CN119530350B - An in situ imaging method for visualizing specific modifications of target nucleic acids within cells - Google Patents
An in situ imaging method for visualizing specific modifications of target nucleic acids within cellsInfo
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Abstract
本发明提供一种可实现细胞内目标核酸特定修饰可视化的原位成像方法。该方法仅需三条具有不同功能的单链核酸：装配有识别核酸特定修饰组件的单链核酸支架、能特异性靶向目标核酸序列的单链核酸引物‑1、单链核酸引物‑2，便可完成对特定修饰与特定核酸序列的靶向并组成滚环扩增的信号放大体系，具有简便经济的特点，具有极高的商业化潜力。在原位滚环扩增后形成一条序列重复的长单链核酸，可富集特定的荧光探针进行成像，该处成像信号点即为目标核酸特定修饰位点所在的空间位置。本发明可与蛋白免疫荧光共用、可实现多种核酸的同时成像、可实现不同亚空间定位的核酸成像，使得该方法成为能够应用在多场景的多功能解决方案。
The present invention provides an in situ imaging method that can realize the visualization of specific modifications of target nucleic acids in cells. This method only requires three single-stranded nucleic acids with different functions: a single-stranded nucleic acid scaffold equipped with a component for recognizing specific modifications of nucleic acids, a single-stranded nucleic acid primer-1 that can specifically target the target nucleic acid sequence, and a single-stranded nucleic acid primer-2, so as to complete the targeting of specific modifications and specific nucleic acid sequences and form a signal amplification system for rolling circle amplification. It is simple and economical and has extremely high commercial potential. After in situ rolling circle amplification, a long single-stranded nucleic acid with a repeated sequence is formed, which can enrich specific fluorescent probes for imaging, and the imaging signal point therein is the spatial position of the specific modification site of the target nucleic acid. The present invention can be used in conjunction with protein immunofluorescence, can realize the simultaneous imaging of multiple nucleic acids, and can realize the imaging of nucleic acids located in different subspaces, making this method a multifunctional solution that can be applied in multiple scenarios.
Description
Technical Field
The invention belongs to the field of nucleic acid chemistry, and in particular relates to an in-situ imaging method capable of realizing specific modification visualization of target nucleic acid in cells.
Background
After completion of the human genome project, the sequence information of the genes is known, but we still cannot read the genetic information hidden in the sequence information well. We have found that there are a number of different modified nucleosides in nucleic acids that play different biological roles in the body, participate in a number of biological processes, and add a new layer to the regulation of genetic information. Research into these modified nucleosides opens up the field of epigenetic science. Currently, researchers have been able to map precisely and quantitatively the sites of various nucleoside modifications in genomes and transcriptomes, greatly advancing our understanding of the function of nucleoside modifications in underlying physiological and pathological processes. However, our spatial positional information of these nucleoside modifications in cells is currently not well understood, which limits our research on modified nucleosides.
Recently, liquid-liquid phase separation and membraneless organelles have been discovered, indicating that the distribution of substances in cells is not uniform and that different subcellular localization is often directly related to specific biological functions. If the spatial information of the modified nucleoside is obtained, the dynamic change and the function of the modified nucleoside under the complex biological background can be deeply analyzed. However, the current positioning of modified nucleosides is mainly based on an immunostaining method of an antibody/fluorescent secondary antibody, which can globally image nucleoside modifications, but cannot distinguish nucleoside modifications in specific nucleic acids, so that the requirement of researchers on the functional study of specific nucleoside modifications of target nucleic acids is difficult to meet. If a tool for in-situ imaging of nucleoside modification in specific nucleic acid can be developed, the nucleoside modification in specific nucleic acid can be precisely positioned, research on the nucleoside modification by researchers is promoted, understanding of the nucleoside modification is deepened, and therefore a plurality of fields such as etiology research, disease prediction, new medicine development, personalized medicine and the like are better guided, and development of the medical and biotechnology fields is promoted.
Disclosure of Invention
The invention aims to provide an in-situ imaging method capable of realizing the visualization of specific modification of target nucleic acid in cells.
The invention adopts the following technical scheme:
an in situ imaging method for visualizing specific modifications of a target nucleic acid in a cell, comprising the steps of:
(1) Grafting the single-stranded nucleic acid bracket of the modification site, namely incubating the single-stranded nucleic acid bracket assembled with the specific modification component of the identification nucleic acid with the fixed and permeabilized cells, or incubating the specific modification component of the identification nucleic acid with the permeabilized cells and then incubating the same with the single-stranded nucleic acid bracket, so that the single-stranded nucleic acid bracket is grafted at the specific modification site to obtain the treated cells;
(2) The identification of target nucleic acid, namely incubating a single-stranded nucleic acid primer-1 and a single-stranded nucleic acid primer-2 which can specifically target a target nucleic acid sequence with the cells treated in the step (1), so that the single-stranded nucleic acid primer-1 and the single-stranded nucleic acid primer-2 are combined with the target nucleic acid;
(3) Signal amplification, namely cyclizing the single-stranded nucleic acid primer-1 by ligase with the aid of a single-stranded nucleic acid bracket grafted to a modification site, and then performing rolling circle amplification by taking the single-stranded nucleic acid primer-1 as a primer extension template of the single-stranded nucleic acid primer-2 to amplify a signal of specific modification of target nucleic acid in cells in situ;
(4) And (3) signal visualization, namely incubating the single-stranded nucleic acid primer-1 after cyclization with the cell after rolling circle amplification, wherein the product after rolling circle amplification is enriched with a large amount of fluorescent probes, so that bright signal points which are obviously different from background signals can be observed under a confocal laser microscope, and the subcellular space positioning of specific modification sites on target nucleic acid is reflected.
