CN113514429B - Fluorescent staining method for DNA nano-structure labeled cell target protein - Google Patents

Abstract

The invention belongs to the technical field of super-resolution microscopic imaging, and particularly relates to a fluorescent staining method for labeling target proteins of cells by using a DNA (deoxyribonucleic acid) nanostructure. The method uses streptavidin as an intermediate, and connects a DNA nano structure with an antibody of a target protein modified with biotin together so as to mark the target protein; wherein: the DNA nano structure is simultaneously modified with biotin, a group anchored on hydrogel and more than two fluorescent dye molecules. When the DNA nano structure is used for modifying the same dye, the brightness is very high, and the quality of the obtained microscopic picture can be greatly improved. When the dominant dye of STED super-resolution microscopy is modified, then the expansion microscopy can be well combined with STED super-resolution. When the dye pair for fluorescence resonance energy transfer is modified, the DNA nano structure can have the twinkling property, so that the expansion microscopic technology can be more conveniently combined with the STORM and SOFI super-resolution technology.

Description

Fluorescent staining method for DNA nano-structure labeled cell target protein

Technical Field

The invention belongs to the technical field of optical microscopy, and particularly relates to a fluorescent staining method for a target protein of a DNA nanostructure marked cell.

Background

The swelling microscopic imaging technique is to anchor fluorescent groups on hydrogel by fluorescence-specifically labeling target objects, such as protein, DNA, RNA, etc., when the hydrogel swells in water, fluorescent molecules which are originally indistinguishable close together within the optical diffraction limit swell with the gel, and their distance from each other increases, so that images with higher resolution can be obtained by using common microscopic imaging equipment. Because the expansion microscopic imaging technology is simple to operate and raw materials are easy to obtain, the expansion microscopic imaging technology has wide application in single-cell animals, cell lines, tissues and even animal weights.

The isotropic and uniform expansion of the sample is an important condition for ensuring that the structure of the sample is not distorted. To ensure uniform swelling, the protease should digest as completely as possible the proteins in the sample that maintain the biological structure, to reduce the pulling forces between the structures. However, if digested too much, the fluorescent group of the fluorescent protein is cleaved by the protease, the dye attached to the antibody is also detached, the fluorescent molecule not anchored to the hydrogel is washed away during swelling, and the free radicals generated during gel formation destroy the fluorescent group. These causes result in a large loss of fluorescence signal and the low effective label density of the sample can seriously affect the imaging quality.

Disclosure of Invention

In view of the shortcomings of the prior art, the present invention aims to provide a fluorescent staining method for labeling target proteins of cells by using DNA nanostructures. The method takes streptavidin and biotin as media, utilizes the characteristic that a DNA nanostructure has multiple endpoints (free radicals) and can modify multiple fluorescent dyes and can not be digested by protease to mark target protein, can overcome the defect that a sample is digested by the protease in the conventional swelling microscopy technology to cause a large amount of loss of a sample fluorescent signal, and can greatly improve the brightness of the fluorescent mark, so that the signal-to-noise ratio of a microscopic picture is higher and the picture quality is better. If the same dye is modified, the brightness of the DNA nanostructure is very high, and the quality of the obtained microscopic picture is greatly improved. If the modified dye molecule is the dominant dye of STED super-resolution microscopy, then the expansion microscopy can be combined well with STED super-resolution. If the dye pair of fluorescence resonance energy transfer is modified, the DNA nano structure can also have the property of scintillation, so that the expansion microscopy technology can be more conveniently combined with STORM and SOFI super-resolution technology.

In order to achieve the above purpose, the invention adopts the following technical scheme.

A fluorescence staining method for labeling target protein of cells by a DNA nano structure, which uses streptavidin as an intermediate and connects the DNA nano structure with an antibody of the target protein modified with biotin so as to label the target protein; wherein: the DNA nano structure is simultaneously modified with biotin, a group anchored on hydrogel and more than two fluorescent dye molecules.

In the present invention, the DNA nanostructure is a DNA double-stranded structure, a DNA tetrahedral structure, or other more complex spatial structure.

