阅读说明：本技术 一种单细胞dna表观遗传修饰空间定位与邻近分布的可视化区分方法 (Visual distinguishing method for single-cell DNA epigenetic modification space positioning and adjacent distribution ) 是由 赵永席 陈锋 薛静 张进 于 2020-06-29 设计创作，主要内容包括：本发明公开了一种单细胞DNA表观遗传修饰空间定位与邻近分布的可视化区分方法,属于分析化学领域,具体涉及通过叠氮衍生物探针1,3-吲二酮(AI)对5-醛基胞嘧啶(5-fC)进行叠氮(N<Sub>3</Sub>)官能团的化学标记、利用T4噬菌体β葡萄糖基转移酶(β-GT)对5-羟甲基胞嘧啶(5-hmC)进行叠氮(N<Sub>3</Sub>)官能团的酶介导标记；成对的临近位点的引物共同对有缺口的环模板实现原位临近连接,剩余不成对的5-fC和5-hmC修饰位点,引物通过链置换反应被释放,接着杂交对应的特异性环模板；并结合核酸扩增、荧光探针杂交,实现单细胞DNA表观遗传修饰空间定位与邻近分布的单分子可视化区分。(The invention discloses a visual distinguishing method for single-cell DNA epigenetic modification space positioning and adjacent distribution, belongs to the field of analytical chemistry, and particularly relates to azide (N) of 5-aldehyde cytosine (5-fC) through an azide derivative probe 1, 3-inddione (AI) 3 ) Chemical labeling of functional groups, azidation (N-C) of 5-hydroxymethylcytosine (5-hmC) with T4 bacteriophage beta glucosyltransferase (beta-GT) 3 ) Enzyme-mediated labeling of functional groups; the paired primers of the adjacent sites jointly realize in-situ adjacent connection on the ring template with the gap, the remaining unpaired 5-fC and 5-hmC modified sites are released through strand displacement reaction, and then the corresponding specific ring template is hybridized; and combines nucleic acid amplification and fluorescent probe hybridization to realize the single-cell DNA epigenetic modification spaceLocalization is distinguished from single molecule visualization of adjacent distribution.)

1. A visual distinguishing method for single-cell DNA epigenetic modification space orientation and adjacent distribution is characterized by comprising the following steps:

1) marking the DNA epigenetic modification sites in the single cells by using a specific marking method;

2) covalently connecting the mark points with corresponding primers through click chemistry, wherein paired primers close to the modification sites can realize in-situ close connection on the ring template with the gap to obtain a complete ring template Pad 1;

3) for the remaining unpaired modification sites, the primers are released through strand displacement reaction, and then specific loop templates Pad2 and Pad3 are utilized to respectively hybridize the modification sites, so that the spatial positioning and adjacent distribution characteristic coding of the modification sites are realized;

4) After the coding is finished, single-cell DNA epigenetic modification space positioning and single-molecule visual distinguishing of adjacent distribution are realized through nucleic acid amplification and fluorescent probe hybridization.

2. The method for visually distinguishing the spatial localization and the adjacent distribution of the epigenetic modification of the single-cell DNA according to claim 1, wherein the modification sites are 5-aldehyde cytosine and 5-hydroxymethyl cytosine.

3. The method for visually distinguishing the spatial localization and the adjacent distribution of the epigenetic modification of the single-cell DNA as claimed in claim 2, wherein in the step 1), the azide derivative probe 1, 3-indolidione is used for chemically labeling the azide function of the 5-aldehyde cytosine, and the T4 bacteriophage beta glucosyltransferase is used for enzyme-mediated labeling of the azide function of the 5-hydroxymethyl cytosine.

4. The visual distinguishing method for single-cell DNA epigenetic modification spatial localization and adjacent distribution according to claim 2, characterized in that in step 2), after 5-aldehyde cytosine and 5-hydroxymethylcytosine modification sites in a single cell are sequentially subjected to specific labeling and click chemistry covalent connection with corresponding RCA primers P1-5-aldehyde cytosine and P2-5-hydroxymethylcytosine, paired primers adjacent to the sites are jointly subjected to in-situ adjacent connection with a ring template with a gap, so as to obtain a complete ring template Pad 1.

Technical Field

The invention belongs to the field of analytical chemistry, and relates to a visual distinguishing method for single-cell DNA epigenetic modification space positioning and adjacent distribution.