Further, the in-situ imaging method for visualizing specific modifications of intracellular target nucleic acids comprises single imaging, simultaneous imaging of multiple nucleic acids, simultaneous imaging of multiple modifications, imaging of different subspace localization, or co-imaging with protein immunofluorescence.
Further, the target nucleic acid is single-stranded DNA or RNA.
Further, the specific modification of the nucleic acid in the step (1) is natural or synthetic base modification, ribose modification or phosphate backbone modification.
Further, the single-stranded nucleic acid scaffold equipped with the recognition nucleic acid specific modification module in the step (1) specifically comprises the following modules:
a modification recognition component a for recognizing and binding to a specific nucleic acid modification;
A single-stranded nucleic acid scaffold B complementarily paired with the head-to-tail sequence of the single-stranded nucleic acid primer-1 for assisting the ligase to circularize the single-stranded nucleic acid primer-1;
A connecting component A which is directly combined with the modification recognition component A to form a component for recognizing specific modification of nucleic acid;
and a connecting component B which is directly combined with the single-stranded nucleic acid bracket B to form a single-stranded nucleic acid bracket, and is combined with the connecting component A to combine the component for identifying the specific modification of the nucleic acid with the single-stranded nucleic acid bracket.
Further, the recognition nucleic acid specific modified component is incubated with the permeabilized cell and then incubated with the single-stranded nucleic acid scaffold, and in the grafting process, the recognition nucleic acid specific modified component and the single-stranded nucleic acid scaffold respectively comprise the following components:
The component for identifying the specific modification of the nucleic acid comprises a modification identification component A, a connecting component A, a modification identification component A and a modification detection component A, wherein the modification identification component A is used for identifying the specific modification of the nucleic acid and combining with the specific modification;
the single-stranded nucleic acid scaffold comprises a single-stranded nucleic acid scaffold B, a connecting component B and a connecting component A, wherein the single-stranded nucleic acid scaffold B is in complementary pairing with the head-tail sequence of the single-stranded nucleic acid primer-1 and is used for assisting ligase to cyclize the single-stranded nucleic acid primer-1, the connecting component B is directly combined with the single-stranded nucleic acid scaffold B to form the single-stranded nucleic acid scaffold, and the connecting component A is combined with the connecting component A and is used for combining a component for identifying specific modification of nucleic acid with the single-stranded nucleic acid scaffold.
Still further, the modified recognition module a may be a binding protein or binding domain thereof, an antibody or antibody fragment, an artificial protein, a peptide fragment, or a modified enzyme or catalytic domain thereof, a de-modified enzyme or catalytic domain thereof, a natural amino acid or residue thereof, a non-natural amino acid or residue thereof, a natural protein modification or a non-natural protein modification, which is covalently bound to a specific modification.
Still further, the linker component a may be a nucleic acid binding protein or binding domain thereof, a HaloTag protein or reaction domain thereof, streptavidin, an antibody or antibody fragment, a natural amino acid or residue thereof, an unnatural amino acid or residue thereof, a natural protein modification or an unnatural protein modification.
Still further, the linker component B may be a specific nucleic acid sequence capable of binding to a nucleic acid binding protein or binding domain thereof, a HaloTag ligand, biotin, an antigen capable of binding to an antibody or antibody fragment, a specific chemical group capable of reacting with a natural amino acid or residue thereof, a specific chemical group capable of reacting with a non-natural amino acid or residue thereof, a specific chemical group capable of reacting with a natural protein modification or a non-natural protein modification.
Still further, the single-stranded nucleic acid scaffold B may be single-stranded DNA, single-stranded RNA, single-stranded LNA with or without nucleoside modifications or phosphate backbone modifications.
Furthermore, a single-stranded nucleic acid scaffold for identifying a specific modification module of nucleic acid is assembled at a specific modification site, the specific modification module of the identification nucleic acid is incubated with the cell after permeabilization and then incubated with the single-stranded nucleic acid scaffold, so that the single-stranded nucleic acid scaffold is grafted at the specific modification site, and the specific method comprises the following steps:
(a) Constructing a module for identifying specific modification of nucleic acid, designing and constructing a fusion protein expression plasmid containing the modification identification module A and the connecting module A, and purifying after protein expression.
(B) Constructing a single-stranded nucleic acid scaffold, designing and solid-phase synthesizing a nucleic acid comprising the single-stranded nucleic acid scaffold B and the connecting component B, and purifying, or connecting the single-stranded nucleic acid scaffold B and the connecting component B and purifying.
(C) The single-stranded nucleic acid scaffold is ligated at the specific modification site by means of the recognition nucleic acid specific modification module by incubating the fusion protein comprising the recognition nucleic acid specific modification module with the nucleic acid comprising the single-stranded nucleic acid scaffold with the cell in sequence, under appropriate conditions at the modification site.
Further, the single-stranded nucleic acid scaffold assembled with the recognition nucleic acid specific modification module is incubated with the cells after the fixation and permeation treatment, thereby grafting the single-stranded nucleic acid scaffold at the specific modification site by the following specific methods:
(a) Constructing a module for identifying specific modification of nucleic acid, designing and constructing a fusion protein expression plasmid containing the modification identification module A and the connecting module A, and purifying after protein expression.