In the present invention, the fluorescent dye molecules are of the same species or different species. Compared with other DNA molecules for swelling, the DNA nanostructure has the advantage of multiple vertexes, and except that biotin and acrylamide occupy two vertexes, the other vertexes can be modified with fluorescent dyes. If the same dye is modified, the brightness of the DNA nanostructure is very high, and the quality of the obtained microscopic picture is greatly improved. If the modified dye molecule is the dominant dye of STED super-resolution microscopy, then the expansion microscopy can be combined well with STED super-resolution. If the dye pair of fluorescence resonance energy transfer is modified, the DNA nano structure can also have the property of scintillation, so that the expansion microscopy technology can be more conveniently combined with STORM and SOFI super-resolution technology.

In the present invention, the group anchored on the hydrogel in the DNA nanostructure is not limited to an acrylamide group or the like.

In the invention, the immunofluorescence process is involved, but the antibody band is not a fluorescent group but biotin, and the primary antibody of the target protein to be marked is connected with biotin or the secondary antibody is connected with biotin.

The fluorescence staining method for the target protein of the DNA nanostructure marked cells comprises the following specific steps:

1) Synthesizing a DNA nano structure simultaneously modified with biotin, a group anchored on the hydrogel and more than two fluorescent dye molecules;

2) Cell culture and staining

Plating cells on a cell slide, fixing the cells after several hours, perforating, blocking, diluting the antibody of the target protein with 1% BSA solution and incubating on the blocked cells, rinsing, diluting the biotin-conjugated secondary antibody and incubating on the cells, rinsing, incubating streptavidin diluted with PBS on the cells, rinsing, incubating the DNA nanostructure solution diluted with PBS on the cells;

3) Expansion microscopy sample processing

Treating the cells incubated with the DNA nanostructures with a monomer solution at room temperature; preparing mother liquor from ammonium persulfate APS and tetramethylethylenediamine by using water, and diluting the mother liquor into gel solution by using monomer solution; adding the gel solution into a polytetrafluoroethylene round hole, then placing the cover glass cell face downwards on the gel solution, standing at room temperature until the gel solution is completely solidified, and digesting with a protease K solution after the gel solution is solidified; then, putting the sample into deionized water for expansion, and changing water until the sample is completely expanded;

4) Microscopic imaging of expanded samples

The expanded samples were cut to size and placed in glass-bottom petri dishes and subjected to Airyscan imaging using a microscope.

Compared with the traditional expansion microscope technology, the invention has the following advantages and beneficial effects:

1. the present invention relates to the biotin-streptavidin system, where biotin and streptavidin have a very strong affinity. Each streptavidin can bind four biotins, the streptavidin connects the DNA nanostructure and the antibody together, and the DNA nanostructure can mark the cell structure to be researched;

2. the DNA nanostructures are not digested by the protease and the acrylamide groups thereon allow the DNA nanostructures to be anchored to the hydrogel so that the swollen sample can be completely digested by the protease and thus uniformly swollen. The traditional expansion sample has the defects that the fluorescence signal loss of the sample is huge and the quality of an imaging picture is poor due to too long digestion time, but the sample is not uniformly expanded due to short digestion time and is distorted.

3. No anchor agent is needed in the sample preparation process, such as Glutaraldehyde (GA), succinimidyl ester of 6- ((acyloyl) amino) hexanoic acid (acyloyl-X, SE), and methacrylic acid N-hydroxysuccinimidyl ester (MA-NHS), and these anchor agents can anchor substances such as protein, DNA, etc. in the sample in the hydrogel. But the anchoring agent also does not completely digest the sample, so that the sample does not swell uniformly.