Background

Different DNA epigenetic markers exist in the mammalian genome. These epigenetic markers have profound effects on both gene expression and chromatin structure and are implicated in the pathology of many diseases (e.g., cancer, etc.). Among the epigenetic markers of genes, 5-methylcytosine (5-mC) and its oxidized derivatives, 5-hmC and 5-fC, have been studied more and found to be not only an intermediate product of the 5-mC demethylation pathway, but also to have good stability. Both DNA epigenetic markers are produced by the 10-11 translocation family of dioxygenases.

The spatial localization of epigenetic markers in single cells is also fundamental to understanding their function, and different epigenetic markers, if they are spatially closely distributed, may interfere with each other or with the proteins they express. For these modified bases, there are currently several deep sequencing strategies for whole genome analysis that can reveal their important functions in normal biological processes and in disease development processes. However, these methods of deep sequencing are all based on the average cell population analysis and cannot achieve the visualization of the subcellular distribution of modified bases.

In research methods directed to the spatial proximity distribution of DNA epigenetic markers, traditional fluorescence in situ hybridization-based cellular imaging is limited to detecting sequences of interest and is not sufficient to assess base modifications; immunoassays are limited to the use of antibodies in naked DNA in combination with DNA modifications (e.g. 5hmC) and when the size of the antibody protein is large, the base sites covered by the DNA binding protein or condensed chromatin can affect the recognition of the immune protein, resulting in a reduced recognition rate. Moreover, such antibodies are non-covalent to DNA, have low affinity-based recognition specificity and binding efficiency, and cannot be studied for spatial proximity of bases with different epigenetic markers; in contrast, chemical enzyme or chemical based methods can more accurately and more fully achieve the labeling of DNA epigenetic sites when labeling 5-hmC or 5-fC. However, this method has not been applied in a real intracellular environment because analogs containing a large number of modified bases in the intracellular environment interfere with recognition. Therefore, current research methods cannot obtain information on the spatial proximity distribution of DNA epigenetic markers.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a visual distinguishing method for single-cell DNA epigenetic modification space positioning and adjacent distribution, which can realize the sequential in-situ specific labeling of 5-fC/5-hmC in a single cell, has high specificity in the labeling process, can effectively avoid interference, and has high reaction efficiency, simple reaction conditions and high reaction speed.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a visual distinguishing method for single cell DNA epigenetic modification space positioning and adjacent distribution, which comprises the following steps:

1) marking the DNA epigenetic modification sites in the single cells by using a specific marking method;

2) covalently connecting the mark points with corresponding primers through click chemistry, wherein paired primers close to the modification sites can realize in-situ close connection on the ring template with the gap to obtain a complete ring template Pad 1;

3) for the remaining unpaired modification sites, the primers are released through strand displacement reaction, and then specific loop templates Pad2 and Pad3 are utilized to respectively hybridize the modification sites, so that the spatial positioning and adjacent distribution characteristic coding of the modification sites are realized;

4) After the coding is finished, single-cell DNA epigenetic modification space positioning and single-molecule visual distinguishing of adjacent distribution are realized through nucleic acid amplification and fluorescent probe hybridization.

Preferably, the modification sites are 5-aldehyde cytosine and 5-hydroxymethylcytosine.

Further preferably, in step 1), the azide derivative probe 1, 3-indolidione is used for chemical labeling of the azide function of 5-aldehyde cytosine, and T4 bacteriophage beta glucosyltransferase is used for enzyme-mediated labeling of the azide function of 5-hydroxymethyl cytosine.

The specific operation is as follows: mu.L of 1 XMES buffer containing 10mM AI and 10% dimethyl sulfoxide was added to the reaction chamber and incubated at 37 ℃ for 24 h. Subsequently, 1 XPBS buffer containing 100nM dibenzocyclooctyne DBCO-modified primer probe P1-fC was added to the reaction chamber using a click chemistry reaction and reacted at 37 ℃ for 1 h. mu.L UDP-N containing 1 XNEBuffer 4, 50. mu.M was added to the reaction chamber₃Reaction of-Glu and 5U of T4 β -GT, incubated at 37 ℃ for 2 h. Finally, 100nM P2-hmC probe was added to the reaction chamber and the reaction was carried out at 37 ℃ for 1 h. The reaction solution of the previous step was removed before each addition of the reaction solution to the reaction chamber, and the reaction chamber was washed three times with PBS.