(B) Constructing a single-stranded nucleic acid scaffold, designing and solid-phase synthesizing the nucleic acid comprising the single-stranded nucleic acid scaffold B and the connecting component B, and purifying.
(C) Mixing the products of steps (a) and (B), and combining the recognition nucleic acid-specific modified module with the single-stranded nucleic acid scaffold under the specific interaction of the connection module A and the connection module B to form the single-stranded nucleic acid scaffold assembled with the recognition nucleic acid-specific modified module.
Further, the single-stranded nucleic acid scaffold B comprises a 5 'complementary pairing region complementarily pairing with the 5' of the single-stranded nucleic acid primer-1, and a3 'complementary pairing region complementarily pairing with the 3' of the single-stranded nucleic acid primer-1.
Further, the single-stranded nucleic acid primer-1 in step (2) may be a single-stranded DNA, a single-stranded RNA, a single-stranded LNA with or without a nucleoside modification or a phosphate backbone modification.
Further, the single-stranded nucleic acid primer-1 described in step (2) comprises the following portions:
a 5' complementary pairing region complementary to a corresponding region of the single-stranded nucleic acid scaffold;
A primer complementary pairing region which can be complementarily paired with the corresponding region of the single-stranded nucleic acid primer-2;
nucleic acid targeting region 1, which is complementarily paired with a specific target nucleic acid;
the 3' complementary pairing region can be complementarily paired with the corresponding region of the single-stranded nucleic acid scaffold.
Further, the single-stranded nucleic acid primer-2 in the step (2) may be a single-stranded DNA, a single-stranded RNA, a single-stranded LNA with or without a nucleoside modification or a phosphate backbone modification.
Further, the single-stranded nucleic acid primer-2 described in step (2) comprises the following portions:
a primer complementary pairing region which can be complementarily paired with the corresponding region of the single-stranded nucleic acid primer-1;
nucleic acid targeting region 2-a region that can be complementarily paired with a specific target nucleic acid.
Further, the condition for combining the single-stranded nucleic acid primer-1 and the single-stranded nucleic acid primer-2 with the target nucleic acid in the step (2) is that 1-1000 nM single-stranded nucleic acid primer-1, 1-1000 nM single-stranded nucleic acid primer-2, formamide, SSC mixed solution and cell sample are incubated and hybridized overnight at 30-50 ℃.
Further, the ligase in the step (3) may be single-stranded DNA ligase, double-stranded DNA ligase, single-stranded RNA ligase or double-stranded RNA ligase. The conditions for the cyclization of the ligase are that the reaction is carried out for 1.5 to 5 hours at room temperature, and if the reaction temperature is low, the reaction time can be prolonged appropriately.
Further, the rolling circle amplification condition in the step (3) is that the nucleic acid polymerase reacts for 3-8 hours at the temperature of 30-50 ℃. The nucleic acid polymerase required for rolling circle amplification may be DNA polymerase or RNA polymerase, including Phi29DNA polymerase, equiPhi DNA polymerase, DNApolymerase, T7 RNApolymerase, SP RNApolymerase, T3 RNApolymerase, etc.
Further, the condition of the fluorescent probe incubation in the step (4) is that 0.1-10 mu M of fluorescent group modified single-stranded nucleic acid probe, formamide, SSC mixed solution and cell sample are incubated for 0.5-3 hours at room temperature.
The invention has the beneficial effects that:
The invention provides an in-situ imaging method capable of realizing visualization of specific modification of target nucleic acid in cells, and by simultaneously providing targeting of specific modification and specific nucleic acid sequence, accurate positioning of nucleoside modification in specific nucleic acid is realized, research on nucleoside modification by researchers can be promoted, and understanding of nucleoside modification is deepened. The method can finish the targeting of specific modification and specific nucleic acid sequence and form a rolling circle amplification signal amplification system by only three nucleic acid probes (single-stranded nucleic acid bracket, single-stranded nucleic acid primer-1 and single-stranded nucleic acid primer-2), and has the characteristics of simplicity, convenience and economy and extremely high commercialization potential.
The targeting of nucleic acid is easily interfered by nonspecific binding, and the method utilizes the single-stranded nucleic acid primer-1 and the single-stranded nucleic acid primer-2 to double-target specific nucleic acid sequences, so that the specificity of the method is greatly improved. In this method, single-stranded nucleic acid primer-1 is used as a template for rolling circle amplification, single-stranded nucleic acid primer-2 is used as a primer for rolling circle amplification, and rolling circle amplification is not performed in a non-specifically bound nucleic acid because the two components cannot be aligned only when both are simultaneously recruited to a specific nucleic acid. This design greatly reduces background noise, making the method very high in signal-to-noise ratio. Due to the high specificity of the method, the method can realize simultaneous imaging of multiple target nucleic acid specific modifications or simultaneous imaging of multiple modifications of the same nucleic acid in cells by designing multiple probes, and has wide application value.
The invention utilizes primer design to improve imaging specificity to identify target nucleic acid specific modification sites, provides sequence information and space information of the target nucleic acid specific modification sites, can realize simultaneous imaging of multiple nucleic acids and nucleic acid imaging with different subspace positioning, and makes the method a multifunctional solution which can be applied to multiple scenes.
