WO2022188496A1

WO2022188496A1 - Dna nanostructure dye for expansion super-resolution imaging, and application thereof

Info

Publication number: WO2022188496A1
Application number: PCT/CN2021/138675
Authority: WO
Inventors: 马炯; 姚龙芳; 糜岚; 张丽; 陈丽雯
Original assignee: 光华临港工程应用技术研发(上海)有限公司
Priority date: 2021-03-08
Filing date: 2021-12-16
Publication date: 2022-09-15
Also published as: CN113004885A

Abstract

The present invention relates to the technical field of super-resolution microimaging, and in particular to a DNA nanostructure dye for expansion super-resolution imaging, and an application thereof. According to the dye of the present invention, three key molecules are modified at terminals of a DNA molecule, comprising: a group for specific recognition, such as benzylguanine (BG), an acrylamide group that anchors a whole probe to a hydrogel, and a fluorescent dye molecule. Compared with other DNA molecules for expansion, for a DNA probe, the group for specific recognition, such as BG, is small in molecular size, and a target protein can be labelled by means of gene expression, such that an error between a fluorescent group and an actual position of the target protein is greatly reduced, and compared with the target protein labelled with an antibody, the type of proteins that can be labelled is also greatly expanded.

Description

A DNA nanostructure dye for expansion super-resolution imaging and its application

technical field

The invention belongs to the technical field of optical microscopy, and in particular relates to a DNA nanostructure dye used for expansion super-resolution imaging and its application.

Background technique

Swelling super-resolution technology is a new technology developed in recent years. This technology does not change the microscope hardware. Instead, the fluorescently labeled biological sample is placed in a swellable hydrogel to solidify, and then the sample is coagulated with polypropylene. The amide hydrogel swells and the whole is uniformly magnified, so that super-resolution images can be obtained with ordinary microscopic imaging equipment.

At present, there are two main fluorescent labeling methods used in expansion super-resolution, one is using a primary antibody to connect a fluorescent-conjugated secondary antibody, and the other is a fluorescent protein fusion target protein. However, labeling the target protein with the primary antibody and the secondary antibody greatly increases the error between the position of the fluorophore and the actual target protein position after expansion. Although fluorescent proteins can also be used for expansion super-resolution, the fluorescent groups of fluorescent proteins are destroyed before the cells are completely digested by protease.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a DNA nanostructure dye for expansion super-resolution imaging and its application. When the DNA nanostructure dye provided by the present invention is combined with a tag protein for fluorescent labeling expansion super-resolution imaging, the error between the actual position of the target protein of the sample and the position of the fluorescent group in the expansion microscopy technique can be reduced; The target protein can improve the fluorescence signal of the swollen sample, so that the signal-to-noise ratio of the microscopic image is higher and the image quality is better.

In order to achieve the above objects, the present invention adopts the following technical solutions.

A DNA nanostructure dye for expansion super-resolution imaging, the DNA nanostructure is used as the carrier of the dye, the DNA nanostructure is the backbone, and the free groups of the DNA nanostructure are modified with fluorescent groups for specific localization. The group that recognizes the tag protein and the group that can be anchored on the hydrogel; wherein: the base pair in the DNA nanostructure does not exceed 40 pairs; the tag protein can be gene-edited, and the tag protein can interact with the target in the cell proteins form fusion proteins.

The above-mentioned DNA nanostructures are DNA double-stranded structures, DNA tetrahedral structures or other more complex spatial structures; the base pairs in the DNA nanostructures are between 10 and 30 pairs.

The above-mentioned fluorescent groups include but are not limited to Alexa Fluor 488, Cy3, Cy5, ATTO 647N, QD655 and the like. The number of the above-mentioned fluorescent groups is more than one, and the plurality of fluorescent groups may be of the same type, or may be fluorescence resonance energy transfer dye pairs. Fluorescent groups can make the target protein to be studied and the cellular structure it represents visible under a fluorescence microscope.

The above-mentioned groups used for specific localization and identification of tagged proteins include, but are not limited to, benzylguanine BG group for SNAP-tagged protein, benzylcytosine (BC) group for CLIP-tagged protein, and chlorine for Halo-tagged protein. Alkyl, etc. The BG group is the substrate of the tag protein SNAP. The gene sequence of the target protein is connected with the gene sequence of SNAP, and the fusion protein of the target protein and SNAP can be expressed by transfection into cells.

The above-mentioned groups that can be anchored on the hydrogel include but are not limited to acrylamide groups, etc., so that the labeled samples can be expanded.

The above-mentioned groups that can be anchored on the hydrogel are acrylamide groups, so that the labeled samples can be expanded.