Preferably, in the step 2), after the 5-aldehyde cytosine and the 5-hydroxymethyl cytosine modification sites in the single cell are sequentially subjected to specific labeling and click chemistry covalent connection with the corresponding RCA primer P1-5-aldehyde cytosine and P2-5-hydroxymethyl cytosine, the paired primers adjacent to the sites jointly realize in-situ adjacent connection on the ring template with the gap, and the complete ring template Pad 1 is obtained.

The specific operation is as follows: after 5-fC and 5-hmC modification sites in the single cell are sequentially subjected to specific labeling and click chemistry covalent connection of corresponding RCA primers (P1-fC and P2-hmC), primers of paired adjacent sites jointly realize in-situ adjacent connection of the gapped ring template, and a complete ring template (Pad 1) is obtained. That is, L-proxi and Pad-proxi probes containing 10U T4DNA ligase were added to 1 XT 4DNA ligase buffer solution and in situ hybridized and ligated with two primer DNA probes, P1-fC and P2-hmC, respectively. Realizing the adjacent distribution characteristic coding of 5-fC and 5-hmC.

Preferably, in step 3), the primers are released by a strand displacement reaction for the remaining unpaired modification sites, comprising the following operations: after in situ ligation of primers at adjacent sites, unpaired 5-fC and 5-hmC modified sites remained and primers were released by strand displacement reaction, i.e., after in situ hybridization and ligation at adjacent sites, excess probe at 5-fC or 5-hmC sites was displaced out using 200nM Disp-fC and Disp-hmC at 37 ℃.

Preferably, in step 3), specific loop template hybridization is performed on the epigenetic modification sites after the strand displacement reaction, comprising the following operations: after the excess probe at the 5-fC or 5-hmC site has been displaced, the site is hybridized using the corresponding specific loop templates (Pad 2 and Pad 3). That is, 200nM of the prepared 5-fC/5-hmC specific circular DNA barcode probe was added to the reaction chamber and incubated at 37 ℃ for 2 h. And the spatial positioning coding of 5-fC and 5-hmC is realized.

Preferably, in step 4), RCA amplification, hybridization of the fluorescent probe and fluorescence imaging are performed, including the following operations: a1 Xφ 29DNA polymerase buffer containing 10U φ 29DNA polymerase and 2mM dNTP was added to the reaction chamber and reacted at 37 ℃ for 2 h. Finally, the fluorescent probe was hybridized with the RCA amplification product in 2 XSSC buffer containing 20% formamide. After hybridization of the fluorescent probes, nuclei were stained with DAPI and imaged by laser scanning confocal microscopy (TCS SP8 STED 3X, Leica). The reaction solution of the previous step was removed before each addition of the reaction solution to the reaction chamber, and the reaction chamber was washed three times with PBS.

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

the invention discloses a visual distinguishing method for single-cell DNA epigenetic modification space positioning and adjacent distribution, which carries out azide (N) on 5-aldehyde cytosine (5-fC) through an azide derivative probe 1, 3-inddione (AI) ₃) Chemical labeling of functional groups, azidation (N-C) of 5-hydroxymethylcytosine (5-hmC) with T4 bacteriophage beta glucosyltransferase (beta-GT)₃) Enzyme-mediated labeling of functional groups; the paired primers of the adjacent sites jointly realize in-situ adjacent connection on the ring template with the gap, the remaining unpaired 5-fC and 5-hmC modified sites are released through strand displacement reaction, and then the corresponding specific ring template is hybridized; and nucleic acid amplification and fluorescent probe hybridization are combined to realize single cell DNA epigenetic modification space positioning and single molecule visual distinction of adjacent distribution. The method has the advantages that:

1. the 5-fC/5-hmC in the single cell is sequentially subjected to in-situ specific labeling, and the difference visualization of the 5-fC/5-hmC spatial positioning and the proximity degree of the 5-fC/5-hmC in the single cell are realized by combining in-situ proximity ligation, strand displacement reaction, specific loop template hybridization and rolling circle amplification. This multi-level spatial information will facilitate the in-depth study of the apparent genetically modified base regulation function and mechanism.