In addition, the method can be compatible with protein immunofluorescence staining, realizes the modification of specific nucleic acid and the simultaneous positioning of specific protein in unified cells, breaks the barrier between nucleic acid research and protein research, and provides a feasible solution for researching the relationship between specific modification on target nucleic acid and protein physiological function from a spatial angle.
Drawings
FIG. 1 is a schematic diagram of an in situ imaging method for visualizing a specific nucleic acid modification of a target nucleic acid in a cell, as exemplified by the m 6 A modification on RNA, according to one embodiment of the present invention;
FIG. 2 is an in vitro validation of the eukaryotic expression of YTH-HaloTag fusion proteins;
FIG. 3 is a screen result of YTH-HaloTag optimal incubation conditions;
FIG. 4 is a schematic representation of the efficiency of YTH-HaloTag incubation in HeLa cells;
FIG. 5 is a schematic diagram of construction and application of a single-stranded nucleic acid scaffold recognizing a specific nucleic acid modification module in a technical route of antibody-protein A-streptavidin-biotin-RNA as a single-stranded nucleic acid scaffold recognizing a specific nucleic acid modification module;
FIG. 6 shows the results of denaturing polyacrylamide gel electrophoresis and Coomassie brilliant blue staining of protein A and streptavidin fusion proteins;
FIG. 7 shows the results of non-denaturing gel electrophoresis and 2D gel electrophoresis for binding verification of fusion proteins to antibodies;
FIG. 8 is a Dot-blot validation result of binding of RNA, antibodies and fusion proteins;
FIG. 9 is the results of gel migration experiments for antibodies, fusion proteins, and biotin-RNA binding;
FIG. 10 is a demonstration of the specificity of the invention in HeLa cells.
FIG. 11 is an image of a specific m 6 A site on RNA (ACTB and MALAT 1) with different subcellular localization within HeLa cells;
FIG. 12 is a schematic representation of an ACTB RNA imaging probe design;
FIG. 13 is a schematic representation of co-localization of the m 6 A modification site on ACTB and the ACTB RNA at that site in HeLa cells;
FIG. 14 is a variation of specific m 6 A modification sites within heat-shock treated MEF cells;
FIG. 15 is a change in modification site after modification knockdown treatment of specific m 6 A site on target ACTB RNA in HeLa cells;
FIG. 16 is a co-imaging of specific m 6 A and protein immunofluorescence on LINE-1RNA of interest in HeLa cells;
FIG. 17 is a graph of simultaneous imaging of multiple nucleic acid sites in HeLa cells and protein immunofluorescence co-imaging.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. They are not to be construed as limiting the scope of the invention.
The invention provides an in-situ imaging method capable of realizing visualization of specific modification of target nucleic acid in cells, and by simultaneously providing targeting of specific modification and specific nucleic acid sequence, accurate positioning of nucleoside modification in specific nucleic acid is realized, research on nucleoside modification by researchers can be promoted, and understanding of nucleoside modification is deepened. The tool can complete the targeting of specific modification and specific nucleic acid sequence and form a rolling circle amplification signal amplification system only by three nucleic acid probes (a single-stranded nucleic acid bracket, a single-stranded nucleic acid primer-1 and a single-stranded nucleic acid primer-2 which are assembled with a specific modification component for identifying nucleic acid), and has the characteristics of simplicity, convenience, economy and extremely high commercialization potential.
In the three nucleic acid probes of the invention, the single-stranded nucleic acid bracket assembled with the specific modification component for identifying the nucleic acid specifically comprises four functional components of the modification identification component A, the connection component B and the single-stranded nucleic acid bracket B, but the mode for realizing the functions is flexible.
Taking the modification of m 6 A nucleic acid as an example, it is possible to use the YTH domain of the m 6 A modified binding protein as modification recognition module A, fusion of the HaloTag protein (YTH-HaloTag) as linker module A, dibenzocyclooctene (DBCO-Halolinker) with halogen modification as linker module B, and single-stranded DNA with azide modification (i.e.single-stranded nucleic acid scaffold B) to form a single-stranded nucleic acid scaffold equipped with recognition nucleic acid specific modification module.
Fig. 1 is a schematic diagram of the technical route.
FIG. 2 shows the results of in vitro verification after eukaryotic expression of the intermediate linker YTH-HaloTag, which is required for the technical route. Wherein (A) is the result of polyacrylamide gel electrophoresis of YTH-HaloTag fusion protein, the band of YTH-HaloTag is between 50kDa and 75kDa, which is consistent with the real molecular weight 59kDa of the protein, and (B) the YTH-HaloTag, DBCO-Halolinker connecting component and Azide-488 are incubated in vitro, and polyacrylamide gel electrophoresis and membrane transfer are carried out, so that the YTH-HaloTag protein is verified to have the connecting activity, and the specificity of the connecting mode is demonstrated by a control group.
The preferred results of this technical route on protein incubation conditions are shown in FIG. 3. Wherein (a) is incubated under 50mM HEPES (ph=7.9), (B) is incubated under 25mM MES (ph=6.4), and (C) a solution with neutral pH is used as the incubation conditions. From the results, incubation conditions of 25mM MES (pH=6.4) had the best YTH-HaloTag binding efficiency.
As shown in fig. 4, the control test of YTH-HaloTag binding efficiency was performed using the optimal incubation conditions, i.e., 25mM MES (ph=6.4), and the experimental group showed the highest binding efficiency from the results.
The following examples will illustrate the manner in which the tool is used, taking the holder as an example.