4. Various dyes can be modified on the DNA nano structure, so that the fluorescence brightness of the marked sample is greatly improved. The diversity of fluorescent molecules enables the expansion microscopy technique to be effectively combined with a variety of super-resolution techniques. The DNA nanostructure is modified with STED dominant dye molecules, such as ATTO647N, and the expansion sample marked by the DNA nanostructure can be well applied to a STED super-resolution microscope. The traditional expansion microscopic technology can only increase the laser power of the STED microscope to improve the picture quality because the fluorescence brightness of the sample is not enough, but the expansion sample can be damaged and deformed due to the overhigh laser power. High fluorescence brightness even with STED dominant dyes, high quality STED pictures can be obtained using low power lasers. If the dye molecule modified by the nano structure is a dye pair of fluorescence resonance energy transfer, the DNA nano structure has the twinkling property, so that the SOFI imaging can be carried out, and the STORM super-resolution imaging can also be carried out without adding a STORM buffer solution.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of DNA nanostructure modification according to the present invention. In the case of DNA tetrahedra, there are 8 endpoints, one for each of biotin and acrylamide groups. The remaining endpoints are dyes.

FIG. 2 shows the dyeing principle of the present invention. With the antibody of the protein of interest, the linker on the primary antibody (FIG. 2 (a)) or the secondary antibody (FIG. 2 (b)) is biotin, 4 subunits of streptavidin are capable of binding 4 biotins, and the four biotins, except for 1 biotin on the antibody, the remaining 3 are those on the tetrahedra. While one antibody can carry 10-20 biotin, so that 1 antibody carrying biotin can bind 30-60 DNA tetrahedra and 180-360 dye molecules at most.

FIG. 3 is a diagram of the present invention, which uses tubulin as the target protein, and the expansion microscopy technique is obtained.

Reference numbers in the figures: 1 is the DNA tetrahedron of FIG. 1, 2 is streptavidin, 3 is the primary antibody, 4 is the target protein, and 5 is the secondary antibody.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

1. Design and sequence synthesis of DNA tetrahedral nanostructures

The 4 strands of the tetrahedral DNA nanostructure are synthesized by Biotechnology (Shanghai) Inc., which modifies biotin, fluorescent dye and acrylamide groups during synthesis. The 4-strand sequence is as follows:

A：

5’-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA-3’

(SEQ ID NO: 1), alexa Fluor 488 was modified at both the 5 'and 3' ends;

B：

5’-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3’

(SEQ ID NO: 2), alexa Fluor 488 on the 5 'end, biotin-TEG on the 3' end;

C：

5’- TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3’

(SEQ ID NO: 3), the 5' -end is modified with Acrydite;

D：

5’-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3’

(SEQ ID NO: 4), alexa Fluor 488 was modified at both the 5 'and 3' ends.

2. Structural formation of DNA tetrahedron

A. Mixing the four single chains B, C and D in TM buffer (20Mm Tris,50Mm MgCl) ₂ pH8.0), a DNA tetrahedral nano-structure with the final concentration of 1 mu M is prepared. And then putting the prepared sample into a PCR instrument for 10 min at 95 ℃, quickly cooling to 4 ℃, and keeping the temperature for more than 10 min at 4 ℃ to obtain the tetrahedral DNA nano structure. FIG. 1 is a schematic diagram of DNA nanostructure modification according to the present invention. In the case of a DNA tetrahedron, there are 8 endpoints in total, one for each of biotin and acrylamide groups. The remaining endpoints are dyes.

3. Cell culture and staining

BS-C-1 cells were plated on a cell slide 1.5 cm in diameter, after 16 hours, the cells were fixed, perforated, and after blocking, antibodies to a-tubulin were diluted with 1% bsa solution and incubated on the blocked cells for 1 hour, and after rinsing, secondary antibodies conjugated with biotin were diluted and incubated on the cells for 45 minutes. After rinsing, streptavidin diluted with PBS was incubated on the cells for 30 minutes. After rinsing, the DNA tetrahedral nanostructure solution diluted with PBS was incubated on the cells, reacted for 30 minutes, rinsed out as clean as possible.

FIG. 2 illustrates the staining principle of the present invention, and the background in FIG. 2 shows the hydrogel. With the antibody of the protein of interest, the linker on the primary antibody (FIG. 2 (a)) or the secondary antibody (FIG. 2 (b)) is biotin, 4 subunits of streptavidin are capable of binding 4 biotins, and the four biotins, except for 1 biotin on the antibody, the remaining 3 are those on the tetrahedra. While one antibody can carry 10-20 biotins, so that 1 antibody carrying biotin can bind 30-60 DNA tetrahedrons and 180-360 dye molecules at most.

