Space-time resolution single cell m6A dynamic modification analysis method and application thereof

Abstract

The invention discloses a space-time resolution single cell m6A dynamic modification analysis method and application thereof, and the method comprises the following steps: 1, using propargyl-L-selenium homocysteine (PSH) to carry out metabolism labeling on living cells, so that the m6A modification site newly modified by RNA is specifically introduced into a propargyl functional group; 2, covalently linking a DNA probe with an azide group to the propargyl functional group through a click chemical reaction; step 3, performing signal amplification on the DNA probe through an adjacent connection and rolling circle amplification technology; and 4, carrying out space imaging by adopting a fluorescence in-situ hybridization or fluorescence in-situ sequencing technology so as to realize space-time dynamic analysis on m6A modification in the single cell. The method disclosed by the invention has space-time resolution capability on m6A dynamic modification.

Description

Technical Field

[0001] This invention relates to a spatiotemporally resolved single-cell m 6 A dynamic modification analysis method. Background Technology

[0002] In the 1970s, N 6 -Methyladenosine (N 6 -methyladenosine, m 6 A) Modifications were first discovered, and with advancements in specific antibody technology and high-throughput sequencing technology, m-modifications have been found in almost all RNA types. 6 A modification. Next-generation sequencing (NGS) technology revealed m 6 The distribution of A in the transcriptome reveals a crucial role in the regulation of mRNA in eukaryotes. 6 The methylation process of protein A involves three main participants: methyltransferases, demethylases, and methylation writers. Methyltransferases (Writers) are primarily driven by a complex composed of METTL3 / 14. 6 In RNA modification, METTL3 catalyzes methylation via S-adenosylmethionine (SAM) as a methyl donor, selectively inducing methylation of the GAC and AAC base sequences. Demethylases FTO and ALKBH5, on the other hand, can remove m... 6 A modifies. Additionally, m 6 Readers, such as the YTH domain protein family, can recognize m 6 A modifiers and regulate gene expression, playing a crucial role in cellular function.

[0003] Research m 6 Methods involving A-base modification can be broadly categorized into two types: sequencing-based and spatial imaging-based. Sequencing-based methods include antibody-based methods, enzyme digestion methods, enzyme conversion methods, and chemical modification methods, which can provide m-base resolution. 6 A. Modification information and modification abundance. Imaging-based methods (such as DART-FISH, m...) 6 A-PHPEA, PREEM, DSA-m 6 (A et al.), utilizing antibody enrichment or APO1-YTH protein expression-dependent targeting technologies, can provide m 6 Static information on the modification site and its spatial location. However, current methods for detecting and quantifying m... 6 Most techniques that modify A can only obtain data at a specific point in time, m. 6 Static information modified by A cannot truly reflect m. 6The A modification exhibits dynamic changes across time and space, and the method's sensitivity is low due to the antibody recognition site. Therefore, for m 6 Precise localization and spatiotemporal dynamics of A-modification remain challenging to study. Mapping more precise m... 6 A modified spatiotemporal dynamic map enables in-depth analysis of m in cells. 6 RNA regulation related to A is of great significance in relation to related diseases.

[0004] In recent years, researchers have developed a variety of m 6 A modified imaging method (such as DART-FISH, m) 6 A-PHPEA, PREEM, DSA-m 6 A). Where m 6 AISH-PLA technology has, for the first time, achieved single-molecule m 6 A spatial imaging study of RNA was conducted, and the effects of heat shock stress on m were investigated. 6 Changes in A modification levels. This method utilizes proximity probes and antibody recognition to target m, respectively. 6 A specific RNA sequence and m 6 A methylation site was then subjected to neighbor-to-neighbor ligation and in situ rolling circle amplification (RCA) to reveal m at single-base resolution in individual cells. 6 The spatial location of RNA. The main technical solution of this method is as follows:

[0005] m 6 AISH-PLA is used for m 6 RNA imaging: (1) Fix cells in blocking buffer. (2) Prepare for oligonucleotide hybridization. (3) Ligation reaction. (4) Perform rolling circle amplification (RCA) reaction. (5) Stain cell nuclei, and then perform confocal microscopy imaging.

[0006] However, this method has the following shortcomings:

[0007] 1. No temporal resolution information

[0008] Because this method relies on antibody recognition m 6 A-modified sites cannot distinguish between newly modified sites and existing m-modified sites. 6 Modified sites (A) can only provide spatial distribution information at a single time point, but due to their inherent "snapshot" characteristics and quantitative limitations caused by antibody binding, they are difficult to provide m... 6 A reliable information on the rate of methylation modification, temporal changes, or dynamic responses to short-term stimuli.

[0009] 2. The method has low sensitivity and high cost.

[0010] This method is used in the study of m 6A has certain limitations in terms of dynamic modification and random characteristics, and antibody recognition m 6 The A-site method has drawbacks such as low sensitivity and high cost, therefore there is an urgent need to develop new, low-cost, highly sensitive, and efficient methods for detecting m. 6 A modifies.

[0011] 3. Can only detect a single site.

[0012] m 6 AISH-PLA is limited by its probe structure design. Although different fluorescence channels can be used for differentiation, the number of channels is limited, and the spectral overlap of each channel and the microscopic imaging conditions also limit the number of parallel labels. Therefore, it can only detect a single site in the same sample and cannot simultaneously detect multiple sites of multiple genes in the same sample.