Alternatively, an antibody targeting the modification of m 6 A may be used as the modification recognition module A, a fusion protein of protein A which specifically binds to the antibody with streptavidin may be used as the linking module A, and RNA with biotin modification may be used as the linking module B and single-stranded nucleic acid scaffold B to form a single-stranded nucleic acid scaffold recognizing the specific modification module of nucleic acid. This stent has been experimentally verified to determine feasibility.
FIG. 5 is a schematic diagram showing construction and application of a single-stranded nucleic acid scaffold for identifying specific modification components of nucleic acids in the technical scheme. First, a fusion protein of protein A and streptavidin was constructed, and then RNA with biotin was synthesized and bound thereto. In use, the m 6 a modification is first targeted with an antibody, and then RNA is ligated to the m 6 a modification using protein a-antibody interactions.
FIG. 6 shows the results of protein expression of fusion proteins of protein A and streptavidin. After expression in an E.coli expression system, purifying by using a Ni column and a molecular sieve column to obtain target protein, and characterizing the purification result by using the Ni column and the molecular sieve column by using denaturing polyacrylamide gel electrophoresis and coomassie brilliant blue staining. The purified streptavidin fusion protein forms tetramers which cannot be completely denatured, and thus tetramer bands appear in denaturing polyacrylamide gel electrophoresis. From the result of denaturing polyacrylamide gel electrophoresis, the fusion protein of protein A and streptavidin was successfully obtained.
FIG. 7 is a diagram showing the binding verification of the fusion protein to the antibody. The rabbit IgG is used for representing the antibody, the antibody is combined with the fusion protein, and then non-denaturing gel electrophoresis is carried out, so that the antibody band is seen to be upwards moved, and the fusion protein band is light, which represents the combination phenomenon of the antibody band and the fusion protein band. Then, 2D gel electrophoresis was performed to further verify that the bound antibody bands were successfully separated from the fusion protein bands using denaturing gel electrophoresis in the second direction, and the binding was verified.
Dot-blot verification of binding of RNA, antibodies and fusion proteins is shown in FIG. 8. RNA with modification m 6 A was bound to cellulose membrane, incubated with antibody targeting m 6 A, followed by addition of fusion protein and imaging of signal with biotin with HRP. Using RNA without the m 6 a modification as a control, antibodies were confirmed to be specific for the m 6 a modification and the fusion protein could bind to the antibody normally. Using a secondary HRP-bearing antibody targeting the m 6 a antibody as a control, the binding capacity of the fusion protein to the antibody was confirmed to be similar to that of the secondary antibody, verifying the feasibility of RNA-antibody-fusion protein binding.
FIG. 9 shows gel migration experiments of antibodies, fusion proteins, and biotin-RNA binding. And respectively combining biotin-RNA, biotin-RNA+fusion protein and rabbit IgG, and then carrying out gel migration experiments to confirm the feasibility of combining the biotin-RNA-fusion protein and rabbit IgG.
FIG. 10 is a graph showing the imaging specificity verification of the invention in HeLa cells. Any connectors and probes in the experimental set were correct and complete. One of the control groups lacks a single-stranded nucleic acid scaffold (DNASPLINT), and the sequence of one single-stranded nucleic acid Primer-1 (Primer-1) is mismatched with the target RNA sequence. Imaging results showed that only the experimental group had obvious signal points, showing the specificity of the invention in imaging.
FIG. 11 is a graph showing the imaging effect of specific modifications on target RNAs with different subcellular localization using the present invention in HeLa cells. The subcellular localization reflected by imaging is consistent with literature reports, and the m 6 A modification site on ACTB is concentrated in the cytoplasm and the m 6 A modification site on MALAT1 is concentrated in the nucleus.
FIG. 14 is a graph showing the visualization of the change in modification sites after knockdown treatment for modification of a specific m 6 A site (chr 7: 5527743) on ACTB RNA in HeLa cells using the method of the present invention. From the figure, the methylation modification at a specific site is knocked down, and imaging signal points are reduced, so that the methylation level can be qualitatively measured by the method.
FIG. 15 is a schematic diagram showing the visualization of the change in the m 6 A site (chr 17: 34958253) on Hspa1b RNA in MEF cells after heat shock treatment using the method of the present invention. From the figure, it is clear that the methylation level increases after heat shock treatment, consistent with the modification changes before and after heat shock at the site reported in the literature.
