CN114350757B - Intracellular paired chromatin modification imaging method based on DNA adjacent combination coding amplification - Google Patents

Abstract

The invention discloses an intracellular paired chromatin modification imaging method based on DNA adjacent combination coding amplification, and belongs to the field of cell imaging. Comprising the following steps: by combining chemical enzyme recognition and DNA nanotechnology, a closed loop reaction is activated through adjacent connection to form a complete annular template, so that rolling circle amplification reaction is initiated, and simultaneous coding imaging analysis of chromatin modification combination is realized. The method overcomes the technical defect that simultaneous detection of multiple paired chromatin modifications cannot be performed.

Description

Intracellular paired chromatin modification imaging method based on DNA adjacent combination coding amplification

Technical Field

The invention belongs to the field of cell imaging, and relates to an intracellular paired chromatin modification imaging method based on DNA adjacent combination coding amplification.

Background

The main components of chromatin are histones and DNA, and chromatin modification mainly refers to chemical modification of histones and DNA. Post-translational modifications of histones (Post-translational Modification, PTM) include methylation, acetylation, phosphorylation, ubiquitination modifications, and the like, which together constitute the histone code. To date, a variety of DNA base modifications have been found, the most notable of which in the mammalian genome are 5-methylcytosine (5 mC) and its oxidized derivatives 5-hydroxymethylcytosine (5-hmC), 5-aldehyde cytosine (5-fC) and 5-carboxyl cytosine (5-carboxylcytosine, 5-caC). These chromatin modifications can regulate the actions of binding proteins such as transcription factors and the like on chromatin, influence chromatin conformation and gene expression, and are widely involved in cellular processes and pathogenic mechanisms, and their abnormal changes are closely related to various malignant tumors.

In recent years, journals such as Nature and Cancer Cell report that different chromatin modifications have complex correlations and roles, and that DNA modifications at certain genetic loci and histone PTM may coexist or be mutually exclusive, exerting antagonistic or synergistic regulatory effects. The above studies indicate that different chromatin modification proximity combination patterns reflect different chromatin states or conformations, revealing a richer, more complex manner of regulation. Therefore, the analysis of different chromatin modification proximity combinations and associated information at the single cell level is of great significance to the deep understanding of epigenetic regulation mechanisms, the exploration of disease mechanisms, diagnosis and treatment schemes and the like.

Cell imaging can observe the internal structure of cells and spatially locate target molecules, which has important significance for clarifying molecular action mechanisms, biological functions and disease occurrence and development rules. However, intracellular chromatin modification proximity types are multi-functional, and the difficulties of multiple modification types, low abundance and the like are added, so that simultaneous imaging analysis of multiple pairs of chromatin modification proximity combination types in a single cell remains a significant challenge.

Disclosure of Invention

The invention aims to provide an intracellular paired chromatin modification imaging method based on DNA adjacent combination coding amplification, which can solve the technical problem that in the prior art, a plurality of intracellular paired chromatin modifications cannot be detected specifically and simultaneously.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses an intracellular paired chromatin modification imaging method based on DNA adjacent combination coding amplification, which comprises the following steps:

the connecting probe is pulled to the 5 'end and the 3' end by two adjacent chromatin modification bar code probes through hybridization reaction, and ring closure reaction is activated to form an annular probe;

and the annular probe is used as a template, and the rolling circle amplification reaction is initiated to realize fluorescent signal amplification and the imaging analysis of a plurality of pairs of chemical modification adjacent combinations on cell chromatin.

Preferably, the method specifically comprises the steps of:

1) Fixing and permeabilizing the cells, and marking a DNA bar code probe corresponding to the 5-hmC on a 5-hmC chemical modification molecule through specific enzyme recognition and click chemical reaction;

2) Selecting two histone modifications of H3K4me3 and H3K27Ac, and marking the bar code probes corresponding to the two histone modifications on the H3K4me3 and H3K27me3 respectively through the processes of recognizing the histone modifications by the first antibody and recognizing the first antibody by the second antibody-nucleic acid cross-linked bar code probe according to the sequence of the H3K4me3 and the H3K27me 3;

3) Ligating the hybridized DNA with the probe;

4) When two chemical modification distances are adjacent, the bar code probes of the two chemical modification distances are hybridized with two chains of the DNA ligation probes, the 5 'end and the 3' end of the two chemical modification distances are pulled up, and the two 5 'end and the 3' end are ligated in the presence of ligase to form a closed annular probe;

5) The annular probe is used as a template to trigger rolling circle amplification reaction to realize fluorescent signal amplification;

5) Finally, observing and analyzing by a laser confocal fluorescence microscope.