The present invention further provides the application of the above-mentioned DNA nanostructure dye for expansion super-resolution imaging, which specifically binds to a tag protein for fluorescent staining. In the application process, it also involves the process of plasmid construction, the translation sequence of the target protein is cloned, and SNAP or other tag protein gene sequences are added in its coding frame; it also involves the cell expression system, which will construct the target protein and tag protein. The plasmid was transfected into cells for expression. Preferably, in the staining process, a detergent is used to make a small amount of holes on the cell membrane, and the cells are fixed after staining with the above-mentioned DNA nanostructure dye for expansion super-resolution imaging.

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

1. Compared with the immunofluorescence labeling method, the molecular weight of the antibody is relatively large, and the error between the position of the fluorescent group and the actual position of the target protein is large, while the molecular weight of the labeled protein is small, and the DNA nanostructure where the fluorescent dye is located is also small. The position of the cluster is relatively close to the actual distance of the target protein, which reduces the distance error.

2. Compared with the immunofluorescence labeling method, our labeling method is suitable for the protein expressed by the labelled transgenic marker, and there are many kinds of proteins that can be labeled; due to the complexity of the production method of antibodies, the kinds of proteins that can be labeled are limited, and the antibodies Due to the similarity of the antigenic determinants of the labeled samples, some antibodies can label other proteins except the target protein, so that the final sample made by the immunofluorescence labeling method has a strong background fluorescence.

3. Compared with the direct fluorescent protein labeling technology, our labeling method maintains the advantages of endogenous protein labeling, and at the same time can overcome the shortcomings of fluorescent proteins that are not resistant to protease digestion. better than fluorescent proteins.

4. During the preparation of the expanded sample, the acrylamide groups on the DNA oligos are enough to anchor the DNA nanostructures on the hydrogel, and the sample preparation process does not require glutaraldehyde (GA), succinimidyl ester of 6 -((acryloyl)amino)hexanoic acid(acryloyl-X, SE), methacrylic acid N-hydroxysuccinimidyl ester(MA-NHS) and other reagents, which makes the preparation process of the swollen samples easier. At the same time, because the entire molecule is based on DNA, it will not be digested by proteases. DNA nanostructures are not digested by proteases.

Description of drawings

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of the DNA nanostructure modification of the present invention. Taking the DNA double strand as an example, there are 4 endpoints on it, and the fluorescent dye molecule, BG and acrylamide group each occupy one endpoint.

Figure 2 shows the dyeing principle of the present invention. The fusion protein of the SNAP-tagged protein and the target protein is expressed in the cells, and then the cells are stained with the DNA nanostructure in Figure 1, and the SNAP-tagged protein is recognized by the BG on the DNA double-strand, so that the target protein has a fluorescent signal.

Fig. 3 is the practical application of the present invention, taking filaggrin as the target protein, and obtaining the picture of staining.

Labels in the figure: 1 is DNA double strand, 2 is BG, 3 is acrylamide group, 4 is fluorescent dye molecule, 5 is SNAP-tagged protein, and 6 is target protein.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below with reference to the preferred embodiments and accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

An acrylamide group is modified at one end of the DNA oligo, which can anchor the DNA oligo to the hydrogel during the preparation of the swollen sample. Because the entire molecule is based on DNA, it cannot be digested by proteases.

1. Design and sequence synthesis of DNA double-stranded structures

The two strands of the DNA double-stranded structure were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and BG, fluorescent dyes and acrylamide groups were modified during synthesis. The sequence of the 2 strands is as follows:

A: 5'-GACGATGTATGCTTAGGGTCT-3'

(5' end modified with acrylamide group, 3' end modified with Alexa Fluor 647)

B: 5'-GACCCTAAGCATACATCGTCTT-3'

(5' end modified with BG)

2. The structure of DNA double-stranded

The two single strands A and B were mixed in equal proportions in the annealing buffer to prepare a DNA double-stranded structure with a final concentration of 50 μM. Then put the prepared sample into the PCR machine and cool down from 98°C to 16°C per minute to obtain the DNA double-stranded structure.

3. Cell Culture and Staining

HeLa cells were transfected with SNAP-LifeAct and plated on 1.5 cm diameter cell slides after 48 hours. After 16 hours, the cells were treated with import buffer (20 mM Hepes, 110 mM KOAc, 5 mM NaOAc, 2 mM MgOAc, 1 mM EGTA, pH 7.3). Wash twice, 40μg/ml digitonin was dissolved in import buffer, treated cells for 30 seconds, then 1.5% polyvinylpyrrolidone (PVP, 360kDa) was dissolved in import buffer, washed twice, and then the PVP solution with 500nM DNA double strands was added to Cells were stained for 5 minutes, then washed twice with import buffer, fixed with 4% paraformaldehyde, and rinsed three times with PBS.

