上海交通大学医学院附属仁济医院 | Virus-like DNA polyhedral framework structure and preparation method and application thereof |
| CN114984032B (en) * | 2022-06-27 | 2023-07-07 | 四川大学 | DNA tetrahedron framework nucleic acid-chlorogenic acid complex and application thereof in preparation of medicines for treating hepatic fibrosis |
| CN116077670A (en) * | 2022-07-06 | 2023-05-09 | 福州大学 | Targeting drug-carrying system based on DNA tetrahedral nanoclusters and construction method and application thereof |
| CN116004771A (en) * | 2023-03-10 | 2023-04-25 | 广西师范大学 | Method for miRNA detection based on competitive hybridization ratio single cell counting strategy |
| CN117304920A (en) * | 2023-08-23 | 2023-12-29 | 祥符实验室 | Near infrared fluorescence tetrahedral probe and preparation method and application thereof |
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