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WO2025184998A1 - 一种基于实现超分辨成像的荧光原位杂交方法 - Google Patents

一种基于实现超分辨成像的荧光原位杂交方法

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Publication number
WO2025184998A1
WO2025184998A1 PCT/CN2024/097317 CN2024097317W WO2025184998A1 WO 2025184998 A1 WO2025184998 A1 WO 2025184998A1 CN 2024097317 W CN2024097317 W CN 2024097317W WO 2025184998 A1 WO2025184998 A1 WO 2025184998A1
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cancer
abnormal
situ hybridization
sample
fluorescence
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French (fr)
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WO2025184998A8 (zh
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高军涛
李亚书
田丰
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Tsinghua University
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Tsinghua University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples

Definitions

  • the present application relates to the field of biological detection, and in particular to a fluorescence in situ hybridization method based on achieving super-resolution imaging.
  • Fluorescence in situ hybridization is a technique that uses fluorescently labeled nucleic acid fragments as probes to complementally pair with the DNA or RNA to be tested in tissues, cells or chromosomes, combining into specific nucleic acid hybrid molecules, and then displaying the location of the nucleic acid to be tested in tissues, cells or chromosomes through a fluorescence detection system.
  • a hybrid of the target nucleic acid and the nucleic acid probe can be formed after denaturation, annealing, and renaturation.
  • a specific nucleotide in the nucleic acid probe is labeled with a reporter molecule, such as biotin or directly labeled with fluorescein.
  • This experimental method allows for qualitative, semi-quantitative, or relative localization analysis of the nucleic acid under a microscope, utilizing an immunochemical reaction between the reporter molecule and a specific avidin labeled with fluorescein, or directly using a fluorescence detection system.
  • RNA-FISH fluorescent probes there are two main methods for producing RNA-FISH fluorescent probes. One is to entrust the company to design and synthesize, which is relatively expensive; the other is to add the promoter required for RNA transcription to the template through PCR, and then transcribe to obtain the probe. This method is cumbersome.
  • Expansion microscopy is a novel super-resolution imaging technique that improves image resolution by physically magnifying the organism itself, capable of increasing the volume of cells and tissues by 8,000 times.
  • the core of this technique is to uniformly embed biological samples in swellable hydrogels, followed by mechanical homogenization. Next, the gel expands through folding linear expansion, thereby also expanding the organism. This process provides anchoring for key biomolecules within the cell, such as organelles and the cytoskeleton.
  • Expansion microscopy has significant application value in improving the resolution of optical microscopes.
  • Using conventional fluorescence microscopy to image the expanded sample can also achieve super-resolution imaging. For example, it can be used to examine cells on a single-molecule basis, making the final observations more reliable and accessible.
  • the process begins by filling the entire biological sample with acrylate and polymerizing it to form a gel. As the gel absorbs water and expands, biomolecules that were previously adjacent are pulled apart, but their relative positions remain unchanged.
  • the resolution of the sample can be greatly improved, making it possible to achieve super-resolution imaging using ordinary microscopes.
  • the brightness of the fluorescence signal may be diluted after the sample expands, and the fluorescence signal is weak, which is not conducive to detection.
  • Zhang Xu, Gao Juntao, Zhang Qiwei and others provided a method for preparing DNA probes in "Method for preparing probes for target nucleic acid targets”. This method is based on Tn5 and is very suitable for analyzing chromatin interactions within a distance of 100Kb or less.
  • the present application provides a fluorescence in situ hybridization method based on achieving super-resolution imaging.
  • the fluorescence in situ hybridization method based on achieving super-resolution imaging of the present application includes the steps of expanding the sample to be tested and performing in situ hybridization with the sample to be tested using a probe targeting a target.
  • the expansion treatment of the sample to be tested includes the following a1) and/or a2):
  • the expansion treatment method a1) is as follows: anchoring and polymerizing the sample to be tested, and then placing it in water until it is fully expanded.
  • the above-mentioned full expansion means that the volume of the sample no longer increases.
  • the anchoring and polymerization method comprises placing the fixed sample in a paraformaldehyde/acrylamide buffer, allowing it to stand, removing the sample, placing it in a mixed solution containing a monomer solution, tetramethylethylenediamine, and acrylamide, and incubating it; the volume ratio of the monomer solution, tetramethylethylenediamine, and acrylamide in the mixed solution is 90:5:5; the monomer solution is a 1 ⁇ PBS buffer containing 23% (w/v) sodium acrylate, 10% (w/v) acrylamide, and 0.1% (w/v) N,N′-methylenebisacrylamide; and the paraformaldehyde/acrylamide buffer is a 1 ⁇ PBS buffer containing 0.7% paraformaldehyde and 1% acrylamide.
  • the expansion treatment method a2) is as follows: adding water to the sample after in situ hybridization, allowing the sample to swell a second time, and discarding the water after the expansion is complete.
  • the preparation method of the probe for the target is:
  • the above-mentioned method for preparing probes for the target is independent or minimally dependent on the specificity of the initial RNA sequence and can effectively remove regions of unwanted sequences (especially repetitive sequences), thereby making the method independent of species-specific Cot-1 DNA for blocking repetitive fragments.
  • the amount of cDNA template required for probe preparation in this method is approximately 50 ng (e.g., 30 ng, 35 ng, 40 ng, 45 ng, 55 ng, 60 ng), which is much lower than the 1 ⁇ g required for traditional FISH.
  • the amount of cDNA template required for probe preparation in this method is approximately 50 ng (e.g., 30 ng, 35 ng, 40 ng, 45 ng, 55 ng, 60 ng), which is much lower than the 1 ⁇ g required for traditional FISH.
  • the process is simple, efficient, and cost-effective.
  • the target cDNA sequence can be derived from any sample containing the target cDNA.
  • sample is used in its broadest sense. In a kind of meaning, it means including cells (for example, people, bacteria, yeast and fungi), tissues or living bodies or samples or cultures and biological samples obtained from any source.
  • Biological samples can be obtained from animals (including people) and refer to biological materials or compositions found therein, including but not limited to bone marrow, blood, serum, platelets, plasma, interstitial fluid, urine, cerebrospinal fluid, nucleic acid, RNA, tissue and its purification or filtered form.
  • these examples should not be construed as limiting the sample types that can be used for the application.
  • the sample is whole genome RNA.
  • the transposase is hyperactive.
  • nucleic acid refers to any molecule comprising nucleic acid, including but not limited to DNA or RNA.
  • the term encompasses sequences comprising any known base analogs of DNA and RNA, including but not limited to: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine,
  • the target cDNA sequence of the target of interest is obtained by excluding unwanted sequence regions from the initial sequence.
  • region of unwanted sequences refers to a region that is substantially free (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% free) of unwanted nucleic acids.
  • Unwanted nucleic acids include, but are not limited to, repetitive nucleic acids, non-conservative sequences, conserved sequences, GC-rich sequences, AT-rich sequences, secondary structures, non-coding sequences (e.g., promoters, enhancers, etc.), or coding sequences.
  • the unwanted region is selected from repetitive sequences.
  • the method of eliminating is to amplify the target cDNA sequence of the target
  • the amplification is PCR amplification.
  • the region of unwanted sequence is at least 100bp; or 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1500bp, 2000bp, 3000bp, 4000bp, 5000bp, 6000bp, 7000bp, 8000bp, 9000bp, 10000bp, 20000bp, 30000bp, 40000bp, or 50000bp.