Further preferably, in step 1), the 5' -end of the DNA barcode probe corresponding to the 5-hmC is modified with a dibenzocyclooctyne functional group, and after the T4 beta-GT enzyme specifically binds uridine diphosphate glucose with azide modification on the 5-hmC site, the barcode probe modified with the dibenzocyclooctyne functional group-5 hmC is marked at the 5-hmC site in situ through click chemistry reaction.

Still more preferably, in step 2), the two kinds of histone modification corresponding barcode probes are an H3K4me3 barcode probe and an H3K27me3 barcode probe, and the 5' ends of the H3K4me3 barcode probe and the H3K27me3 barcode probe are both modified with azide functional groups for reacting with dibenzocyclooctyne molecules on the secondary antibody, and the secondary antibody-nucleic acid barcode probe is prepared by click chemistry reaction.

Further preferably, in step 3), there are three pairs of DNA ligation probes, including a first pair of Pad1-DNA, pad2-DNA, a second pair of Pad3-DNA, pad4-DNA, and a third pair of Pad5-DNA, pad6-DNA.

Still more preferably, the first pair of Pad1-DNA, pad2-DNA is attached to adjacent 5-hmC and H3K4me3 barcode probes, the second pair of Pad3-DNA, pad4-DNA is attached to adjacent 5-hmC and H3K27me3 barcode probes, and the third pair of Pad5-DNA, pad6-DNA is attached to adjacent H3K4me3 and H3K27me3 barcode probes.

Still more preferably, each DNA ligation probe has two sequences, each of which is modified at the 5 'and 3' ends with a phosphate group and a hydroxyl group, respectively.

Compared with the prior art, the invention has the following beneficial effects:

according to the method, a connecting probe can be pulled up to the 5 'end and the 3' end by two adjacent chromatin modification bar code probes through hybridization reaction, so that a ring-closing reaction is activated to form a complete annular probe, and then the annular probe is used as a template to trigger rolling circle amplification reaction to realize fluorescent signal amplification. Finally, imaging analysis of multiple pairs of chemically modified proximity combinations on cellular chromatin is achieved by the DNA proximity ligation amplification method. The method combines the chemical enzyme recognition and the DNA nanotechnology, activates the ring-closure reaction through adjacent connection to form a complete ring-shaped template, thereby triggering the rolling circle amplification reaction and realizing simultaneous coding imaging analysis of chromatin modification combination. The method overcomes the technical defect that simultaneous detection of various paired chromatin modifications cannot be carried out, and has the specific advantages that:

1) Single cell single molecule imaging can be achieved.

2) The reaction system is simple, the reaction efficiency is high, and simultaneous, rapid and accurate imaging of a plurality of pairs of adjacent molecules on intracellular chromatin can be realized;

3) The method can simultaneously study a plurality of pairs of adjacent chemical modification molecules on chromatin in a single cell, and find that the adjacent distribution of different post-translationally modified histones is different.

Furthermore, three chemical modifications for regulating genome expression function are selected as targets, one is DNA base modification, and the cytosine oxidized derivative is 5-hydroxymethylcytosine (5-hmC), and two kinds of histone modification are H3K4me3 and H3K27Ac respectively. At this time, there are three possible modification proximity pairs within a single cell, including a 5-hmC and H3K4me3 proximity pair, a H3K4me3 and H3K27Ac proximity pair, and a 5-hmC and H3K27Ac proximity pair. Thus, three different closed loop probes exist after corresponding proximity ligation, triggering a multicolor rolling circle amplification reaction, and finally realizing the coded imaging analysis of chromatin modification proximity combination.