[0013] First, the overall processing steps of this technology are numerous, cumbersome, and complex, requiring a considerable amount of time and potentially leading to RNA degradation, thus increasing the risk of failure. Second, this technology requires the use of dual antibodies—anti-m antibodies. 6 Antibody A and anti-rabbit antibodies have high experimental costs and low sensitivity; finally, this technology can only provide spatial information at a certain moment, lacking temporal resolution information, and can only obtain m 6 A single piece of information about a modified site is insufficient to detect multiple sites in the same sample simultaneously.

[0014] 4. The processing steps are complex.

[0015] m 6 AISH-PLA combines in situ hybridization (ISH) and neighbor-to-neighbor linkage (PLA) technologies. The process requires the use of anti-m... 6 Incubation with antibodies such as A antibody, anti-rabbit antibody, streptavidin, biotin-labeled neighbor probe-b, neighbor probe-a, circular template-1, and circular template-2 involves numerous and complex processing steps, takes a long time, and is prone to mRNA degradation. Summary of the Invention

[0016] The main objective of this invention is to provide a spatiotemporally resolved single-cell m 6 A dynamic modification analysis method. This aims to solve at least one of the above problems.

[0017] The technical solution of the present invention is as follows:

[0018] A spatiotemporally resolved single-cell m 6 A dynamic modification analysis method includes the following steps:

[0019] Step 1: Metabolic labeling of live cells is performed using propargyl-L-selenocysteine ​​(PSH), which enables the newly modified RNA to form m 6Site A specifically introduces a propargyl functional group; the metabolic marker is used at different time points t2, t3, ..., t... n New modifications m in marker RNA 6 Site A allows for the new modification of RNA to m 6 Site A contains a propargyl functional group, indicating time information; preferably, propargyl-L-selenocysteine ​​(PSH) at a concentration of 0.1 mM to 5 mM is used to perform metabolic labeling of live cells for 30 minutes to 24 hours, allowing newly modified RNA to be labeled. 6 A site-specific introduction of the propargyl functional group;

[0020] Step 2: The DNA probe with the azide group is covalently linked to the propargyl functional group by a click chemical reaction;

[0021] Step 3: Amplify the signal of the DNA probe using proximity ligation and rolling circle amplification techniques;

[0022] Step four: Spatial imaging is performed using fluorescence in situ hybridization or fluorescence in situ sequencing techniques, and the magnified m is observed under a laser confocal microscope. 6 A site signal to indicate m 6 Spatial information of A; thereby enabling the analysis of m within a single cell. 6 Spatiotemporal dynamic analysis of A-modification; labeling each cell m with encoded probes. 6 A, which includes the combination of m 6 The targeting sequence of A and the readout sequence of the fluorescently labeled readout probe; each m 6 A is encoded by a specific combination of readout sequences; using sequential hybridization and imaging, each round uses a different readout probe to identify each m. 6 The A site binds to the readout sequence and decodes the RNA; the readout probe is used to decode the bases, and the fluorescent probe converts the decoded sequence information into a fluorescent signal; at least two probes are matched with the target DNA and then ligated by a ligase to generate fluorescence for imaging.

[0023] Further, in step one, the PSH metabolic labeling step includes:

[0024] Step S1: Starve the cells in a methionine-free medium; preferably for 15 to 60 minutes.

[0025] Step S2: Add PSH for incubation within a specific time window to mark newly occurring m events during that time period. 6 A modification. Preferably, 1 mM to 3 mM of PSH is added for incubation for 2 to 24 hours to mark newly occurring m-type markers during this period. 6 A modifies.

[0026] Further, in step two, the click chemistry reaction is a copper(I)-catalyzed azido-yne cycloaddition reaction. Preferably, the click chemistry reaction is a copper(I)-catalyzed azido-yne cycloaddition reaction, and the reaction conditions are: at 30°C-37°C, using 400 μM to 1 mM ascorbic acid as a reducing agent, and reacting for 20 minutes to 1 hour.

[0027] Furthermore, in step three, the signal amplification step includes:

[0028] Neighbor hybridization was performed using one or more pairs of circular DNA templates; circularization ligation was performed using T4 DNA ligase.

[0029] Rolling circle amplification was performed using Phi29 DNA polymerase to generate DNA nanospheres that could be recognized by fluorescent probes.

[0030] Preferably, the signal amplification step includes:

[0031] Neighborhood hybridization was performed using circular DNA templates at concentrations of 0.05 μM to 0.5 μM at a temperature of 36–38°C for 45 minutes to 2 hours.

[0032] Circularization ligation was performed using T4 DNA ligase at 10 U / μL to 30 U / μL at 36-38°C for 20 minutes to 1 hour.

[0033] Rolling circle amplification was performed using 0.5 U / μL to 2 U / μL of Phi29 DNA polymerase at 36–38°C for 1–3 hours to generate DNA nanospheres that could be recognized by fluorescent probes.

[0034] Furthermore, in step four, fluorescence in situ sequencing is used for multi-target detection, specifically including:

[0035] For different m 6 A probe set with barcode sequences was designed based on the target RNA.

[0036] Perform multiple cycles of hybridization, imaging, and chemical quenching; preferably, perform 3 to 6 cycles of hybridization, imaging, and chemical quenching, each cycle using 50 mM to 100 mM KOH solution to wash for 3 to 10 minutes at room temperature to strip the fluorescent probe;

[0037] By decoding the fluorescence signal sequence, it is possible to detect multiple m in the same sample. 6 Simultaneous identification and spatial localization of site A.

[0038] Furthermore, the barcode of the fluorescence in situ sequencing is encoded by a combination of AG, GA, and TC base pairs.