  • the transposase is selected from one or a combination of any two or more of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9, Tn10, Tn551, Tn971, Tn916, Tn1545, Tn1681, Tgf2, Tol2, Himar1, and HARBI1.
  • Tgf2 and Tol2 are from the hAT family
  • Himar1 is from the Tcl/Mariner family
  • HARBI1 is from the PIF/Harbinger family.
  • label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein.
  • Labels include, but are not limited to, dyes; radioactive labels, such as 32 P; binding moieties such as biotin; haptens such as digoxin; luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). Labels can provide signals that can be detected by fluorescence, radioactivity, colorimetry, gravimetric analysis, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
  • Labels can be charged moieties (positive or negative) or, alternatively, can be charge neutral. Labels can include nucleic acid or protein sequences or combinations thereof, as long as the sequence comprising the label is detectable. In some embodiments, nucleic acids are directly detected (e.g., directly reading the sequence) without labeling.
  • the label is a fluorophore, a colorimetric label, a quantum dot, biotin, and other label molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cyclic olefins for click reactions, and initiator groups for polymer labeling), and can also be selected from polypeptides/protein molecules, LNA/PNA, non-natural amino acids and their analogs (such as peptoids), non-natural nucleic acids and their analogs (nucleotide mimics) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced emission molecules, rare earth ion ligand molecules, polymetallic oxygen clusters, etc.).
  • label molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cyclic olefins for click reactions, and initiator groups for polymer labeling), and can also be selected from polypeptides/protein molecules
  • the label is a fluorophore.
  • the fluorophore can be selected from fluorescein-based dyes, rhodamine-based dyes, and cyanine dyes.
  • the fluorescein dye includes standard fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc.
  • FITC fluorescein isothiocyanate
  • FAM hydroxyfluorescein
  • TET tetrachlorofluorescein
  • the rhodamine dyes include R101, tetraethyl rhodamine (RB200), and carboxytetramethylrhodamine (TAMRA).
  • the cyanine dye is mainly selected from two categories, one is thiazole orange (TO), oxazole orange (YO) series and dimer dyes thereof, and the other is polymethine series cyanine dyes.
  • the fluorophore may also be selected from the following dyes: diphenylethylene, naphthalimide, coumarins, acridines, pyrenes, and the like.
  • Fluorophores are typically labeled at the 5' end of the primer or probe sequence, but can also be placed at the 3' end by changing the modification bond (e.g., -OH or -NH bond).
  • the method of generating the probe comprises amplification, cloning, synthesis, or a combination thereof.
  • amplification refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide when the term "nucleic acid” is used together, typically starting from a small amount of polynucleotide (e.g., as little as a single polynucleotide molecule), wherein the amplified product or amplicon is typically detectable.
  • the amplification of polynucleotides includes a variety of chemical and enzymatic methods. Producing multiple DNA copies from one or several copies of a target cDNA or template DNA molecule during polymerase chain reaction (PCR), rolling circle amplification (RCA), or ligase chain reaction (LCR) is a form of amplification.
  • Amplification is not limited to the strict duplication of the starting molecule.
  • using reverse transcription RT-PCR to produce multiple cDNA molecules from a limited amount of RNA in a sample is a form of amplification.
  • producing multiple RNA molecules from a single DNA molecule during the transcription process is also a form of amplification.
  • the method for generating the probe is: amplifying the fragmented DNA sequence using primers that can bind to the linker sequence.
  • primer refers to an oligonucleotide, whether naturally occurring in a purified restriction digest or synthetically produced, which, when placed under conditions that induce the synthesis of primer extension products complementary to a nucleic acid chain (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH), can serve as a starting point for synthesis.
  • the primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its chains before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • primer should be long enough to initiate the synthesis of extension products in the presence of an inducing agent.
  • the precise length of the primer will depend on many factors, including the use of temperature, primer source, and method.
  • primers range from 10-100 or more nucleotides (e.g., 10-300, 15-250, 15-200, 15-150, 15-100, 15-90, 20-80, 20-70, 20-60, 20-50 nucleotides, etc.).
  • primers comprise additional sequences that do not hybridize to target nucleic acids.
  • term " primer” includes chemically modified primers, fluorescently modified primers, functional primers (fusion primers), sequence-specific primers, random primers, primers with specific and random sequences, and DNA and RNA primers.
  • the primer is labeled.
  • the label is defined as the term "label" above;
  • the label is selected from a fluorophore, a colorimetric label, a quantum dot, or biotin; preferably a fluorophore.
  • hybridization is used to refer to the pairing of complementary nucleic acids.
  • Hybridization and the strength of hybridization are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the Tm of the hybrid formed, and the G:C ratio within the nucleic acids.
  • a single molecule containing a pair of complementary nucleic acids within its structure will be "self-hybridizing.”
  • the in situ hybridization is to perform 3D FISH labeling of the probe and the expanded target cells.
  • the present application also provides a method for RNA signal localization, using the above-mentioned fluorescence in situ hybridization method based on achieving super-resolution imaging.
  • the present invention also provides a method for detecting diseases caused by abnormal RNA expression, including the above-mentioned RNA signal localization method.
  • the diseases caused by abnormal RNA expression include: diseases caused by abnormal MYC mRNA expression, diseases caused by abnormal TP53 mRNA expression, diseases caused by abnormal EGFR mRNA expression, diseases caused by abnormal BRAF mRNA expression, diseases caused by abnormal HER2 mRNA expression and/or diseases caused by abnormal SRC mRNA expression.
  • the diseases with abnormal MYC mRNA expression include lymphoma, lung cancer, breast cancer, gastric cancer, colorectal cancer, cervical cancer, leukemia, neuroblastoma, retinoblastoma, and sarcoma;
  • Diseases with abnormal TP53 mRNA expression include breast cancer, lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, hematological malignancies, and gliomas;
  • Diseases with abnormal EGFR mRNA expression include non-small cell lung cancer, head and neck cancer, colorectal cancer, brain cancer, and pancreatic cancer;
  • Diseases with abnormal BRAF mRNA expression include malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, pancreatic cancer, test tube cancer, esophageal cancer, breast cancer, and ovarian cancer;
  • Diseases with abnormal HER2 mRNA expression include breast cancer and gastric cancer;
  • Diseases with abnormal SRC mRNA expression include breast cancer, colorectal cancer, lung cancer, prostate cancer, gastric cancer, osteosarcoma, soft tissue sarcoma, renal cancer, familial macrognathia, and cardiovascular disease.
  • FIG1 is a schematic diagram of a process flow of an embodiment of the present application.
  • FIG2 shows the mRNA signals of POLR2A detected in the nucleus and cytoplasm using the Tn5-RNA-FISH method and the commercial probe in Example 1.
  • Figure 3 shows the lncRNA signal of MALAT1 RNA detected in the cell nucleus using the Tn5-RNA-FISH method and using a commercial probe;
  • Figure 4 shows the use of Tn5-RNA-FISH probe combined with expansion microscopy to detect the signals of POLR2A and MALAT1 RNA in cells.
  • the following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.
  • the experimental methods in the following examples, unless otherwise specified, are conventional methods.
  • the experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
  • the quantitative tests in the following examples were performed in triplicate, and the results were averaged.