2. The paired primers adjacent to the epigenetic modification sites are subjected to in-situ proximity connection, after the 5-fC and 5-hmC modification sites in a single cell are sequentially subjected to specific labeling and click chemistry covalent connection with corresponding RCA primers (P1-fC and P2-hmC), the paired primers adjacent to the sites jointly realize in-situ proximity connection on a ring template with a gap, so that a complete ring template can be obtained, the adjacent distribution characteristic coding of the 5-fC and 5-hmC is realized, and then the adjacent distribution information of the epigenetic modification bases 5-fC and 5-hmC can be obtained. And the labeling process has high specificity, so that interference can be avoided.

3. The primers of the unpaired epigenetic modification sites of the invention undergo strand displacement reaction, and excess probes on non-adjacent 5-fC or 5-hmC sites can be displaced by utilizing excess probes. After in situ ligation of primers at adjacent sites, the primers at the remaining unpaired 5-fC and 5-hmC modified sites were released using 200nM Disp-fC and Disp-hmC at 37 ℃. The process is a reaction with high yield, simple reaction conditions and high reaction speed.

4. The epigenetic modification sites after the strand displacement reaction are subjected to specific ring template hybridization, and the sites after the strand displacement reaction are hybridized by utilizing corresponding specific ring templates (Pad 2 and Pad 3), so that the space positioning coding of 5-fC and 5-hmC can be realized. By using the method, more comprehensive subcellular distribution information can be provided for deeper epigenetic study.

Drawings

FIG. 1 is a schematic diagram of the present invention;

FIG. 2 shows the test tube validation experimental results of visualization of the pairwise proximity distribution of 5-fC and 5-hmC; wherein A is mass spectrometry of 5-fC and AI specific labeled products; b, performing sequencing analysis after treating different dsDNA samples by using AI; c is N ₃-mass spectrometry results of 5-gmC; d is the result of pairwise adjacent visualization of 5-fC and 5-hmC on the coverslip;

FIG. 3 shows the spatial localization of single cells 5-fC and 5-hmC; wherein, A is representative cell images of 5-fC, 5-hmC and H3K4me1 in different cell lines; b is co-localization assay of 5-fC/5-hmC, 5-fC/H3K4me1 and 5-hmC/H3K4me1 in different cell lines (N50); c is a co-localization assay of 5-fC or 5-hmC with 5 histone modifications in MCF-10A cells (N ═ 50). N represents the number of statistical cells;

FIG. 4-1 is a schematic diagram of the design of visualization of pairwise proximity distributions of 5-fC and 5-hmC within a single cell;

fig. 4-2 is a graph of the results of statistical analysis of fluorescence intensity and RCA amplification product RCP spot counts within each channel of a single cell (N ═ 55);

FIGS. 4-3 are graphs of the results of DAPI signal intensity (blue), paired proximal sites (green), unpaired 5fC (yellow) or 5hmC (red) as a function of distance from the nuclear center to the nuclear membrane;

fig. 4-4 show the corresponding Pearson correlation results (N55) for each signal with DAPI.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or 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 accompanying drawings:

referring to fig. 1, which is a schematic diagram of the present invention, the method for visually distinguishing the spatial localization and the adjacent distribution of the epigenetic modification of single-cell DNA disclosed in the present invention comprises the following steps:

1) marking the DNA epigenetic modification sites in the single cells by using a specific marking method;

2) Covalently connecting the mark points with corresponding primers through click chemistry, wherein paired primers close to the modification sites can realize in-situ close connection on the ring template with the gap to obtain a complete ring template Pad 1;

3) for the remaining unpaired modification sites, the primers are released through strand displacement reaction, and then specific loop templates Pad2 and Pad3 are utilized to respectively hybridize the modification sites, so that the spatial positioning and adjacent distribution characteristic coding of the modification sites are realized;

4) after the coding is finished, single-cell DNA epigenetic modification space positioning and single-molecule visual distinguishing of adjacent distribution are realized through nucleic acid amplification and fluorescent probe hybridization.

Specifically, N-cleavage of 5-fC by azide derivative probe AI₃Chemical labelling of functional groups, N-labelling of 5-hmC with beta-GT₃Enzyme-mediated labeling of functional groups; and covalently connecting corresponding RCA primers to the labeled sites respectively by using click chemistry. The paired primers of the adjacent sites jointly realize in-situ adjacent connection on the ring template with the gap, the remaining unpaired 5-fC and 5-hmC modified sites are released through strand displacement reaction, and then the corresponding specific ring template is hybridized; and combines nucleic acid amplification and fluorescent probe hybridization to realize single molecule visualization of single cell DNA epigenetic modification space positioning and adjacent distribution And (6) distinguishing.