[0039] This invention also provides the method for studying m in cellular stress response. 6 A. Application in the dynamic control mechanism; characterized in that the application includes any one or more of the following:

[0040] Application in a hypoxic stress model: Cells were cultured in a hypoxic environment, and the method described above was used to analyze the newly modified m at different time points under hypoxic stress conditions. 6 Abundance variations and spatial distribution of site A to reveal the effects of hypoxic stress on m 6 A modifies the dynamic regulatory effect;

[0041] Application in drug screening: The method is applied to cancer model cells or disease model cells, by simultaneously detecting multiple disease-related genes. 6 A modifies the dynamics, plotting m under different drug action times. 6 A-modified spatiotemporal dynamic maps are used to screen for disease treatment drugs.

[0042] The present invention also provides a kit for implementing the method, comprising: propargyl-L-selenocysteine ​​(PSH); a DNA probe with an azide group; a click chemistry reaction buffer and a catalyst; an enzyme and substrate required for rolling circle amplification; and a fluorescent probe required for fluorescence in situ hybridization or sequencing.

[0043] Furthermore, the kit includes a barcode probe set, ligase, and quenching reagent for multi-round fluorescence in situ sequencing.

[0044] In traditional molecular biology research, bulk-level RNA analysis has long been dominant. However, this averaging approach masks the heterogeneity of gene expression at the single-cell level and fails to investigate the dynamic processes of RNA modification within individual cells. Furthermore, m 6 A-modification, the most common internal modification in eukaryotic mRNA, plays a crucial role in RNA splicing, transport, stability, and translation. However, m... 6 The spatiotemporal dynamics of gene A within a single cell, and how it plays a precise role in gene expression regulation, remain largely unknown. To overcome this challenge, this application proposes m... 6 A-Dynam technology—a low-cost, highly sensitive single-cell m 6 A novel RNA multiple spatial resolution detection technology can detect m 6 A combined RNA metabolic labeling strategy and in situ sequencing technology enables single-molecule level mRNA assays in single cells. 6 A highly specific detection of RNA. The advantages of this invention are as follows:

[0045] 1. Achieving spatiotemporal resolution and indicating dynamic modification information through PSH metabolic labeling: First, this technique uses the SAM precursor methionine analogue propargyl-L-selenocysteine ​​(PSH) at different time points t2, t3, ..., t... n New modifications m in marker RNA 6 Site A allows for the new modification of RNA to m 6 Site A contains a propargyl functional group, indicating temporal information; secondly, spatial resolution is achieved through PSH metabolic labeling, proximity linkage (PLA) technology, rolling circle amplification (RCA), and fluorescent probe recognition: This technique generates nanospheres after RCA and incorporates fluorescent probes to induce fluorescence, allowing magnified nanospheres to be observed under a laser confocal microscope. 6 A site to indicate m 6 A's spatial information; this method can observe each cell within a continuous time frame m 6 Monitor and reveal changes in the amount of modification A, and identify new modifications m. 6 The dynamic changes of site A.

[0046] 2. High detection sensitivity: First, the methionine small molecule analog PSH successfully marks the propargyl group at the methylation site of its mRNA by mimicking the natural methylation modification pathway. Then, the newly modified RNA carrying the propargyl functional group... 6 Site A undergoes a click chemistry reaction with a probe tipped with an azide, significantly enhancing m 6 The detection limit at site A is improved, while also reducing non-specific adsorption issues caused by cross-reactivity in traditional antibody-dependent methods; secondly, specificity is further enhanced through the specific design of the probe sequence; this dual protection significantly improves m 6 Limit of detection for signal at site A.

[0047] 3. Achieving heterogeneity in the modification of different genes: Labeling each cell m with encoding probes. 6 A, which includes the combination of m 6 The targeting sequence of A and the readout sequence of the fluorescently labeled readout probe. Each m 6 A is encoded by a specific combination of readout sequences. When using sequential hybridization and imaging, each round uses a different readout probe to identify each m. 6 The A-site binds to the readout sequence and decodes the RNA. The readout probe decodes the bases, and the fluorescent probe converts the decoded sequence information into a fluorescent signal. Two short probes are precisely matched with the target DNA and ligated using T4 ligase to generate fluorescence for imaging. This method allows observation of each cell over a continuous time period... 6 Monitor and reveal changes in the amount of modification A, and identify new modifications m. 6 The dynamic changes of site A.

[0048] 4. A simplified procedure for spatial imaging with single-base resolution was developed: DNA strands containing azide groups were subjected to copper (I)-catalyzed azido-yne cycloaddition (CuAAC) with PSH-labeled m 6 This covalent ligation step replaces the complex multi-step antibody incubation procedure, reducing operational procedures and steps, shortening reaction time, improving RNA stability, and lowering experimental costs. Attached Figure Description

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0050] Figure 1 This is a schematic diagram illustrating the experimental principle of the present invention.

[0051] Figure 2 For hypoxia-induced m 6 Description of A and mRNA site changes analysis.

[0052] Figure 3 For subcellular resolution pulse tracking m 6 Spatial distribution of RNA and nucleocytoplasmic ratio distribution of 15 genes.

[0053] Figure 4 For PSH synthesis steps Detailed Implementation

[0054] Example 1

[0055] like Figure 1 As shown, the method of the present invention is as follows:

[0056] 1) PSH metabolic markers and rolling circle amplification

[0057] This invention uses the SAM precursor methionine analogue propargyl-L-selenocysteine ​​(PSH) to label newly modified m in RNA. 6 Site A. By newly modifying m 6 Metabolic labeling of the A site with a propargyl functional group, using copper (I)-catalyzed azido-acetylic acid cycloaddition (CuAAC), combines the modified azido group single-stranded DNA sequence with PSH-labeled m 6 A covalent connection.