  • DMEM culture medium purchased from GIBCO
  • streptomycin/penicillin double antibody purchased from GIBCO
  • trypsin purchased from GIBCO
  • FBS purchased from GIBCO
  • RNA extraction kit purchased from Life Technology
  • reverse transcription kit purchased from Thermo Scientific
  • Qubit RNA high sensitivity kit purchased from Life Technology
  • Qubit DNA high sensitivity kit purchased from Life Technology
  • AntiFade mounting medium containing DAPI, purchased from Life Technology
  • Fixogum purchased from Marubu
  • Tn5 transposase kit purchased from Vazyme
  • HS-Taq purchasedd from Takara
  • PCR product purification kit purchased from Zymo
  • 37% hydrochloric acid purchasedd from Sinopharm
  • Tris-HCl purchasedd from Sigma
  • Triton-X100 purchasedd from Sigma
  • ethanol purchasedd from Sigma
  • a) dextran sulfate purchased from GIBCO
  • A549 cells (Wuhan Purnosai Biotechnology Co., Ltd.), POLR2A mRNA (NCBI Gene ID: 5430), and MALAT1 RNA (NCBI Gene ID: 378938) in the following examples.
  • the method for preparing a probe for target RNA in this application comprises the following steps:
  • Primer Design Design amplification primers for the target RNA and send them to a synthesis company for synthesis. Fluorescently labeled primers are synthesized according to the sequence provided in the Tn5 kit.
  • RNA extraction For each cell type, 1 ⁇ 10 6 cells were collected and extracted according to the experimental procedures of the RNA extraction kit. The extracted RNA was quantified using Qubit.
  • cDNA Preparation Add 1 ⁇ g of total RNA, reverse transcription primer, dNTPs, 5 ⁇ reverse transcription buffer, RNase inhibitor, and reverse transcriptase to a 50 ⁇ l PCR tube in a 20 ⁇ l reaction volume. Reverse transcription conditions: 25°C for 10 minutes, then 50°C for 30 minutes.
  • Probe Template cDNA Amplification Take 2 ⁇ l of the reverse transcription product from Step 3 as the template and add the diluted primers (amplification primers from Step 1) to a 50 ⁇ l PCR tube for PCR.
  • PCR conditions were: 98°C for 3 min, followed by 30 cycles of (98°C for 30 s, 55°C for 30 s, 72°C for 3 min), 72°C for 5 min, and a 4°C hold.
  • PCR products were purified using a recovery kit, quantified using a Qubit assay, and stored at -20°C.
  • Tn5 Fragmentation Take 50 ng of the DNA product from step 4 and add Tn5 enzyme and reaction buffer to a total volume of 50 ⁇ l. Heat in a 55°C water bath for 10 minutes. Purify the DNA using a PCR product recovery kit.
  • PCR amplification and fluorescent labeling All products from step 5 were subjected to PCR amplification.
  • the PCR conditions were 75°C for 5 min, (98°C for 30 s, 55°C for 30 s, 72°C for 30 s) ⁇ 30 cycles, 72°C for 5 min, and maintained at 4°C.
  • the DNA product was purified using a PCR product purification kit, and then 50 ng was taken as a template for PCR amplification and labeling using the fluorescently labeled primers in step 1).
  • the PCR conditions were 98°C for 3 min, (98°C for 30 s, 55°C for 30 s, 72°C for 30 s) ⁇ 30 cycles, 72°C for 5 min, and maintained at 4°C.
  • Probe denaturation Before use, take the probe out of the refrigerator, place it in a PCR instrument, heat it at 95°C for 5 minutes, and then immediately place it on ice for later use.
  • the method for using the above-mentioned probe to perform FISH detection of the RNA to be tested in cells based on expansion microscopy technology in this application comprises the following steps:
  • Fluorescence imaging and processing The sealed slides were photographed using a fluorescence microscope, confocal microscope, or super-resolution microscope.
  • a Zeiss confocal microscope (model: LSM780) was used.
  • the image was acquired using 405, 488, 568, 594, and 647 lasers and corresponding filter combinations, and a 63 ⁇ ApoPLAN NA1.4 oil-immersed objective lens.
  • ZEN SP2.3 software was used for image acquisition, and FIJI (ImageJ core version 1.52h) was used for image processing.
  • the method for performing FISH detection (not based on expansion microscopy) of the RNA to be tested in cells using the above-mentioned probe or commercial probe in this application comprises the following steps:
  • Cell fixation Fix the cells with 4% paraformaldehyde at room temperature for 10 minutes, wash with 0.1M Tris-HCl for 10 minutes, then permeabilize the membrane and digest RNA with 0.5% Triton-X100 containing 10 ⁇ g/mL RNase A, treat in a water bath at 37 degrees for 30 minutes, wash three times with PBS, and treat with 0.1M hydrochloric acid solution at room temperature for 30 minutes; after washing three times with PBS, treat in 50% deionized formamide 2 ⁇ SSC solution at room temperature for 30 minutes, dehydrate with gradient ethanol, and air-dry.
  • Fluorescence Imaging and Processing Seal the slides and image them using a fluorescence microscope or confocal microscope.
  • This application used a Zeiss confocal microscope (model: LSM780) equipped with 405, 488, 568, 594, and 647 lasers and corresponding filter combinations, and a 63 ⁇ ApoPLAN NA1.4 oil immersion lens.
  • ZEN SP2.3 software was used for image acquisition, and FIJI (ImageJ core version 1.52h) was used for image processing.
  • the technical route of one embodiment of the present application can be briefly summarized as follows: 1) transcribe total RNA into cDNA; 2) amplify the target cDNA using amplification primers; 3) fragment the target cDNA using transposase Tn5 to obtain target DNA fragments; 4) amplify the target DNA fragments using fluorescently labeled primers to obtain fluorescent probes; 5) expand the sample (cell) to be tested; 6) perform in situ hybridization between the fluorescent probe and the expanded sample; 7) perform fluorescence detection on the hybridized sample. (The sample can also be expanded a second time before fluorescence detection.)
  • probes prepared using the Tn5 method were used to detect RNA localization in unexpanded samples to verify the accuracy and sensitivity of the probes prepared using the Tn5 method.
  • the Tn5-RNA-FISH probe was then compared with a commercial RNA-FISH probe (kit purchased from Stellaris) in A549 cells targeting POLR2A mRNA to verify the site-specific labeling of Tn5-RNA-FISH.
  • Tn5-RNA-FISH RNA localized in the nucleus in unexpanded samples. Compared to commercial probes, Tn5-RNA-FISH uses longer probes. Successful nuclear entry of the probes can be a potential concern in applications.
  • a Tn5-FISH probe targeting MALAT1 RNA was used for validation.
  • MALAT1 RNA is a lncRNA primarily localized in the nuclear speckle region of the cell nucleus.
  • Expansion microscopy has important application value in improving the resolution of optical microscopes. Using a conventional fluorescence microscope to image expanded samples can also achieve super-resolution imaging. However, after sample expansion, the brightness of the fluorescence signal may be diluted, which places high demands on the probe signal intensity and the tightness of the probe binding to the target sequence. Existing commercial probes cannot meet this requirement.
  • the Tn5-RNA-FISH probe was used in conjunction with expansion microscopy to detect intracellular POLR2A and MALAT1 RNA.
  • Probes are prepared for target RNA, detecting the target RNA while simultaneously expanding the sample. This effectively increases the accessibility of the target molecule and the strength of the detection signal without disrupting the RNA structure. This process also makes RNA information, which is previously difficult to distinguish due to its close proximity, easier to analyze, resulting in more intuitive and clear test results.