Example 1 Co-localization visualization of a specific m 6 A site on target ACTB and ACTB RNA at which the site is located in HeLa cells
1. YTH-HaloTag fusion protein construction and expression
The construction of YTH-HaloTag plasmid, which is characterized in that FlagTag is arranged at the N end for subsequent purification, a HaloTag sequence is added at the downstream of FlagTag, a flexible segment composed of 15 amino acids is arranged at the downstream of the HaloTag sequence, GSSGGGGSGGGGSSG, the interference of two functional regions of fusion protein is avoided, and YTH structural domains from YTHDF are added at the downstream of the flexible segment. The above sequences were constructed into pcDNA3 vectors to allow expression of the proteins in eukaryotic systems. The correctness of the protein bands was checked with coomassie brilliant blue. Storage conditions 50% glycerol was stored at-20 ℃;
2. chemical synthesis of DBCO-Halolinker
DBCO-Halolinker serves as an intermediate linking component, the HaloTag ligand Halolinker portion of which is used for linking to HaloTag, and the DBCO portion is used for linking to single-stranded nucleic acid scaffold B with azide modification. The preparation method of DBCO-Halolinker is divided into two steps. In the first step, 4- (11, 12-didehydrodibenzo [ b, f ] azaoct-5 (6H) -yl) -4-oxo-butyrate (compound 1) is synthesized by mixing 5- (11, 12-didehydrodibenzo [ b, f ] azaoct-5 (6H) -yl) -4-oxo-butyric acid, N-hydroxysuccinimide and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride under the protection of nitrogen, and adding an anhydrous dichloromethane solvent for dissolution. The reaction mixture was stirred at room temperature for 3.5 hours. The reaction solution was washed with 5% aqueous citric acid, 5% aqueous sodium bicarbonate and saturated brine in this order. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. Compound 1 was obtained by a silica gel column purification method. And characterizing the product by nuclear magnetism and mass spectrometry to obtain a correct pure product, and then carrying out the next reaction. In the second step, the compound 1, N-Diisopropylethylamine (DIPEA) and 2- (2- (6-chlorohexyloxy) ethoxy) ethylamine hydrochloride were dissolved in DMF, reacted at room temperature for 20 minutes, washed four times with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. DBCO-Halolinker is obtained by a silica gel column purification method. Nuclear magnetism and mass spectrometry means characterize DBCO-Halolinker.
The first step of chemical synthesis reaction formula:
The second step of chemical synthesis reaction formula:
3. imaging nucleic acid primer design
Co-ACTB-Primer and Co-ACTB-pad, wherein Co-ACTB-pad has a phosphorylation modification, and can be looped under the action of a splint of Co-ACTB-Primer as shown in FIG. 12, and primers for imaging the target m 6 A site (chr 7: 5527602) on ACTB RNA, namely single-stranded nucleic acid Primer-1, single-stranded nucleic acid Primer-2, named ACTB-Primer-1 and ACTB-Primer-2 in this example, wherein ACTB-Primer-1 has a phosphorylation modification, and can be looped under the action of a splint of single-stranded nucleic acid scaffold (named DNA SPLINT), wherein DNA SPLINT has an azide modification and a base dT (preventing rolling circle amplification of DNASPLINT), and hybridization patterns of DNA SPLINT, ACTB-Primer-1 and ACTB-Primer-2 are shown in FIG. 1. The primer sequences and modifications are shown in Table 1.
TABLE 1ACTB RNA and m 6 A site imaging primers
4. Pretreatment of cell samples
This example relates to the use of RNA as nucleic acid, and in order to inhibit RNA degradation, nuclease-FREE WATER (DEPC treated water) was used instead of deionized water, and RNase inhibitor was added from this step to the reaction.
(1) HeLa cells were seeded onto confocal dedicated dishes, the example used an 8-well dish, so that following treatment all followed by 100-200. Mu.L of the system, medium was aspirated when the culture was 80% full, and the medium was removed by washing twice with PBS to terminate the culture. The cell sample detergents required for this example are shown in Table 2;
(2) Cells were fixed with 4% Paraformaldehyde (PFA) at room temperature, PFA was removed after 15 min, and fixation was terminated by washing twice with 200 μl PBS;
(3) Adding pre-cooled methanol at-80 ℃ to further strengthen the fixation of cytoplasm and intima structure, removing methanol after 10 minutes, and washing twice with PBSR to terminate;
(4) Cells were permeabilized with 0.2% Triton-X100 at room temperature, after 10 minutes 0.2% Triton-X100 was removed and permeabilized was stopped by washing twice with PBSR;
TABLE 2 cell sample detergents
5. DNA SPLINT grafting
(1) Incubating the pretreated cell sample with the YTH-HaloTag fusion protein at room temperature, washing off the excess YTH-HaloTag with PBSR after 1 hour, so that the m 6 A modification site is recognized by the YTH-HaloTag and binds to the on-band HaloTag, and the incubation system is shown in Table 3;
TABLE 3 protein incubation System (200. Mu.L System)
(2) Incubating the cell sample with DBCO-Halolinker at 37℃and washing off excess DBCO-Halolinker with PBSR after 0.5 hours, dehalogenating Halolinker with HaloTag to bring the DBCO group to the m 6 A modification site, the reaction system being shown in Table 4;
TABLE 4DBCO-Halolinker reaction System (200. Mu.L System)
(3) DNA SPLINT is added, the azide and DBCO are subjected to click reaction, and finally successful grafting of the single-stranded DNA branched chain is realized on the m 6 A modified site, the reaction system is shown in Table 5, and the single-stranded DNA branched chain is washed twice for 5 minutes each time by PBSR, so that the interference of the unbound single-stranded DNA branched chain on the subsequent results is avoided;
TABLE 5 azide-modified DNA branched reaction System (100. Mu.L System)
(4) Optional procedure the sample can be optionally secondarily immobilized with 4% PFA at room temperature for 5min to avoid the subsequent overnight reaction from decrosslinking.