Drawings

FIG. 1 is a schematic diagram of the present invention; wherein, (a) is a schematic diagram of an intracellular paired chromatin modification imaging method based on DNA proximity combination encoding amplification; (B) a process for preparing a secondary antibody-nucleic acid strand; (C) The principle of 5-hmC chemoenzymatic recognition and nucleic acid probe labeling on DNA; (D) Two modified antibody recognition and nucleic acid probe labeling principles on histones;

FIG. 2 is a graph showing the results of specific recognition imaging of chromatin modification in cells. Fluorescent imaging channel of nuclear dye DAPI; (ii) neighboring 5-hmC and H3K4me3 imaging results; (iii) 5-hmC and H3K27me3 imaging results for the neighborhood; (iv) imaging results for adjacent H3K3me3 and H3K27me 3; (V) is the result of the DAPI channel and three pairs of adjacent combined channels being superimposed on each other.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

referring to FIG. 1, a schematic diagram of the present invention, wherein (A) is a schematic diagram of an intracellular paired chromatin modification imaging method based on DNA proximity combination encoding amplification; (B) a process for preparing a secondary antibody-nucleic acid strand; (C) The principle of chemical enzyme method identification and nucleic acid probe marking for 5-hmC on DNA; (D) Principle of antibody recognition and nucleic acid probe labeling for two modifications on histones. The method takes a plurality of pairs of chemical modification combinations on intracellular chromatin as a research object, and realizes simultaneous single-molecule imaging analysis of the plurality of pairs of chemical modification combinations on intracellular chromatin.

The method comprises the following three parts:

a first moiety, binding uridine diphosphate glucose with azide modification to the 5-hmC site by T4 beta-GT enzyme, followed by labeling the barcode probe-5 hmC by click chemistry;

the second part, in the order of H3K4me3 and H3K27me3, respectively marking the bar code probes corresponding to 2 kinds of histone modification on H3K4me3 and H3K27me3 through the process of recognizing the first antibody by the first antibody recognition histone modification and the second antibody-nucleic acid crosslinking bar code probe recognition;

a third part, the hybridized DNA ligation probe (two ligation probes are shown here, each modified at their 5 'and 3' ends with a phosphate and hydroxyl group, respectively); when two chemical modification distances are adjacent, the bar code probes of the two chemical modification distances are hybridized with two chains of the connecting probes, the 5 'end and the 3' end of the bar code probes are pulled up, and the two 5 'end and the 3' end are connected in the presence of ligase, so that a closed annular probe is finally formed; and then, taking the annular probe as a template, and initiating a rolling circle amplification reaction to realize fluorescent signal amplification.

Example 1

The method of the invention is used for realizing DNA adjacent connection coding amplification imaging analysis of a plurality of pairs of modification combinations on cell chromatin

Cells were fixed with 4% (mass/volume) paraformaldehyde for 10 minutes at room temperature using MDA-231 cell line as a basic model, washed 3 times with PBS, and permeabilized with 0.5% (volume/volume) Triton X-100 for 5 minutes at room temperature; first, DNA base modified 5-hmC labeling was performed, and cells were incubated with 50. Mu.M azide modified uridine diphosphate glucose and 5U T4. Beta. -glycosyltransferase enzyme at 37℃for 2 hours. Subsequently, 250 nM DBCO (dibenzocyclooctyne) -modified bar-coded probe-5 hmC and 0.5. Mu.g/mL herring sperm DNA were added and a copper-free click reaction was performed at 37℃for 60 minutes.

After 3 times washing of cells with PBS, cells were incubated with QuickBlock-type blocking buffer for 1 hour at room temperature, a primary antibody recognizing post-translationally modified histone H3K4me3 was added and incubated overnight at 4 ℃. After PBST (PBS containing 0.05% Tween-20) was washed 3 times, the secondary antibody-nucleic acid H3K4me3 bar code probe was added and incubated at room temperature for 1 hour.