  • the above-mentioned method of performing FISH detection on the RNA to be tested in cells can be used to detect samples of various diseases caused by abnormal RNA expression, including but not limited to the following diseases:
  • Detection of disease samples with abnormal MYC mRNA expression such as lymphoma, lung cancer, breast cancer, gastric cancer, colorectal cancer, cervical cancer, leukemia, neuroblastoma, retinoblastoma, sarcoma, etc.
  • Detection of disease samples with abnormal TP53 mRNA expression such as breast cancer, lung cancer (especially non-small cell lung cancer), pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, ovarian cancer, hematological malignancies (acute myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome), glioma, etc.
  • Detection of disease samples with abnormal EGFR mRNA expression such as non-small cell lung cancer, head and neck cancer, colorectal cancer, brain cancer, pancreatic cancer, etc.
  • Detection of disease samples with abnormal BRAF mRNA expression such as malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, pancreatic cancer, test tube cancer, esophageal cancer, breast cancer, ovarian cancer, etc.
  • HER2 mRNA expression such as breast cancer, gastric cancer, etc.
  • Detection of disease samples with abnormal SRC mRNA expression such as breast cancer, colorectal cancer, lung cancer, prostate cancer, gastric cancer, osteosarcoma, soft tissue sarcoma, kidney cancer, familial macrognathia, cardiovascular disease, etc.
  • the probe preparation method used in this application is simple and fast, does not require expensive instruments and equipment, and can obtain highly accurate fluorescent probes for RNA-FISH through simple steps; the probe production cost is low, and the cost is reduced by 5-10 times compared to commercial probes or traditional methods of preparing probes.
  • the volume of the cells expands to about 60 times the original size after expansion, which can achieve the resolution effect of super-resolution microscopy and more accurate detection sites. At the same time, it avoids the problem of weak fluorescence signals of the original commercial probes and the inability to obtain good fluorescence detection results.

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Abstract

公开了一种基于实现超分辨成像的荧光原位杂交方法，所述方法包括对待测样品的膨胀处理，以及使用针对目标靶标的探针与待测样品进行原位杂交的步骤。所述对待测样品的膨胀处理包括如下a1)和/或a2)：a1)在原位杂交前，对待测样品进行膨胀处理；a2)在原位杂交后，对样品进行二次膨胀。将针对目标靶标的探针与膨胀显微技术结合，细胞膨胀后，体积扩大至原来的60倍左右，可以实现超分辨显微镜的分辨效果，检测位点更为准确，同时，避免了原有商业探针的荧光信号弱，无法获得较好的荧光检测结果的问题。

Description

一种基于实现超分辨成像的荧光原位杂交方法
相关申请的交叉引用
本申请要求申请日为2024年3月6日的中国专利申请(申请号为202410254373.5)的优先权，该专利申请的全部内容在此被援引加入本文。
技术领域
本申请涉及生物检测领域，具体的说，涉及一种基于实现超分辨成像的荧光原位杂交方法。
背景技术
荧光原位杂交(fluorescence in situ hybridization，FISH)是一种利用荧光标记的核酸片段为探针，与组织、细胞或染色体上待测DNA或RNA互补配对，结合成专一的核酸杂交分子，通过荧光检测系统将待测核酸在组织、细胞或染色体中的位置显示出来的技术。
如果待检测的细胞或组织切片上的靶核酸与所用的核酸探针是同源互补的，二者经变性-退火-复性，即可形成靶核酸与核酸探针的杂交体。将核酸探针的某一种核苷酸标记上报告分子如生物素或直接标记荧光素，可利用该报告分子与荧光素标记的特异亲和素之间的免疫化学反应或直接经荧光检测体系在镜下对待测核酸进行定性、半定量或相对定位分析的一种实验方法。
目前来看，现有RNA-FISH荧光探针制作方法主要有两种，一种是交由公司设计合成，成本较高；而另一种是通过PCR给模板添加上RNA转录所需要的启动子，再进行转录获得探针，该方法过程繁琐。