[0058] The specific operation method is as follows: (1) Starvation treatment: human cervical cancer cells (HeLa) slides are starved for 30 min in methionine-free DMEM (Dulbecco's Modified Eagle Medium) medium; (2) PSH metabolic labeling: propargyl-L-selenocysteine ​​(PSH, 1.5 mM) is added for metabolic labeling for 12 h; (3) Fixation: the original medium is discarded, and the slides are washed twice / 5 min with diethyl pyrocarbonate-phosphate buffered saline (DPBS), followed by fixation with 1.6% paraformaldehyde (PFA) for 10 min. After fixation, the paraformaldehyde (PFA) is discarded, and the slides are washed twice / 5 min with DPBS at room temperature; (4) Permeabilization: the slides are permeated with methanol at -20 °C at -80 °C for 30 min, followed by permeabilization with hydrochloric acid (HCl, 0.1 M) for 5 min. min, after permeation, remove the droplets, wash 3 times with DPBST; (5) Click chemistry: Add N3-modified Probe B (2 μM, Table 4), click chemistry reaction buffer (1.5 × Lumiprobe click buffer), and ascorbic acid solution (L-ascorbic acid; 800 μM) to the cell crawling slide, react at 37℃ for 0.5 h, then wash 3 times / 5 min with 1× hybridization buffer (1× Hyb Buffer; 2× Sodium Citrate Buffer, 20% formamide) at room temperature, and finally wash 3 times / 2 min with DEPC-PBST at room temperature; (6) Probe hybridization: Add diethyl pyrocarbonate treated water (DEPC-H2O), 6× Sodium Citrate Buffer (Saline Sodium Citrate Buffer), 15% formamide and Probe B (0.1 μM, Table 4) to the crawling slide, react at 37℃ for 2 h, wash after reaction, and wash according to DEPC-PBST, 1× HybBuffer and DEPC-PBST were washed 3 times / 3 min at room temperature; (7) Cyclic hybridization: diethyl pyrocarbonate treated water (DEPC-H2O), bovine serum albumin (BSA, 2 μg / μL) glycerol solution, cyclic probe 1 (circle 1, Table 1), probe 2 (circle 2, Table 2), 1×T4 DNA ligase buffer (T4 ligase buffer), sodium chloride (NaCl, 0.25 M), and Tween-20 (Tween-20, 0.25 M) were added.2%), ribonuclease inhibitor (RiboLock RNase inhibitor; 1 U / μL) solution were mixed and added to the cell crawling slide for cyclic hybridization. The reaction was carried out at 37 ℃ for 1 h. After the reaction was completed, the cells were washed 3 times with DEPC-PBST (RT / 3 min); (8) Cyclic ligation: Diethyl pyrocarbonate treated water (DEPC-H2O), bovine serum albumin (BSA, 2 μg / μL) glycerol solution, adenosine triphosphate (ATP, 1 mM), T4 DNA ligase (T4 DNA ligase, 20 U / μL), 1×T4 DNA ligase buffer (T4 ligase buffer), sodium chloride (NaCl, 0.25 M), Tween-20 (Tween-20, 0.2%), ribonuclease inhibitor (RiboLock RNase inhibitor, 1 U / μL) solution were mixed and added to the cell crawling slide. The reaction was carried out at 37 ℃ for 0.5 h. After the reaction was completed, the cells were washed 3 times with DEPC-PBST (RT / 3 min). (9) Rolling circle amplification (RCA): Diethyl pyrocarbonate-treated water (DEPC-H2O), bovine serum albumin (BSA, 2 μg / μL) glycerol solution, deoxyribonucleoside triphosphate mixture (dNTPs Mix, 1 mM), Phi29 DNA polymerase (Phi29 polymerase, 1 U / μL), 1×Phi29 DNA polymerase buffer (1×Phi29 buffer), glycerol (Glycerol, 5%), and ribonuclease inhibitor (RiboLockRNase inhibitor, 1 U / μL) were mixed and added to the cell crawling slide. The reaction was carried out at 37 °C for 2 h. After the reaction was completed, the cells were washed 3 times with DEPC-PBST (RT / 3 min); (10) Fixation: Paraformaldehyde (1.6% PFA) was used for fixation for 10 min. After fixation, the PFA was discarded, and the cells were washed twice with DPBS at room temperature for 5 min; (11) Fluorescence in situ hybridization: 2× hybridization buffer (2×Hyb Buffer) was added to the cell crawling slide. ; 4× sodium citrate buffer, 40% formamide), m. 6 A in situ hybridization fluorescent probe (m 6 After mixing A-FISH-Cy5, add it to the slide and perform fluorescence in situ hybridization. React at 37 °C for 0.5 h. After the reaction is complete, wash 3 times with DEPC-PBST (RT / 3 min); (12) Imaging: After air drying, add 20 μL Slowfade [DAPI (0.5 μg / mL)], mount the slide, and perform imaging in a high-sensitivity laser confocal microscope Leica Sp8.

[0059] Note: Cyclic probes 1 and 2 need to be phosphorylated. The specific procedure is as follows: Mix diethyl pyrocarbonate treated water (DEPC-H2O), Circle 1 (Table 1) or Circle 2 (Table 2), T4 phage polynucleotide kinase (T4 PNK kinase; 0.2 U / μL), 1× T4 phage polynucleotide buffer (1× PNK Buffer), and adenosine triphosphate (ATP, 1 mM) and perform polymerase chain reaction (37 ℃ / 2 h, 65 ℃ / 10 min, 4 ℃ ∞).