6. Identification of target RNA sequences
The mixed solution with the probe is hybridized with the cell sample overnight, and the reaction is carried out for 8-12 hours at 40 ℃, and the hybridization system is shown in Table 6
TABLE 6 Probe overnight hybridization System (200. Mu.L System)
7. Rolling circle amplification template cyclization
The overnight hybridized cell samples were washed twice with PBSR a 10 min each to remove unbound probes. The reaction system containing T4 DNA ligase was added to the washed cell sample at 200. Mu.L per well. The reaction was gently swirled at room temperature for 2 hours. If it is desired to lengthen the reaction time, the temperature of the ligation reaction may be appropriately lowered. The ligation reaction system containing T4 DNA ligase is shown in Table 7;
TABLE 7T4 DNA ligation System (200. Mu.L System)
8. Rolling circle amplification
The cyclized cell sample was washed twice with PBSR to remove the ligation system. A rolling circle amplification system containing Phi29 DNA polymerase was added to the cell samples at 200. Mu.L per well at 30℃for 5 hours. Rolling circle amplification system containing Phi29 DNA polymerase is shown in Table 8.
TABLE 8 Rolling circle amplification System (200. Mu.L System)
9. In situ fluorescent probe hybridization
The rolling circle amplified cell samples were washed 2 times with PBS. And incubating the cell sample by using a probe modified by a fluorescent group, and incubating for 0.5-1 hour at room temperature with slight shaking. The cell samples after the fluorescent probe addition are all required to be protected from light in the subsequent process. The incubation system with fluorescent probes is shown in table 9.
TABLE 9 fluorescent probe hybridization System (200. Mu.L System)
10. Laser confocal microscope observation
The fluorescence hybridized cell samples were washed twice with PBST for 5 minutes each. Nuclei were stained with 1. Mu.g/mL 4', 6-diamidino-2-phenylindole (DAPI) for 10min at room temperature and washed with PBST for two 5 min. Observations were made with a confocal laser microscope. The imaging effect is shown in figure 13. The m 6 A modification on most ACTB RNA and ACTB RNA have a co-localization relationship in space, which accords with the imaging expectation.
Example 2 subcellular localization imaging and protein immunofluorescence Co-imaging of specific m 6 A sites on LINE-1RNA of interest in HeLa cells
1. Imaging primer design
The primers used in this example were LINE-1Primer-1 and LINE-1Primer-2, which image the m 6 A site of interest (chr 19: 1948573) on LINE-1 RNA. The primer sequences and modifications are shown in Table 10.DNA SPLINT probe sequences were as in example 1.
TABLE 10LINE-1RNA m 6 A site imaging primer
2. Pretreatment of cell samples as in example 1
3. Grafting with DNA SPLINT in example 1
4. Identification of target RNA sequences
The mixed solution with the probe was hybridized with the cell sample overnight, and reacted at 40℃for 8-12 hours, and the hybridization system is shown in Table 11.
TABLE 11 probe overnight hybridization System (200. Mu.L System)
5. Cyclization with Rolling circle amplification template of example 1
6. Rolling circle amplification as in example 1
7. Hybridization of in situ fluorescent probes in example 1
8. Protein immunofluorescence imaging
Immunofluorescence imaging was performed on SAFB proteins in this example. The immunofluorescence co-imaging principle of this example is shown in fig. 16. The method mainly comprises the following steps:
(1) The cell sample hybridized with the fluorescent probe was washed twice with PBST for 5 minutes to wash off the excess free fluorescent probe.
(2) Cell samples were blocked with 6% Bovine Serum Albumin (BSA) for 1 hour at room temperature.
(3) Primary antibody SAFB Rabbit pAb was added and incubated overnight at 4 ℃ with gentle shaking. Cell samples after overnight washing with PBST were washed three times for 5 minutes each to remove non-specific binding.
(4) The secondary antibody with a fluorescent group (Alexa Fluor 647donkey anti-Rabbit IgG) was diluted 1:200 with 1% BSA and incubated for 1 hour at room temperature. The cell samples were washed three times with PBST for 5 minutes each, and non-specifically bound secondary antibodies were washed away.
9. Laser confocal microscope observation
As in example 1. The imaging effect is shown in figure 16. It can be seen that SAFB proteins are located in the nucleus and LINE-1RNA is also mainly located in the nucleus, and the subspace distribution of both are consistent with literature reports. The scheme can be used for researching the spatial position relation between nucleic acid and protein.
EXAMPLE 3 Simultaneous imaging of multiple nucleic acid sites in HeLa cells and protein immunofluorescence Co-imaging
1. Imaging primer design
Primers required for this example were HPS6 Primer-1 and HPS6 Primer-2, which image the m 6 A site of interest (chr 10: 102066921) on HPS6 RNA. Primers for imaging the m 6 A site (chr 10: 72275238) of interest on DDIT4 RNA, DDIT4 Primer-1 and DDIT4 Primer-2. The primer sequences and modifications are shown in Table 12.DNA SPLINT probe sequences were as in example 1.
TABLE 12 Simultaneous imaging primers for different nucleic acid sites
2. Pretreatment of cell samples as in example 1
3. Grafting with DNA SPLINT in example 1
4. Identification of target RNA sequences
The mixed solution with the probe was hybridized with the cell sample overnight, and reacted at 40℃for 8-12 hours, and the hybridization system is shown in Table 13.
TABLE 13 probe overnight hybridization System (200. Mu.L System)
5. Cyclization with Rolling circle amplification template of example 1
6. Rolling circle amplification as in example 1
7. In situ fluorescent probe hybridization
The incubation system with fluorescent probes is shown in table 14.
TABLE 14 fluorescent probe hybridization System (200. Mu.L System)
8. Protein immunofluorescence imaging
The present example performed immunofluorescence imaging of YTH-HaloTag proteins. Since YTH-HaloTag protein has Flag tag, the primary antibody is selected Flag tag Mouse McAb, and the secondary antibody is selected Alexa Fluor Plus 488goat anti-Mouse IgG, and the specific operation procedure is the same as that of the protein immunofluorescence imaging of example 2.