膨胀显微术(Expansion microscopy，ExM)是一种新型超分辨成像技术，它可以通过物理放大生物体本身来提高最终图像分辨率，能够实现将细胞和组织体积扩大8000倍。该技术的核心是使用可膨胀水凝胶将生物样品均匀地包埋在其中，然后进行机械均质化处理。接下来，凝胶通过折叠线性膨胀而膨胀，从而也使生物体膨胀，这一过程为细胞内的关键生物分子(例如细胞器、细胞骨架等)提供了锚定。
膨胀显微术在提高光学显微镜的分辨率方面具有重要的应用价值。利用常规的荧光显微镜对膨胀后的样品进行成像，同样可实现超分辨的成像效果。例如，它可用于在单分子基础上检测细胞，从而使最终的观察结果更加可靠和容易获得。整个流程中首先将丙烯酸盐充满整个生物样品内部，并使其聚合形成凝胶。凝胶吸水膨胀后，原本临近的生物分子被拉远，但相对位置保持不变。
借助膨胀显微术，能够大大提高样品的分辨率，从而可以实现使用普通显微镜实现超分辨成像。
虽然利用常规的荧光显微镜对膨胀后的样品进行成像，同样可实现超分辨的成像效果，但样品膨胀后，荧光信号的亮度可能会被稀释，荧光信号较弱，不利于检测。
张旭、高军涛、张奇伟等人，在《针对目标核酸靶标的探针的制备方法》中提供了一种制备DNA探针的方法，这一方法基于Tn5，十分适合分析100Kb及以内距离的染色质相互作用。
发明公开
本申请提供一种基于实现超分辨成像的荧光原位杂交方法。
本申请的基于实现超分辨成像的荧光原位杂交方法，包括对待测样品的膨胀处理，以及使用针对目标靶标的探针与待测样品进行原位杂交的步骤。
其中，所述对待测样品的膨胀处理包括如下a1)和/或a2)：
a1)在原位杂交前，对待测样品进行膨胀处理；
a2)在原位杂交后，对样品进行二次膨胀。
其中，所述a1)的膨胀处理方法为：将待测样品进行锚定与聚合，之后置于水中，直至膨胀完全。上述膨胀完全是指样品体积不再变大。
其中，所述锚定与聚合的方法为：将固定后的样品置于多聚甲醛/丙烯酰胺缓冲液中，静置，取出样品，置于含有单体溶液、四甲基乙二胺和丙烯酰胺的混合溶液中，孵育；所述混合溶液中单体溶液、四甲基乙二胺和丙烯酰胺的体积比为90:5:5；单体溶液为含23％(w/v)的丙烯酸钠、10％(w/v)的丙烯酰胺、0.1％(w/v)N，N′-亚甲基双丙烯酰胺的1×PBS缓冲液；多聚甲醛/丙烯酰胺缓冲液为含0.7％多聚甲醛、1％丙烯酰胺的1×PBS。
其中，所述a2)的膨胀处理方法为：向原位杂交后的样品中，加入水，静置使样品二次膨胀，膨胀结束后将水弃净。
其中，所述针对目标靶标的探针的制备方法为：
b1)获取目标靶标RNA对应的靶cDNA序列；
b2)使用转座酶，将所述靶cDNA序列进行片段化的同时，在片段化的DNA序列两端加上接头序列；
b3)利用所述接头序列，获取所述片段化的DNA序列，以产生探针。
上述针对目标靶标的探针的制备方法，不依赖或极少依赖于初始RNA序列的特异性，能有效去除不想要的序列的区域(尤其是重复序列)，从而使得本方法也不依赖于物种特异的Cot-1DNA对重复片段进行封闭；
一个重要的优点在于，本方法在制备探针时，所需的cDNA模板量约为50ng(例如30ng、35ng、40ng、45ng、55ng、60ng)，远低于传统FISH的1μg；同时，对于一个位点，仅需要做一次Tn5高效转座酶的片段化之后，就可以进行大量的探针制备，流程简洁高效，而且性价比高。
本申请中靶cDNA序列可来源于任何含有靶cDNA的样品。
术语“样品”以其最广泛的含义使用。在一种含义中，其意指包括细胞(例如，人、细菌、酵母和真菌)、组织或活体、或获自任何来源的样本或培养物、以及生物样品。生物样品可以获自动物(包括人)、并且指其中发现的生物材料或组合物，包括但不限于骨髓、血液、血清、血小板、血浆、间隙液、尿液、脑脊液、核酸、RNA、组织、及其纯化或过滤形式。然而，这些实例不应解释为限定可用于本申请的样品类型。
在一些实施方式中，所述样品为全基因组RNA。
在一些实施方式中，所述转座酶是高活性的。
如本文所用，术语“核酸”指任何包含核酸的分子，包括但不限于DNA或RNA。该术语涵盖包含DNA和RNA的任何已知碱基类似物的序列，所述类似物包括但不限于：4-乙酰胞嘧啶、8-羟基-N6-甲基腺苷、吖丙啶基胞嘧啶、假异胞嘧啶、5-(羧基羟基甲基)尿嘧啶、5-氟尿嘧啶、5-溴尿嘧啶、5-羧甲基氨基甲基-2-硫代尿嘧啶、5-羧甲基氨基甲基尿嘧啶、二氢尿嘧啶、肌苷、N6-异戊烯基腺嘌呤、1-甲基腺嘌呤、1-甲基假尿嘧啶、1-甲基鸟嘌呤、1-甲基肌苷、2,2-二甲基鸟嘌呤、2-甲基腺嘌呤、2-甲基鸟嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-甲基腺嘌呤、7-甲基鸟嘌呤、5-甲基氨基甲基尿嘧啶、5-甲氧基氨基甲基-2-硫代尿嘧啶、β-D-甘露糖基Q核苷、5’-甲氧基羰基甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-异戊烯基腺噪呤、尿啼啶-5-氧基乙酸甲酯、尿啼啶-5-氧基乙酸、氧基丁氧基胸苷(oxybutoxosine)、假尿嘧啶、Q核苷、2-硫胞嘧啶、5-甲基-2-硫尿嘧啶、2-硫尿嘧啶、4-硫尿嘧啶、5-甲基尿嘧啶、N-尿嘧啶-5-氧基乙酸甲酯、尿嘧啶-5-氧基乙酸、假尿嘧啶、Q核苷、2-硫胞嘧啶和2,6-二氨基嘌呤。
在一些实施方式中，所述目标靶标的靶cDNA序列为从初始序列中排除掉不想要的序列的区域得到。
如本文所用，术语“不想要的序列的区域”指实质上不含(例如85％、90％、91％、92％、93％、94％、95％、96％、97％、99％或100％不含)不想要的核酸的区域。不想要的核酸包括但不限于重复核酸、非保守序列、保守序列、富含GC的序列、富含AT的序列、二级结构、非编码序列(例如启动子、增强子等)或编码序列。
在一些实施方式中，所述不想要的区域选自重复序列。
在一些实施方式中，所述排除的方法为扩增所述目标靶标的靶cDNA序列；
在一些实施方式中，所述扩增为PCR扩增。
在一些实施方式中，所述不想要的序列的区域至少为100bp；或为120bp、130bp、140bp、150bp、160bp、170bp、180bp、190bp、200bp、250bp、300bp、350bp、400bp、450bp、500bp、600bp、700bp、800bp、900bp、1000bp、1500bp、2000bp、3000bp、4000bp、5000bp、6000bp、7000bp、8000bp、9000bp、10000bp、20000bp、30000bp、40000bp、50000bp。
在一些实施方式中，所述转座酶选自Tn1、Tn2、Tn3、Tn4、Tn5、Tn6、Tn7、Tn9、Tn10、Tn551、Tn971、Tn916、Tn1545、Tn1681、Tgf2、Tol2、Himar1以及HARBI1中的一种或任意多种的组合。
Tgf2和Tol2来自于hAT家族，Himar1来自于Tcl/Mariner家族，HARBI1来自于PIF/Harbinger家族。
在一些实施方式中，所述探针是标记的。
如本文所用的术语“标记”指可用于提供可检测的(优选可定量的)效果且可以连接至核酸或蛋白的任何原子或分子。标记包括但不限于染料；放射性标记，诸如32P；结合部分诸如生物素；半抗原诸如地高辛；发光、发磷光或发荧光部分；和单独的荧光染料或与可以通过荧光共振能量转移(FRET)抑制或移动发射光谱的部分组合的荧光染料。标记可以提供可通过荧光、放射性、比色、重量测定、X射线衍射或吸收、磁性、酶活性等检测的信号。标记可以是带电荷的部分(正电荷或负电荷)或可选地，可以是电荷中性的。标记可以包括核酸或蛋白序列或由其组合，只要包含标记的序列是可检测的。在一些实施方案中，核酸在没有标记的情况下直接检测(例如，直接读取序列)。