After 3 times washing of the cells by PBST, the cells were again incubated with QuickBlock-type blocking buffer for 1 hour at room temperature, and then a primary antibody recognizing post-translationally modified histone H3K27me3 was added and incubated overnight at 4 ℃. After PBST washing the cells 3 times, secondary antibody-nucleic acid H3K27me3 bar code probe was added and incubated for 1 hour at room temperature.

PBST washes cells 3 times, adds a plurality of pairs of ligation probes (three pairs of ligation probes, each pair comprising two sequences, each of which has a phosphate and hydroxyl group modified at the 5 'and 3' ends, pad1-DNA, pad2-DNA, pad3-DNA, pad4-DNA, pad 5-hmC and H3K27me3 barcode probes, pad5-DNA, pad6-DNA, and H3K4me3 and H3K27me3 barcode probes, respectively) to cells; when two chemical modifications are adjacent, the barcode probe captures both strands toward the hybridized ligation probe, pulling their 5 'and 3' ends closer, and ligating the two 5 'and 3' ends in the presence of T4 DNA ligase, eventually forming a closed circular probe, where three chemical modifications are adjacent in three instances, ligating three different closed circular probes, and subsequently initiating a multicolor rolling circle amplification reaction. Specifically, after washing the cells 3 times with 2 XSSC, 10. Mu.L of 1 XPHI 29 buffer containing 5U of phi29 polymerase, 2.5 mM dNTPs and 0.5. Mu.LBSA (10 mg/mL) was added to 37 ^o C for 2 hours. After washing, 20. Mu.L of a hybridization mixture comprising 200 nM of FITC-probe, 200 nM of Cy3-probe, 200 nM of Cy5-probe and 20% formamide was added and incubated at 37℃for 30 minutes; after the cells were washed 3 times with 2 XSSC, the nuclei were stained with 20. Mu.L of DAPI working solution, incubated at room temperature for 10 minutes, and finally subjected to simultaneous encoding amplification imaging analysis on a plurality of pairs of chemical modification combinations on the chromatin by using a confocal laser fluorescence microscope.

The sequences designed for the above examples are shown in table 1 below:

TABLE 1

Name of the name	Sequence (5 '-3')	Sequence Listing numbering
			H3K4me3 bar code probe	azido-AAAAAAAAAAAAAAAAAAAAGACGCTAATAGTTAAGACGCTT	SEQ ID NO:1
5hmC bar code probe	dibenzocyclooctyne-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATGACAGAACTAGACACTCTT	SEQ ID NO:2
			H3K27me3 bar code probe	azido-AAAAAAAAAAAAAAAAAAAATAAGGCTATGCGAAGAGTATCC	SEQ ID NO:3
Pad1-DNA	Phosphate group-CTATTAGCGTCCAGTGAATTATACCCGGTCGCTTCTTTATGCC GTCAAGAGTGTCTA-hydroxy group	SEQ ID NO:4
			Pad2-DNA	P-GTTCTGTCATATACAAGCGTCTAA	SEQ ID NO:5
Pad3-DNA	Phosphate group-GCATAGCCTTACTCGACAGAGCTTACTCACAGCCAGCATCACAAGGTCAAGAGTGTCTA-hydroxy group	SEQ ID NO:6
			Pad4-DNA	Phosphate group-GTTCTGTCATATATGGATACTCTTC-hydroxy group	SEQ ID NO:7
Pad5-DNA	Phosphate group-CTATTAGCGTCGTGGCCTGAGCCTTCCTCGGTACGGTCTGTAAGGTCAGGATACTCTTC-hydroxy group	SEQ ID NO:8
			Pad6-DNA	Phosphate group-GCATAGCCTTAATAAGCGTCTTAA-hydroxy group	SEQ ID NO:9

Fluorescence results referring to fig. 2, the fluorescent imaging channel of the nuclear dye DAPI in fig. 2 (i) shows that the nuclei are uniformly stained; (ii) adjacent 5-hmC and H3K4me3 imaging, (iii) adjacent 5-hmC and H3K27me3 imaging, (iv) adjacent H3K3me3 and H3K27me3 imaging, each single point representing a signal of an adjacent molecule, demonstrating that the method of the invention can achieve single molecule imaging of a combination of chromatin adjacent modifications; (V) is the result of the DAPI channel and three pairs of adjacent combination channels superimposed on each other, and it can be seen that three pairs of chromatin adjacent combinations exist largely in the nucleus.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