[0060] 3) Multiple rounds of fluorescence in situ sequencing

[0061] Multi-round fluorescence in situ sequencing (FISSEQ) is a revolutionary technology that combines high-throughput sequencing with microscopic imaging. Its principle involves in situ reverse transcription of RNA into cDNA within fixed cells or tissues, amplification into clusters, and then decoding the cDNA sequence through multiple cycles of fluorescent probe hybridization, imaging, and chemical quenching. Ultimately, the gene sequence information is precisely mapped to its original spatial location, achieving three-dimensional spatial analysis at the whole transcriptome level and simultaneous detection of multiple genes. This technology provides a powerful tool for developmental biology and disease research.

[0062] The specific procedure is as follows: After the rolling circle amplification reaction, add 50 μL of paraformaldehyde (PFA, 1.6%) to the reaction area for fixation for 10 min, and wash the sample with 50 μL of DEPC-PBST. Add 50 μL of fluorescence in situ sequencing reagent [containing 0.1 μM four-round sequencing fluorescent probe, 0.2 μM ISS-Primer, T4 DNA ligase (T4 DNA Ligase, 20 U / μL), 1 mM adenosine triphosphate (ATP, 2 μg / μL), bovine serum albumin (BSA, 2 μg / μL) glycerol solution, and 1×T4 Ligase Buffer] to the reaction area and incubate at 37 °C for 30 min. After the reaction, wash the sample three times with 50 μL of DEPC-PBST, 5 min each time. After the sample dries, add 20 μL of anti-fluorescence quenching mounting solution containing DAPI for staining, mount with standard coverslips, and perform microscopic imaging. After each round of imaging, the reaction area was soaked and cleaned with DEPC-PBS for 10 min. After removing the coverslip, the reaction area was cleaned three times with DEPC-PBST for 5 min each time to remove excess staining agent. Then, 50 μL of 80 mM KOH solution was added to clean the sample for 5 min to remove the fluorescent probe from the previous round of reaction. Finally, the sample was cleaned three times with DEPC-PBST for 5 min each time to wash away excess KOH, and then the next round of fluorescent in situ sequencing reagent was added.

[0063] This invention is the first to combine temporal and spatial resolution, providing a deeper understanding of the newly modified m. 6 A reversible dynamic modification provides important information on the function and regulation of organisms; (2) improves m through click chemistry and in situ rolling ring amplification reaction. 6 A. Detection sensitivity; (3) Simultaneous detection of multiple target sites in the same sample using in situ sequencing coding strategy; (4) No need for expensive antibodies, greatly reducing costs.

[0064] This invention develops a low-cost, highly sensitive single-cell m 6 A method for detecting multiple spatial resolution RNA—m 6 A-Dynam innovatively combines m 6 A combined RNA metabolic labeling strategy and in situ sequencing technology enables single-molecule level mRNA assays in single cells. 6 A. High-specificity detection of RNA.

[0065] A high-purity metabolic marker, propargyl-L-selenocysteine ​​(PSH), was synthesized via an organic reaction. Within a 12-hour labeling timeframe, PSH specifically labeled newly modified m-molecules in RNA. 6 At site A, m was simultaneously captured. 6 The dynamic change of A modification over time is necessary to achieve m 6 A-modification time-resolved analysis laid the foundation. This was achieved through analysis of intracellular m... 6 Through in-depth research on the modified metabolic pathway, this technique identified propargyl-L-selenocysteine ​​(PSH). PSH is a metabolite produced by mammalian cells... 6 An analogue of an intermediate substance modified with A can be efficiently taken up during cellular metabolism and participate in RNA biosynthesis. This technology introduces PSH into a cell culture system, where PSH specifically labels newly modified m in RNA within a precisely controlled timeframe. 6 Site A. By adjusting the action time of PSH, this technique can accurately capture m 6 A modifies the dynamic changes over time, thereby enabling the control of m. 6 Temporal resolution analysis of the A-modification process. This strategy lays the foundation for further in-depth research on m. 6 The A-modified temporal regulatory mechanism laid the foundation.

[0066] This technique utilizes click chemistry to achieve the desired effect on newly modified m 6 A four-probe system was introduced near site A to form a specific neighbor-to-neighbor hybridization system. Simultaneously, rolling circle amplification was introduced. After condition optimization, m was found in single cells. 6 The detection signal A is significantly enhanced, greatly improving detection sensitivity. To more comprehensively analyze m... 6 Heterogeneity of A-modification, this technique for each m 6 ARNAs are designed with unique barcodes that enable them to generate specific fluorescent signals in four rounds of in situ sequencing. Using bioinformatics analysis, this technology can accurately allocate each mRNA within complex single-cell environments. 6 The spatial location of RNA. This technique can not only obtain the m-location of newly modified genes over a period of time, but also... 6 A abundance can also accurately map the spatial location of these modifications within cells. This comprehensive analytical strategy provides a basis for in-depth research on m 6 The function of A modification in gene expression regulation is supported by comprehensive data.

[0067] m 6 The development of A-Dynam, in m 6 This method is of great significance in the field of A-modification research. It innovatively combines temporal and spatial resolution to deeply explore new modifications with high specificity and sensitivity.6 The function and regulatory mechanisms of dynamic changes at site A in organisms. Compared with traditional methods, m 6 A-Dynam does not rely on antibody cross-linking, avoiding problems caused by antibody specificity and batch-to-batch variability; at the same time, it does not depend on the expression of specific proteins, simplifying the experimental procedure and greatly reducing experimental costs. The establishment of this method provides a completely new technical means for the study of RNA epigenetic regulation.