在一些实施方式中，所述标记是荧光团、比色标记、量子点、生物素以及其他可以用于探测的标签分子(如用于拉曼衍射成像的炔烃基团，用于click反应的环烯烃，用于聚合物标记的引发集团)，也可以选自多肽/蛋白分子，LNA/PNA，非天然氨基酸及其类似物(比如拟肽)，非天然核酸及其类似物(拟核苷酸)和纳米结构(包括无机纳米颗粒，NV-center，聚集/组装诱导发光分子，稀土离子配体分子，多金属氧簇等)。
在一些实施方式中，所述标记是荧光团。
在一些实施方式中，所述荧光团可选自荧光素类染料、罗丹明类染料以及菁染料。
在一些实施方式中，所述荧光素类染料包括标准荧光素及其衍生物，如异硫氰酸荧光素(FITC)、羟基荧光素(FAM)、四氯荧光素(TET)等。
在一些实施方式中，所述罗丹明类染料包括R101、四乙基罗丹明(RB200)和羧基四甲基罗丹明(TAMRA)等。
在一些实施方式中，所述菁染料主要选自两类，一类是噻唑橙(thiazole orange,TO)、噁唑橙(oxazole orange,YO)系列及其二聚体染料，另一类是多甲川系列菁染料。
在一些实施方式中，荧光团还可以选择下述染料：二苯乙烯、萘酰亚胺、香豆素类、吖啶类、芘类等。
荧光团通常标记在引物或探针序列的5'端，但通过改变修饰键(例如-OH或-NH键)也可以将其置于3'端。
在一些实施方式中，在步骤c)中，产生探针的方法包括扩增、克隆、合成或其组合。
术语“扩增(ampIifying或amplification)”在“核酸”此用语的上下文中共同出现时，指产生多个拷贝的多核苷酸、或多核苷酸的部分，通常从少量的多核苷酸开始(例如，少至单个多核苷酸分子)，其中扩增产物或扩增子通常是可检测的。多核苷酸的扩增包括多种化学和酶促方法。在聚合酶链反应(PCR)、滚环扩增(RCA)或连接酶链反应(LCR)过程中从一个或几个拷贝的靶cDNA或模板DNA分子产生多个DNA拷贝是扩增的形式。扩增不限于起始分子的严格复制。例如，使用逆转录RT-PCR从样品中有限量的RNA产生多个cDNA分子是扩增的形式。此外，在转录过程期间从单一DNA分子产生多个RNA分子也是扩增的形式。
在一些实施方式中，在步骤c)中，产生探针的方法为：利用能够结合所述接头序列的引物，扩增所述片段化的DNA序列。
术语“引物”指寡核苷酸，无论其是在经纯化的限制性消化物中天然存在或是合成产生的，所述寡核苷酸当置于诱导与核酸链互补的引物延伸产物合成的条件下时(例如，存在核苷酸和诱导剂诸如DNA聚合酶和在合适的温度和pH下)，所述寡核苷酸能够作为合成的起始点而起作用。引物优选是单链的，以用于扩增的最大效率，但可选地也可以是双链的。如果是双链的，则在用于制备延伸产物前，首先将引物处理以分开其链。优选地，引物是寡脱氧核糖核苷酸。引物应足够长，以在诱导剂存在的情况下引发延伸产物的合成。引物的精确长度将取决于许多因素，包括温度、引物来源和方法的使用。例如，在一些实施方案中，引物范围为10-100或更多个核苷酸(例如10-300、15-250、15-200、15-150、15-100、15-90、20-80、20-70、20-60、20-50个核苷酸等)。
在一些实施方案中，引物包含不与目标核酸杂交的另外的序列。术语“引物”包括化学修饰的引物、荧光修饰的引物、功能引物(融合引物)、序列特异性引物、随机引物、具有特异性和随机序列的引物、及DNA和RNA引物。
在一些实施方式中，所述引物是标记的。
在一些实施方式中，所述标记为上述术语“标记”所限定；
在一些实施方式中，所述标记选自荧光团、比色标记、量子点或生物素；优选荧光团。
如本文所用，术语“杂交”用于指互补核酸的配对。杂交和杂交强度(即核酸之间结合的强度)受此类因素如核酸之间的互补性程度、所涉及的条件的严格性、形成的杂交物的Tm以及核酸内的G:C比率等的影响。在其结构内含有互补核酸的配对的单一分子将是“自杂交的”。
在一些实施方式中，所述杂交测定为原位杂交；
优选的，所述原位杂交为将所述探针与膨胀后的目的细胞进行3D FISH标记。
本申请还提供一种RNA的信号定位方法，使用上述的基于实现超分辨成像的荧光原位杂交方法。
本发明还提供一种RNA表达异常引起的疾病的检测方法，包括上述的RNA的信号定位方法。
其中，所述RNA表达异常引起的疾病包括：MYC mRNA表达异常的疾病、TP53 mRNA表达异常的疾病、EGFR mRNA表达异常的疾病、BRAF mRNA表达异常的疾病、HER2 mRNA表达异常的疾病和/或SRC mRNA表达异常的疾病。
其中，所述MYC mRNA表达异常的疾病包括淋巴瘤、肺癌、乳腺癌、胃癌、结直肠癌、宫颈癌、白血病、神经母细胞瘤、视网膜母细胞瘤、肉瘤；
TP53 mRNA表达异常的疾病包括乳腺癌、肺癌、胰腺癌、结直肠癌、肝癌、胃癌、卵巢癌、血液系统恶性肿瘤、神经胶质瘤；
EGFR mRNA表达异常的疾病包括非小细胞肺癌、头颈部癌、结直肠癌、脑癌、胰腺癌；
BRAF mRNA表达异常的疾病包括恶性黑色素瘤、甲状腺癌、结直肠癌、非小细胞肺癌、胰腺癌、试管癌、食管癌、乳腺癌、卵巢癌；
HER2 mRNA表达异常的疾病包括乳腺癌、胃癌；
SRC mRNA表达异常的疾病包括乳腺癌、结直肠癌、肺癌、前列腺癌、胃癌、骨肉瘤、软组织肉瘤、肾癌、家族性巨颌症、心血管疾病。
附图说明
图1为本申请的一个实施方式的流程示意图。
图2为实施例1中，使用Tn5-RNA-FISH方法与使用商业化探针在细胞核和细胞质中检测到POLR2A的mRNA信号。
图3为使用Tn5-RNA-FISH方法与使用商业化探针在细胞核中检测到MALAT1 RNA的lncRNA信号；
图4为使用Tn5-RNA-FISH探针，结合膨胀显微镜，检测细胞内POLR2A和MALAT1 RNA的信号。
实施发明的最佳方式
以下的实施例便于更好地理解本申请，但并不限定本申请。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下面结合具体实施方式对本申请进行进一步的详细描述，给出的实施例仅为了阐明本申请，而不是为了限制本申请的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本申请的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的涉及仪器主要包括：PCR仪(Biored)，杂交仪(Abbott Thermobite)，水浴锅。
下述实施例中的试剂：DMEM培养基(购于GIBCO)，链霉素/青霉素双抗(购于GIBCO)，胰蛋白酶(购于GIBCO)，FBS(购于GIBCO)，RNA提取试剂盒(购于Life Technology)，反转录试剂盒(购于Thermo Scientific)，Qubit RNA高灵敏度试剂盒(购于Life Technology)，Qubit DNA高灵敏度试剂盒(购于Life Technology)，AntiFade封片剂(含DAPI，购于Life Technology)，Fixogum(购于Marubu)，Tn5转座酶试剂盒(购于Vazyme)，HS-Taq(购于Takara)，PCR产物纯化试剂盒(购于Zymo)，37％盐酸(购于国药)，Tris-HCl(购于sigma)，Triton-X100(购于sigma)，乙醇(购于sigma)，硫酸葡聚糖(购于sigma)，肝糖原(购于Life Technology)，商业化MALAT1RNA-FISH探针(购于Stellaris)，商业化POLR2A RNA-FISH探针(购于Stellaris)，20×SSC(购于Life Technology)，去离子甲酰胺(购于Solarbio)，PBS(购于Solarbio)，3M醋酸钠(购于Solarbio)，4％多聚甲醛(购于Solarbio)，NP-40(购于Solarbio)，AcX(Thermo-Fisher)，Lablel-IT Amine Modifying Reagent(Mirus Bio,LLC)，酰胺(sigma)，N，N′-亚甲基双丙烯酰胺(sigma)，丙烯酸钠(sigma)，四甲基乙二胺(sigma)，过硫酸铵(sigma)，过硫酸铵(sigma)。