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Claims (1)

1. An intracellular paired chromatin modification imaging method based on DNA proximity combination encoding amplification, comprising:

the connecting probe is pulled to the 5 'end and the 3' end by two adjacent chromatin modification bar code probes through hybridization reaction, and ring closure reaction is activated to form an annular probe;

taking the annular probe as a template, triggering rolling circle amplification reaction to realize fluorescent signal amplification and realizing imaging analysis of a plurality of pairs of chemical modification adjacent combinations on cell chromatin;

the method specifically comprises the following steps:

1) Fixing and permeabilizing the cells, and marking a DNA bar code probe corresponding to the 5-hmC on a 5-hmC chemical modification molecule through specific enzyme recognition and click chemical reaction;

the 5' -end of the DNA bar code probe corresponding to the 5-hmC is modified with a dibenzocyclooctyne functional group, and after the T4 beta-GT enzyme specifically binds uridine diphosphate glucose with azide modification on a 5-hmC site, the bar code probe modified by the dibenzocyclooctyne functional group-5 hmC is marked at the 5-hmC site in situ through click chemistry reaction;

2) Selecting two histone modifications of H3K4me3 and H3K27Ac, and marking the bar code probes corresponding to the two histone modifications on the H3K4me3 and H3K27me3 respectively through the processes of recognizing the histone modifications by the first antibody and recognizing the first antibody by the second antibody-nucleic acid cross-linked bar code probe according to the sequence of the H3K4me3 and the H3K27me 3;

the two types of histone modification corresponding bar code probes are an H3K4me3 bar code probe and an H3K27me3 bar code probe, and the 5' ends of the H3K4me3 bar code probe and the H3K27me3 bar code probe are modified with azide functional groups for reacting with dibenzocyclooctyne molecules on the secondary antibody, so as to prepare the secondary antibody-nucleic acid bar code probe through click chemistry reaction;

3) Ligating the hybridized DNA with the probe;

the DNA ligation probes have three pairs, including a first pair of Pad1-DNA and Pad2-DNA, a second pair of Pad3-DNA and Pad4-DNA, and a third pair of Pad5-DNA and Pad6-DNA; the first pair of Pad1-DNA and Pad2-DNA is connected with adjacent 5-hmC and H3K4me3 bar code probes, the second pair of Pad3-DNA and Pad4-DNA is connected with adjacent 5-hmC and H3K27me3 bar code probes, and the third pair of Pad5-DNA and Pad6-DNA is connected with adjacent H3K4me3 and H3K27me3 bar code probes; each DNA connection probe has two sequences, and the 5 'and 3' ends of the two sequences are respectively modified with a phosphate group and a hydroxyl group;

wherein:

the sequence of the H3K4me3 bar code probe is shown as SEQ ID NO. 1;

the sequence of the 5hmC bar code probe is shown as SEQ ID NO. 2;

the sequence of the H3K27me3 bar code probe is shown as SEQ ID NO. 3;

the sequence of the Pad1-DNA is shown as SEQ ID NO. 4;

the sequence of the Pad2-DNA is shown as SEQ ID NO. 5;

the sequence of the Pad3-DNA is shown as SEQ ID NO. 6;

the sequence of the Pad4-DNA is shown as SEQ ID NO. 7;

the sequence of the Pad5-DNA is shown as SEQ ID NO. 8;

the sequence of the Pad6-DNA is shown as SEQ ID NO. 9;

4) When two chemical modification distances are adjacent, the bar code probes of the two chemical modification distances are hybridized with two chains of the DNA ligation probes, the 5 'end and the 3' end of the two chemical modification distances are pulled up, and the two 5 'end and the 3' end are ligated in the presence of ligase to form a closed annular probe;

5) The annular probe is used as a template to trigger rolling circle amplification reaction to realize fluorescent signal amplification;

6) Finally, observing and analyzing by a laser confocal fluorescence microscope.