[0068] Example 1

[0069] 1. Preparation of cell slides

[0070] A uniformly grown human cervical cancer cell line (HeLa cells) was pre-cultured. Adhesion slides were placed in a biosafety cabinet, sprayed with 75% ethanol solution, and irradiated under UV light for 0.5 h before being transferred to a culture dish. HeLa cells were digested with trypsin to form a cell suspension, and 0.5 mL was added to the slide. The culture dish was gently shaken until a cell film formed on the slide. Complete DMEM was then added and cultured for 24 h until the cells reached adherent state.

[0071] 2. PSH metabolic markers

[0072] Preparation of Se-propargyl-L-Homoselenocysteine ​​(PSH): First, anhydrous methanol (30 mL) was added to a round-bottom flask (100 mL), and acetyl chloride (10 mL) was slowly added dropwise at 0 °C while stirring for 10 min. Then, L-selenomethionine (15.3 mmol) was added under nitrogen protection. Figure 4 The reaction mixture was reacted at room temperature for 18 h. After the reaction was complete, the reaction solvent was removed by vacuum distillation to obtain the crude product. The crude product was dissolved in dichloromethane (30 mL), and triethylamine (30.6 mmol) and di-tert-butyl dicarbonate (18.4 mmol) were added sequentially under ice-water bath conditions (0 °C). After reacting at room temperature for 4 h, the mixture was extracted to obtain a clear oil (4.0 g, Figure 4 .1a). Subsequently, the clear oily substance (1.2 g) was dissolved in acetonitrile (15 mL), and potassium carbonate (5.8 mmol), 3-bromopropyne (11.6 mmol), and tetrabutylammonium iodide (5.8 mmol) were added sequentially to the reaction system. The reaction was carried out in an oil bath at 80°C for 18 hours. The reaction solvent was evaporated under reduced pressure, and a pale yellow oily substance was extracted. Figure 4.2a). Finally, the pale yellow oil (1.83 g), dichloromethane (20 mL), and trifluoroacetic acid (4 mL) were added sequentially to a round-bottom flask (100 mL), stirred at room temperature for 2 h, and the reaction solvent was evaporated under reduced pressure. The solvent was then dissolved in tetrahydrofuran (6 mL) under an ice-water bath. The pH was adjusted to alkaline by adding 5 M lithium hydroxide solution, and the reaction was carried out at room temperature for 2 h. After the experiment, the solution was filtered and purified by semi-preparative liquid chromatography to obtain propargyl-L-selenocysteine ​​(PSH, 0.6 g). Figure 4 .3a).

[0073] Weigh 22.10 mg of PSH powder using an electronic analytical balance, dissolve it in 1 mL of enzyme-free water to obtain 100 mM PSH stock solution, and store at 4 °C protected from light. After the cells have grown well, wash the slides three times with diethyl pyrocarbonate-phosphate-buffered saline (DPBS). Incubate in methionine-free complete DMEM for 30 min, wash the slides with D-PBS, add 100 mM PSH stock solution, and perform metabolic labeling for 12 h to induce the cells to produce m... 6 A RNA.

[0074] 3. Hypoxia stimulation

[0075] HeLa cell smears were prepared and placed in 10 cm cell culture dishes for incubation at a constant temperature. The culture dishes were then placed in a clean, self-sealing bag, and a 2.5 L hypoxic gas-generating bag was placed inside. The bag was sealed and incubated at 37 °C for 12 h under hypoxic conditions. Subsequent metabolic labeling was performed according to the PSH metabolic labeling method. Figure 2 ).

[0076] 4. Fixation and permeation

[0077] Fixation and permeabilization were performed in a biosafety cabinet and at -80°C. After removing the culture medium, the slides were washed three times for 3 min each with DEPC-PBST (1×DPBS + 0.1% Tween-20). They were then fixed with 1.6% PFA for 10 min, washed three times for 3 min each with D-PBS, and permeabilized at -80°C for 30 min with anhydrous methanol (stored at -20°C). The slides were then washed twice with diethyl pyrocarbonate-phosphate-buffered saline (DPBS) in a clean bench and air-dried at room temperature. The reaction area was defined using an immunohistochemical pen, permeabilized with hydrochloric acid (HCl, 0.1M) for 5 min, and the droplets were aspirated. The slides were then washed three times for 5 min each with 50 μL DEPC-PBST (1×DPBS + 0.1% Tween-20).

[0078] 5. Click Chemistry reaction

[0079] Add 50 μL of Click Chemistry reagent (containing 2 μM N3-Probe B primer, 800 μM L-ascorbic acid, and 1× Click Chemistry Buffer) to the reaction area and incubate at 37 °C for 30 min. After the reaction, wash the sample three times with 50 μL DEPC-PBST (1×DPBS + 0.1% Tween -20), 5 min each time.

[0080] 6. Probe A in situ hybridization

[0081] After the Click Chemistry reaction was completed, 50 μL of Probe A (Table 3) in situ hybridization reagent (containing 0.1 μM Probe A primer pool (Table 3), 15% formamide, and 6× SSC) was added to the reaction area, and the reaction was carried out in a constant temperature incubator at 37 °C for 2 h. After in situ hybridization, the samples were washed three times with 50 μL DEPC-PBST (1×DPBS + 0.1% Tween-20), three times with 50 μL High Salt Buffer (containing 4× SSC, 10% formamide, 1% Tween-20, 0.1 mg / mL yeast tRNA, and RiboLock RNase inhibitor (0.2 U / µL)), and three times with 50 μL LDEPC-PBST (1×DPBS + 0.1% Tween-20), with each wash lasting 5 min.