4. Expansion microscopy sample processing

Cells incubated with DNA tetrahedral nanostructures were treated with monomer solution (1 XPBS, 2M NaCl,2.5% acrylamide, 0.15% N, N' -methylenebisacrylamide, 8.625% sodium acrylate) for about 1 minute at room temperature. Ammonium Persulfate (APS) 10% and tetramethylethylenediamine 10% were made into a high concentration mother liquor with water, diluted with monomer solution to 0.2% gel solution for gelation, APS was added last in the process. About 100. Mu.l of the gel solution was added to a 1mm deep, 1cm diameter polytetrafluoroethylene circular hole, followed by placing a cover slip cell side down on top of the gel solution. The mixture was allowed to stand at room temperature for 1 hour until it was completely solidified. After coagulation, digestion was performed with proteinase K solution (50 mM Tris (pH 8), 1mM EDTA, 0.5% Triton X-100, 0.8M guanidine HCl,8 units/mL proteinase K) for 20 hours. The sample was then expanded in deionized water, changing water every 30 minutes until full expansion.

5. Microscopic imaging of expanded samples

The swollen samples were cut to the appropriate size and placed in glass-bottomed petri dishes of glass slide No. 0 and subjected to Airyscan imaging with a zeiss LSM880 type microscope. The results are shown in FIG. 3, where (a) is a view of the microtube before expansion and (b) is a view of the microtube after expansion, and the hollow is not visible in the microtube before expansion and the hollow structure is visible after expansion. (c) Is the grey scale value of the underlined parts of the graphs (a) and (b), from (c) it can be seen that the microtubules before swelling were only one peak observed by normal microscopy, and that both peaks can be resolved after using the swelling protocol described herein, where the distance between the peaks is 65nm, corresponding to the diameter of the microtubules plus the distance of the antibody size.

Sequence listing

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Claims (4)

1. A fluorescence staining method for labeling target protein of cells by a DNA nano structure is characterized in that streptavidin is used as an intermediate, and the DNA nano structure is connected with an antibody of the target protein modified with biotin, so that the target protein is labeled; wherein: the DNA nano structure is a DNA tetrahedral structure, the end points of the DNA nano structure are simultaneously modified with biotin and a group anchored on hydrogel, and the other end points are modified with fluorescent dye molecules;

the method comprises the following specific steps:

1) Synthesizing a DNA nano structure modified with biotin, a group anchored on the hydrogel and a fluorescent dye molecule at the same time;

2) Cell culture and staining

Spreading the cells on a cell slide, fixing the cells after several hours, perforating, blocking, diluting the biotin-conjugated antibody of the target protein with 1% BSA solution and incubating on the blocked cells, or diluting the biotin-conjugated secondary antibody after rinsing and incubating on the cells, incubating streptavidin diluted with PBS on the cells after rinsing, and incubating the DNA nanostructure solution diluted with PBS on the cells after rinsing;

3) Expansion microscopy sample processing

Treating the cells incubated with the DNA nanostructures with a monomer solution at room temperature; preparing mother liquor from ammonium persulfate APS and tetramethylethylenediamine by using water, and diluting the mother liquor into gel solution by using monomer solution; adding the gel solution into a polytetrafluoroethylene round hole, then placing the cover glass cell face downwards on the gel solution, standing at room temperature until the gel solution is completely solidified, and digesting with a protease K solution after the gel solution is solidified; then, putting the sample into deionized water for expansion, and changing water until the sample is completely expanded;

4) Microscopic imaging of expanded samples

The expanded samples were cut to size and placed in glass-bottom petri dishes and subjected to Airyscan imaging using a microscope.

2. The fluorescent staining method of claim 1, wherein the fluorescent dye molecules are of the same species or different species.

3. The fluorescent staining method of claim 1, wherein the fluorescent dye molecules are fluorescence resonance energy transfer dye pairs.

4. The fluorescent staining method of claim 1, wherein the group anchored on the hydrogel in the DNA nanostructure is an acrylamide group.

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