9. Laser confocal microscope observation
As in example 1. The imaging effect is shown in figure 17. Different RNAs can be seen to have different imaging signal points, representing that they contain different rates of modification, and specific spatial localization can be seen in the magnified view. And together with immunofluorescence, show that the protocol allows simultaneous investigation of the interrelationship of different nucleic acids, interrelationship of nucleic acids and proteins in the same sample.
Claims (5)
1. An in situ imaging method capable of realizing visualization of specific m 6 a modification of target RNA in cells, comprising the following steps:
(1) Grafting the single-stranded nucleic acid bracket of the modification site, namely incubating the single-stranded nucleic acid bracket assembled with the recognition RNA specific modification component with the fixed and permeabilized cells, or incubating the recognition RNA specific modification component with the permeabilized cells and then incubating the recognition RNA specific modification component with the single-stranded nucleic acid bracket, so that the single-stranded nucleic acid bracket is grafted at the specific modification site to obtain the treated cells;
(2) The identification of target RNA, namely incubating a single-stranded nucleic acid primer-1 and a single-stranded nucleic acid primer-2 which can specifically target a target RNA sequence with the cells treated in the step (1), so that the single-stranded nucleic acid primer-1 and the single-stranded nucleic acid primer-2 are combined with the target RNA;
(3) Signal amplification, namely cyclizing the single-stranded nucleic acid primer-1 by ligase with the aid of a single-stranded nucleic acid bracket grafted to a modification site, and then performing rolling circle amplification by taking the single-stranded nucleic acid primer-1 as a primer extension template of the single-stranded nucleic acid primer-2 to amplify a signal of specific modification of target RNA in a cell in situ;
(4) Signal visualization, namely incubating the single-stranded nucleic acid primer-1 hybridized fluorescent probe with the amplified cell of the rolling circle, capturing a bright signal point which is obviously different from a background signal under a laser confocal microscope, and reflecting subcellular space positioning of a specific m 6 A modification site on target RNA;
in step (1), the single-stranded nucleic acid scaffold equipped with the recognition nucleic acid specific modification module comprises the following modules:
a modification recognition component a for recognizing and binding to a specific nucleic acid modification;
A single-stranded nucleic acid scaffold B complementarily paired with the head-to-tail sequence of the single-stranded nucleic acid primer-1 for assisting the ligase to circularize the single-stranded nucleic acid primer-1;
A connecting component A which is directly combined with the modification recognition component A to form a component for recognizing specific modification of nucleic acid;
A connecting component B which is directly combined with the single-stranded nucleic acid scaffold B to form a single-stranded nucleic acid scaffold, and is combined with the connecting component A to combine the component for identifying the specific modification of the nucleic acid with the single-stranded nucleic acid scaffold;
the construction method of the single-stranded nucleic acid scaffold assembled with the recognition nucleic acid specific modification component comprises the following steps:
(1) Constructing a module for identifying specific modification of nucleic acid, namely designing and constructing a fusion protein expression plasmid containing the modification identification module A and the connecting module A, and purifying after protein expression;
(2) Constructing a single-stranded nucleic acid scaffold, namely designing and solid-phase synthesizing the single-stranded nucleic acid scaffold B comprising the connecting component B and directly purifying, or connecting the single-stranded nucleic acid scaffold B with the connecting component B and purifying;
(3) Mixing the products obtained in the steps (1) and (2), and combining the component for identifying the specific modification of the nucleic acid with the single-stranded nucleic acid bracket under the specific interaction of the connecting component A and the connecting component B to form the single-stranded nucleic acid bracket assembled with the component for identifying the specific modification of the nucleic acid;
The modification recognition component A is YTH, the single-stranded nucleic acid scaffold B is Splint, the connecting component A is HaloTag, and the connecting component B is DBCO-Halolinker.
2. The method of claim 1, wherein the imaging comprises single imaging, simultaneous imaging of multiple nucleic acids, simultaneous imaging of multiple modifications, different subspace localization imaging, or co-imaging with protein immunofluorescence.
3. An in situ imaging method for enabling visualization of intracellular target RNA specific m 6 a modifications according to claim 1, wherein the single stranded nucleic acid scaffold comprises the following components:
a 5 'complementary pairing region complementary to the 5' sequence of the single-stranded nucleic acid primer-1;
3 'complementary pairing region complementary pairing with 3' sequence of single stranded nucleic acid primer-1.
4. The method of in situ imaging for visualizing a specific modification of an intracellular target nucleic acid of claim 1, wherein in step (2), the single stranded nucleic acid primer-1 comprises:
A 5' complementary pairing region complementary to a corresponding region of the single-stranded nucleic acid scaffold;
a primer complementary pairing region complementary pairing with the corresponding region of the single-stranded nucleic acid primer-2;
a nucleic acid targeting region complementarily paired with a specific target nucleic acid;
And 3' complementary pairing region complementary pairing with corresponding region of single-stranded nucleic acid scaffold.
5. The method of in situ imaging for visualization of intracellular target RNA specific m 6 a modifications of claim 1, wherein in step (2), single stranded nucleic acid primer-2 comprises the following parts:
a primer complementary pairing region complementary pairing with the corresponding region of the single-stranded nucleic acid primer-1;
Nucleic acid targeting region, complementary pairing to a specific target nucleic acid.
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