[0082] 7. Circularization and Rolling Circle Amplification

[0083] (1) Circulation: After the in situ hybridization reaction of Probe A (Table 3) was completed, 50 μL of circulation reagent (containing 0.1 μM Circle primers (Circle1+Circle2, Table 1, Table 2), bovine serum albumin (BSA, 2 μg / μL) glycerol solution, 0.25 M NaCl, 0.2% Tween-20, ribonuclease inhibitor (RiboLock RNase inhibitor, 1 U / μL), 1×T4 Ligase Buffer) was added to the reaction area. The reaction was carried out in a constant temperature incubator at 37 °C for 1 h. After the reaction was completed, the sample was washed 3 times with 50 μL DEPC-PBST for 5 min each time.

[0084] (2) Ligation: After the cyclization reaction, add 50 μL of ligation reagent (containing 20 U / μL T4 DNA ligase, 1 mM ATP, 2 μg / μL bovine serum albumin (BSA, 2 μg / μL) glycerol solution, 0.25 M NaCl, 0.2% Tween-20, ribonuclease inhibitor (RiboLock RNase inhibitor, 1 U / μL), 1×T4 Ligase Buffer) to the reaction area, and incubate at 37 °C for 30 min. After the reaction, wash the sample three times with 50 μL DEPC-PBST, 5 min each time.

[0085] (3) Rolling circle amplification: After the ligation reaction is completed, add 50 μL of rolling circle amplification reagent (containing 1 U / μL phi29 DNA polymerase, 1 mM dNTPs, bovine serum albumin (BSA, 2 μg / μL) glycerol solution, 5% glycerol, ribonuclease inhibitor (RiboLock RNase inhibitor, 1 U / μL), 1×phi29 Ligase Buffer) to the reaction area, and react in a constant temperature incubator at 37 °C for 2 h. After the reaction is completed, wash the sample 5 times with 50 μL DEPC-PBST, 5 min each time.

[0086] 8. Fluorescence in situ hybridization (FISH)

[0087] After the rolling circle amplification reaction, 50 μL of fluorescence in situ hybridization reagent (containing 0.1 μM FISH-Cy5 fluorescent probe, 15% formamide, and 6×SSC) was added to the reaction area. The mixture was reacted at room temperature for 30 min. After the reaction, the sample was washed three times with 50 μL LEPC-PBST for 5 min each time. After the reaction area dried, 20 μL of anti-fluorescence quenching mounting solution containing DAPI was added, and the sample was mounted using a standard 18 mm × 18 mm coverslip. The sample was then stored in the dark for microscopic imaging.

[0088] 9. Fluorescence in situ sequencing (FISS)

[0089] After the rolling circle amplification reaction, 50 μL of 1.6% paraformaldehyde (PFA) was added to the reaction area for fixation for 10 min. Then, PFA was discarded for fluorescence in situ sequencing (FISS), and the sample was washed with 50 μL of DEPC-PBST. 50 μL of fluorescence in situ sequencing reagent [containing 0.1 μM sequencing fluorescent probe (Fluo Probe, Table 5), 0.2 μM ISS-Primer (Table 5), 20 U / μL T4 DNA ligase, 1×ISS Buffer (bovine serum albumin (BSA, 0.4 μg / μL) glycerol solution), 4 mM ATP, and 2×T4 Ligase Buffer)] was added to the reaction area. The reaction was incubated at 37 °C for 30 min. After the reaction, the sample was washed three times with 50 μL of DEPC-PBST for 5 min each time. After the sample dried, 20 μL of anti-fluorescence quenching mounting medium containing DAPI was added for staining, and the sample was mounted with a standard coverslip for microscopic imaging. After each round of imaging, the reaction area was soaked and cleaned with DEPC-PBS for 10 min. After removing the coverslip, the reaction area was cleaned three times with DEPC-PBST for 5 min each time to remove excess staining agent. Then, 50 μL of 80 mM KOH solution was added to clean the sample for 5 min to remove the fluorescent probe from the previous round of reaction. Finally, the sample was cleaned three times with DEPC-PBST for 5 min each time to wash away excess KOH, and then the reagent for the next round of fluorescence in situ sequencing was added.

[0090] Experimental results:

[0091] (1) Hypoxia stimulation:

[0092] Fifteen genes (RPLP0, PSMB4, HSP90AB1, MYC, CCNB1, and RPL14) were selected to treat HeLa cells with hypoxia, and m 6 A-Dynam analysis of 15 types of m 6Changes in RNA and total RNA were observed, while a control group cultured under normal oxygen conditions was set up. The hypoxia-induced m were determined by comparison. 6 Changes at site A. The m-site was investigated using multi-round fluorescence in situ sequencing technology. 6 A correlation was found between RNA expression and hypoxia treatment. Results were as follows: Figure 2 As shown, the present invention applies m 6 A-Dynam can accurately detect 15 types of m 6 ARNA and Total RNA showed significant differences. Under hypoxic conditions, 15 RNA molecules... 6 Compared to cells in normal culture, A and Total RNA showed different degrees of changes in gene expression.

[0093] In summary, the results of this invention prove that m 6 A-Dynam can detect m 6 The expression changes of RNA under hypoxia further confirmed m 6 A-Dynam in visual detection of m 6 The important value and potential of RNA pattern changes.

[0094] (2) Pulse tracking

[0095] The applicant, combining pulse tracking strategy, explored m 6 Spatial distribution of RNA at subcellular resolution (A) Figure 3 A). By using pulse marking for 12 hours, followed by tracking for 0, 2, 4, and 8 hours respectively, 15 types of m were successfully identified. 6 A RNA was used for spatiotemporal imaging at subcellular resolution. Following this, four rounds of image alignment, nucleocytoplasmic segmentation, and identification were performed to obtain 15 m... 6 A bar chart of subcellular distribution of RNA ( Figure 3 B, C). Bioinformatics analysis revealed that in the initial stage (0-2 h), most m 6 A RNA completes its transport from the nucleus to the cytoplasm, a process known as "nucleus exit"; in the later stages (4-8 hours), some RNA... 6 A RNA re-enters the nucleus, forming a "nucleoplasmic cycle" (ARNA re-enters the nucleus). Figure 3 D).