下述实施例中的A549细胞(武汉普诺赛生物科技有限公司)，POLR2A的mRNA(NCBI Gene ID:5430)，MALAT1 RNA(NCBI Gene ID:378938)。
下述实施例中的耗材：SuperFrost载玻片(购于ThermoFisher)，ThermoFisher1.5#盖玻片(购于ThermoFisher)。
本申请中针对目标RNA的探针的制备方法包括如下步骤：
1)引物设计：扩增引物，针对目标RNA，设计对应的引物，发给合成公司合成。荧光标记引物，根据Tn5试剂盒提供的序列进行合成。
2)RNA提取：对于每一种细胞，取1×106细胞，按照RNA提取试剂盒的实验步骤进行提取，提取后的RNA用Qubit定量。
3)cDNA获取：将1微克总RNA、反转录引物、dNTP、5×反转录缓冲液、RNA酶抑制剂、反转录酶加入50微升PCR管中，反应体积20微升。反转录条件为25℃10分钟，50℃30分钟。
4)探针模板cDNA扩增：取2微升步骤3的反转录产物作为模板，将稀释好的引物(步骤1中的扩增引物)加入PCR管，反应体积50微升进行PCR。PCR的条件为，98℃3min，(98℃30s，55℃30s，72℃3min)×30循环，72℃5min，4℃保持。PCR产物经回收试剂盒纯化回收，Qubit定量，存于-20℃。
5)Tn5片段化：取50ng步骤4的DNA产物，加入Tn5酶以及反应缓冲液，总体积50微升。55℃水浴加热10分钟，随后用PCR产物回收试剂盒纯化DNA。
6)PCR扩增及荧光标记：步骤5所有产物投入PCR扩增。PCR的条件为，75℃5min，(98℃30s，55℃30s，72℃30s)×30循环，72℃5min，4℃保持。使用PCR产物纯化试剂盒纯化DNA产物，随后取50ng作为模板，用步骤1)中的荧光标记引物进行PCR扩增标记。PCR的条件为，98℃3min，(98℃30s，55℃30s，72℃30s)×30循环，72℃5min，4℃保持。标记后的产物用Qubit定量后，加入2微升肝糖原进行2小时的-80℃乙醇沉淀(0.1倍体积3M醋酸钠，2.5倍体积无水乙醇)。醇沉后的探针用75％乙醇洗3次，挥发干净乙醇，并用杂交液(2×SSC，10％硫酸葡聚糖，50％去离子甲酰胺)重悬，存于-20℃。
7)探针变性：使用前，将探针从冰箱取出，置于PCR仪中，95℃加热5分钟后，立即置于冰上备用。
本申请中使用上述探针对细胞中待测RNA进行基于膨胀显微镜技术的FISH检测的方法包括如下步骤：
1)样品的固定：用4％多聚甲醛室温固定细胞10min，用1×PBS洗两遍，随后加入1毫升70％的酒精，置于4℃通透10分钟，结束后弃去酒精，加入洗涤缓冲液(2×SSC，10％去离子甲酰胺)室温静置2-5分钟。(一般是将细胞8*104个细胞滴在玻片上，培育一天后进行固定。)
2)锚定与聚合：将固定后的样品置于1mL多聚甲醛/丙烯酰胺缓冲液中(含0.7％多聚甲醛、1％丙烯酰胺的1×PBS)，37℃静置5小时。保温结束后，从-20℃冰箱取出预冷的90微升单体溶液(含23％(w/v)的丙烯酸钠、10％(w/v)的丙烯酰胺、0.1％(w/v)N，N′-亚甲基双丙烯酰胺的1×PBS缓冲液)，加入5微升四甲基乙二胺、5微升丙烯酰胺，迅速吹打混匀，滴加到-20℃预冷的孵育小室中。将样品取出，吸去残留液体，将样品置于孵育小室底部，使细胞面与液体接触，将小室在冰上放置5min，随后37℃孵育1小时。
2)样品膨胀：孵育结束后，将盖玻片与凝胶用镊子移出，加入到消化缓冲液中(含1mg/ml蛋白酶K的NEB buffer2)，37℃消化30min。随后移除消化液，将载有凝胶的盖玻片置于50ml水中，每30分钟换一次水，直至膨胀完全(大概3-4次)。选择凝胶上平面光滑处，切下约1cm2的胶块，用于后续杂交。
3)原位杂交：将100微升探针溶液(2ng/μL)滴加到共聚焦小皿底部，凝胶置于杂交液中，使细胞面与探针接触，随后将共聚焦小皿置于杂交仪中，37℃过夜。第二天将杂交液弃去，加入1毫升洗涤缓冲液，37℃避光孵育30分钟。孵育结束后弃去溶液，加入1毫升DAPI染液(10mg/ml),37℃避光孵育30分钟,随后弃去DAPI染液。加入1毫升洗涤缓冲液，室温静置2-5分钟。将缓冲液弃去，用吸水纸小心吸去多余残留液体。
4)二次膨胀：向共聚焦小皿中加入2ml水，轻微晃动后，将水弃净，再重复洗涤1次。随后向皿中加入2ml水，室温放置30min，使样品二次膨胀。膨胀结束后将水弃净，用吸水纸小心吸去多余残留液体。将样品避光保存于4℃或直接拍摄。
5)荧光成像及处理：将封好的片子用荧光显微镜、共聚焦显微镜或者超分辨显微镜进行拍摄。本申请中用的是蔡司的共聚焦显微镜(型号：LSM780)， 配备405，488，568，594和647激光及对应的滤光片组合，镜头为63×ApoPLAN NA1.4油镜。镜油为蔡司Immersion Oil F518，25℃折射率1.515.图片采集软件为ZEN SP2.3，处理软件为FIJI(ImageJ core version：1.52h)。
本申请中使用上述探针或商业探针对细胞中待测RNA进行FISH检测(不基于膨胀显微镜技术的)的方法包括如下步骤：
1.细胞固定：用4％多聚甲醛室温固定细胞10min，用0.1M Tris-HCl洗10min，随后用含有10μg/mL RNase A的0.5％Triton-X100透膜并消解RNA，水浴37度处理30min，PBS洗3遍后用0.1M盐酸溶液室温处理30min；PBS洗3遍后，在50％去离子甲酰胺2×SSC溶液中室温处理30min，梯度乙醇脱水晾干。
2.原位杂交：将10微升探针溶液(2ng/μL)与细胞混合后用Fixogum封于玻片内，置杂交仪杂交(75度5min，37度过夜)。第二天用0.3％NP-40的2×SSC溶液室温洗细胞3次，每次5min，随后用AntiFade封片剂封片，玻片边缘用Fixogum封住溶液，避光保存4度或直接拍摄。
3.荧光成像及处理：将封好的片子用荧光显微镜或共聚焦显微镜进行拍摄。本申请中用的是蔡司的共聚焦显微镜(型号：LSM780)，配备405，488，568，594和647激光及对应的滤光片组合，镜头为63×ApoPLAN NA1.4油镜。镜油为蔡司Immersion Oil F518，25℃折射率1.515.图片采集软件为ZEN SP2.3，处理软件为FIJI(ImageJ core version：1.52h)。