4. Sample processing of expansion microscopy

Cells incubated with DNA double-stranded structures were treated with monomer solution (1×PBS, 2M NaCl, 2.5% acrylamide, 0.15% N,N′-methylenebisacrylamide, 8.625% sodium acrylate) at room temperature for approximately 1 minute. 10% ammonium persulfate (APS) and 10% tetramethylethylenediamine were made into a high concentration mother liquor with water, diluted with monomer solution to 0.2% gel solution for gelation, and APS was added last in this process. About 100 microliters of the gel solution was added to a 1 mm deep, 1 cm diameter Teflon circular hole, and a coverslip was placed cell side down on top of the gel solution. Let stand for 1 hour at room temperature until fully solidified. After solidification it was digested with proteinase K solution (50 mM Tris (pH 8), 1 mM EDTA, 0.5% Triton X-100, 0.8 M guanidine HCl, 8 units/mL proteinase K) for 20 hours. The samples were then swollen in deionized water, changing the water every 30 minutes until fully swollen.

5. Microscopic imaging of expanded samples

The swollen samples were cut into appropriate sizes and placed in a glass bottom petri dish with a No. 0 slide, and imaged with a widefield microscope. The results are shown in Figure 3, (1) is the fluorescence image of EGFP-labeled microfilaments, (2) is an enlarged image of the pseudopodia in (1), and (3) is the fluorescence image of BG-dsDNA-labeled expanded microfilaments , (4) is the enlarged view of the pseudopodia in (3), (5) is the gray value of the dashed part in (2) and (4). As can be seen from Figure 3, the details of the pseudopodia after the expansion of the BG-dsDNA-labeled cells are relatively clear, while the details of the microfilaments labeled with EGFP cannot be seen. As can be seen from Figure 3(5), the BG-dsDNA The distance between the two lines that can be resolved under the label is smaller than that of the EGFP label.

From the above example, it can be seen that we use SNAP-tagged protein to label the target protein, connect the small molecule BG (benzylguanine) of SNAP to the DNA oligo chain of about 20bp, and at the same time modify the acrylamide group on the DNA oligo chain and Fluorescent dye molecules. This greatly reduces the error between the position of the fluorophore and the actual target protein, and also avoids the loss of the fluorophore during digestion.

Claims

A DNA nanostructure dye for expansion super-resolution imaging, characterized in that it takes a DNA nanostructure as the backbone, and the free groups of the DNA nanostructure are modified with fluorescent groups, which are used for specific positioning and identification of tag proteins. and a group that can be anchored on a hydrogel; wherein: the base pair in the DNA nanostructure does not exceed 40 pairs; the tag protein can be gene edited, and the tag protein can form a fusion protein with the target protein in the cell.
The DNA nanostructure dye according to claim 1, wherein the DNA nanostructure is a DNA double-stranded structure or a DNA tetrahedral structure; and the base pairs in the DNA nanostructure are between 10 and 30 pairs.
The DNA nanostructured dye according to claim 1, characterized in that, there are more than one fluorescent group, and the plurality of fluorescent groups are of the same kind, or a pair of fluorescence resonance energy transfer dyes.
The DNA nanostructure dye according to claim 1, wherein the fluorescent group is selected from one or more of Alexa Fluor 488, Cy3, Cy5, ATTO 647N or QD655.
The DNA nanostructure dye according to claim 1, wherein the group for specific localization and identification tag protein is a benzylguanine BG group for SNAP-tagged protein, a benzylcytosine BC group for CLIP-tagged protein groups or chlorinated alkane groups for Halo-tagged proteins.
The DNA nanostructure dye according to claim 1, wherein the group capable of anchoring on the hydrogel is an acrylamide group.
The application of the DNA nanostructure dye for expansion super-resolution imaging according to claim 1, characterized in that it specifically binds to a tag protein to perform fluorescent staining.
The application according to claim 7, characterized in that it comprises a plasmid construction process, wherein the translation sequence of the target protein is cloned, and a tag protein gene sequence is added in its coding frame; it also includes a cell expression system, which will construct a target protein and the tagged protein plasmids were transfected into cells for expression.
The application according to claim 7, characterized in that, in the staining process, a detergent is used to punch a small amount of holes on the cell membrane, and the DNA nanostructure dye for expansion super-resolution imaging according to claim 1 is used for staining and then fixed. cell.