[0096] In summary, m 6 A-Dynam successfully resolved m at subcellular resolution 6 A dynamic regulation, this YTH protein-mediated "nucleoplasmic cycling" mechanism, not only provides m 6The subcellular localization of a RNA provides a precise spatiotemporal control module, and further, by coupling the cell cycle regulator (CCNB1) with the methylation metabolic pathway (MAT2A), it constructs a dual safeguard system for maintaining cell cycle progress and epigenetic modification homeostasis. This discovery provides a basis for understanding m 6 The functional heterogeneity of A-modification in processes such as cell fate determination, proliferation and differentiation provides a novel subcellular localization regulatory perspective.

[0097] Sequence description:

[0098] Table 1: Circle 1 Primer Pool

[0099]

[0100] Table 2: Circle 2

[0101]

[0102] Table 3: Description of the Probe A primer pool

[0103]

[0104] Table 4: Probe B Primer Pool

[0105]

[0106] Table 5: Multi-round imaging probe and primer sequences

[0107]

[0108] Note: All DNA sequences above were designed in-house and synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by high performance liquid chromatography.

[0109] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A spatiotemporally resolved single-cell m 6 A dynamic modification analysis method includes the following steps: Step 1: Metabolic labeling of live cells was performed using propargyl-L-selenocysteine ​​(PSH) to induce new RNA modification in m 6 Site A specifically introduces a propargyl functional group; the metabolic marker is used at different time points t2, t3, ..., t... n New modifications m in marker RNA 6 Site A enables new RNA modifications m 6 Site A contains a propargyl functional group, which is used to indicate time information; Step 2: The DNA probe with the azide group is covalently linked to the propargyl functional group by a click chemical reaction; Step 3: Amplify the signal of the DNA probe using proximity ligation and rolling circle amplification techniques; Step four: Spatial imaging is performed using fluorescence in situ hybridization or fluorescence in situ sequencing techniques, and the magnified m is observed under a laser confocal microscope. 6 A site signal to indicate m 6 Spatial information of A; thereby enabling the targeting of novel modifications m within single cells. 6 Spatiotemporal dynamic analysis of site A; labeling each cell m with coded probes. 6 A, which includes the combination of m 6 The targeting sequence of A and the readout sequence of the fluorescently labeled readout probe; each m 6 A is encoded by a specific combination of readout sequences; Using sequential hybridization and imaging, each round uses a different readout probe to identify each m 6 The A site binds to the readout sequence and decodes the RNA; the readout probe is used to decode the bases, and the fluorescent probe converts the decoded sequence information into a fluorescent signal; at least two probes are matched with the target DNA and then ligated by a ligase to generate fluorescence for imaging.

2. The method according to claim 1, characterized in that, Step one, the PSH metabolic labeling step includes: Step S1: Starve the cells in a methionine-free culture medium; Step S2: Add PSH for incubation within a specific time window to mark newly occurring m events during that time period. 6 A modifies.

3. The method according to claim 1, characterized in that, In step two, the click chemical reaction is a copper (I)-catalyzed azide-alkyne cycloaddition reaction.

4. The method according to claim 1, characterized in that, Step three, the signal amplification step includes: Proximity hybridization was performed using one or more pairs of circular DNA templates; circularization was performed using T4 DNA ligase; and rolling circle amplification was performed using Phi29 DNA polymerase to generate DNA nanospheres that could be recognized by fluorescent probes.

5. The method according to claim 1, characterized in that, In step four, fluorescence in situ sequencing is used for multi-target detection, specifically including: for different m 6 A probe set with barcode sequences was designed based on the target RNA; multiple rounds of hybridization, imaging, and chemical quenching cycles were performed; and the fluorescent signal sequence was decoded to detect multiple RNA molecules in the same sample. 6 Simultaneous identification and spatial localization of site A.

6. The method according to claim 5, characterized in that, The barcodes from the fluorescence in situ sequencing are encoded by a combination of AG, GA, and TC base pairs.

7. The method according to any one of claims 1 to 6 in the study of cellular stress response m 6 A is used in the dynamic control mechanism; its characteristics are as follows: The applications include any one or more of the following: Application in a hypoxic stress model: Cells were cultured in a hypoxic environment, and the method described above was used to analyze the newly modified m at different time points under hypoxic stress conditions. 6 Abundance variations and spatial distribution of site A to reveal the effects of hypoxic stress on m 6 A modifies the dynamic regulatory effect; Application in drug screening: The method is applied to cancer model cells or disease model cells, by simultaneously detecting multiple disease-related genes. 6 A modifies the dynamics, plotting m under different drug action times. 6 A-modified spatiotemporal dynamic maps are used to screen for disease treatment drugs.

8. A kit for implementing the method according to any one of claims 1-6, characterized in that, Include: Propyl-L-selenocysteine ​​(PSH); DNA probes with azide groups; Click on chemical reaction buffers and catalysts; Enzymes and substrates required for rolling circle amplification; Fluorescent probes required for fluorescence in situ hybridization or sequencing.

9. The reagent kit according to claim 8, characterized in that, It further includes a barcode probe set, ligase, and quenching reagent for multi-round fluorescence in situ sequencing.

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