如图1所示，本申请的一个实施例的技术路线可以简单概括为：1)将总RNA转录为cDNA，2)使用扩增引物扩增目标靶标的cDNA；3)使用转座酶Tn5将目标靶标的cDNA片段化处理，得到目标靶标的DNA片段，4)使用荧光标记引物对目标靶标的DNA片段进行扩增，得到荧光探针；5)对待测样本(细胞)进行膨胀处理；6)将荧光探针与膨胀后的样本进行原位杂交；7)对杂交后的样本进行荧光检测。(也可以在荧光检测前对样本进行二次膨胀)。
实施例1
首先，使用Tn5方法制作的探针，检测未膨胀的样品中RNA定位，以验证Tn5方法制作的探针的准确性和灵敏性。将Tn5-RNA-FISH探针与商业化RNA-FISH探针对比(试剂盒购Stellaris)，在A549细胞中，针对POLR2A的mRNA，验证Tn5-RNA-FISH对位点的标记特异性。
已有研究表明，POLR2A的mRNA在细胞核和细胞质中均有分布，使用Tn5-RNA-FISH方法与使用商业化探针检测得到的结果相同，在细胞核和细胞质中都能够检测到POLR2A的mRNA信号，实验结果如图2所示。
实施例2
使用Tn5方法制作的探针，检测未膨胀的样品中定位于细胞核中的RNA。相较于商业化探针，Tn5-RNA-FISH所使用的探针长度更长。在应用中，探针能否顺利入核，是可能存在的一个问题。为了对Tn5-FISH探针的入核能力进行检测，使用靶向MALAT1 RNA的Tn5-FISH探针进行了验证。MALAT1 RNA为lncRNA，主要定位在细胞核的核斑点区。
实验结果如图3所示，在A549细胞中，Tn5-RNA-FISH检测到的MALAT1的RNA信号主要定位在细胞核中，信号定位与之前的研究、商业化探针检测到的信号定位均一致。
实施例3
膨胀显微镜技术膨胀显微术在提高光学显微镜的分辨率方面具有重要的应用价值。利用常规的荧光显微镜对膨胀后的样品进行成像，同样可实现超分辨的成像效果。但样品膨胀后，荧光信号的亮度可能会被稀释，这对探针信号强度、探针与目标序列结合的紧密性具有较高的要求，使用现有的商业化探针无法满足这一要求。使用Tn5-RNA-FISH探针，结合膨胀显微镜，检测细胞内POLR2A和MALAT1 RNA。
实验结果如图4所示，在膨胀后的A549细胞中，Tn5-RNA-FISH检测到的POLR2A和MALAT1的RNA的信号定位，与膨胀前一致。POLR2A的RNA在细胞核与细胞质中均有分布，MALAT1的RNA主要分布在细胞核中。
该发明可用于临床样本的检测，针对靶标RNA制备探针，检测目标RNA，同时将样本膨，能够在不破坏RNA结构的前提下，有效增加靶标分子的可及性和检测信号的强度。这一过程还可以使得原本由于距离较近而难以区分的RNA信息变得易于分析，从而获得更为直观、清晰的检测结果。
上述对细胞中待测RNA进行FISH检测的方法，可以用于多种由RNA表达异常引起的疾病的样本的检测，所述由RNA表达异常引起的疾病包括但不限于如下种类：
MYC mRNA表达异常的疾病样本的检测，例如：淋巴瘤、肺癌、乳腺癌、胃癌、结直肠癌、宫颈癌、白血病、神经母细胞瘤、视网膜母细胞瘤、肉瘤等；
TP53 mRNA表达异常的疾病样本的检测，例如：乳腺癌、肺癌(尤其是非小细胞肺癌)、胰腺癌、结直肠癌、肝癌、胃癌、卵巢癌、血液系统恶性肿瘤(急性髓系白血病、慢性淋巴细胞白血病、骨髓增生异常综合症)、神经胶质瘤等；
EGFR mRNA表达异常的疾病样本的检测，例如：非小细胞肺癌、头颈部癌、结直肠癌、脑癌、胰腺癌等；
BRAF mRNA表达异常的疾病样本的检测，例如：恶性黑色素瘤、甲状腺癌、结直肠癌、非小细胞肺癌、胰腺癌、试管癌、食管癌、乳腺癌、卵巢癌等；
HER2 mRNA表达异常的疾病样本的检测，例如：乳腺癌、胃癌等；
SRC mRNA表达异常的疾病样本的检测，例如：乳腺癌、结直肠癌、肺癌、前列腺癌、胃癌、骨肉瘤、软组织肉瘤、肾癌、家族性巨颌症、心血管疾病等。
以及其他多种由RNA表达异常引起的疾病。
工业应用
本申请中使用的探针制备方法简单快捷，不需要昂贵的仪器设备，能通过简单的步骤流程获得高准确性的用于RNA-FISH的荧光探针；探针制作成本低，相较于商业化探针或传统的制备探针的方式，成本降低5-10倍。将本申请的探针与膨胀显微技术结合，细胞膨胀后，体积扩大至原来的60倍左右，可以实现超分辨显微镜的分辨效果，检测位点更为准确，同时，避免了原有商业探针的荧光信号弱，无法获得较好的荧光检测结果的问题。

Claims (14)

  1. 一种基于实现超分辨成像的荧光原位杂交方法，其特征在于，包括对待测样品的膨胀处理，以及使用针对目标靶标的探针与待测样品进行原位杂交的步骤。
  2. 根据权利要求1所述的基于实现超分辨成像的荧光原位杂交方法，其特征在于，所述对待测样品的膨胀处理包括如下a1)和/或a2)：
    a1)在原位杂交前，对待测样品进行膨胀处理；
    a2)在原位杂交后，对样品进行二次膨胀。
  3. 根据权利要求2所述的基于实现超分辨成像的荧光原位杂交方法，其特征在于，所述a1)的膨胀处理方法为：将待测样品进行锚定与聚合，之后置于水中，直至膨胀完全。
  4. 根据权利要求3所述的基于实现超分辨成像的荧光原位杂交方法，其特征在于，所述锚定与聚合的方法为：将固定后的样品置于多聚甲醛/丙烯酰胺缓冲液中，静置，取出样品，置于含有单体溶液、四甲基乙二胺和丙烯酰胺的混合溶液中，孵育；所述混合溶液中单体溶液、四甲基乙二胺和丙烯酰胺的体积比为90:5:5；单体溶液为含23％(w/v)的丙烯酸钠、10％(w/v)的丙烯酰胺、0.1％(w/v)N，N′-亚甲基双丙烯酰胺的1×PBS缓冲液；多聚甲醛/丙烯酰胺缓冲液为含0.7％多聚甲醛、1％丙烯酰胺的1×PBS。
  5. 根据权利要求2所述的基于实现超分辨成像的荧光原位杂交方法，其特征在于，所述a2)的膨胀处理方法为：向原位杂交后的样品中，加入水，静置使样品二次膨胀，膨胀结束后将水弃净。
  6. 根据权利要求1所述的基于实现超分辨成像的荧光原位杂交方法，其特征在于，所述针对目标靶标的探针的制备方法为：
    b1)获取目标靶标RNA对应的靶cDNA序列；
    b2)使用转座酶，将所述靶cDNA序列进行片段化的同时，在片段化的DNA序列两端加上接头序列；
    b3)利用所述接头序列，获取所述片段化的DNA序列，以产生探针。
  7. 根据权利要求6所述的方法，其特征在于，所述转座酶选自Tn1、Tn2、Tn3、Tn4、Tn5、Tn6、Tn7、Tn9、Tn10、Tn551、Tn971、Tn916、Tn1545、Tn1681、Tgf2、Tol2、Himar1以及HARBI1中的一种或任意多种的组合。
  8. 根据权利要求6所述的方法，其特征在于，所述探针是标记的，所述标记选自荧光团、比色标记、量子点、生物素、用于拉曼衍射成像的炔烃基团、用于click反应的环烯烃、用于聚合物标记的引发集团、多肽/蛋白分子、LNA/PNA、非天然氨基酸及其类似物和非天然核酸及其类似物以及纳米结构。
  9. 根据权利要求6所述的方法，其特征在于，所述引物是标记的，所述标记选自荧光团、比色标记、量子点、生物素、用于拉曼衍射成像的炔烃基团、用于click反应的环烯烃、用于聚合物标记的引发集团、多肽/蛋白分子、LNA/PNA、非天然氨基酸及其类似物、非天然核酸及其类似物以及纳米结构。
  10. 根据权利要求6所述的方法，其特征在于，在步骤b3)中，产生探针的方法包括扩增、克隆、合成或其组合；所述扩增的方法为利用能够结合所述接头序列的引物扩增所述片段化的DNA序列。
  11. 一种RNA的信号定位方法，其特征在于，使用权利要求1-10任一所述的基于实现超分辨成像的荧光原位杂交方法。
  12. 一种RNA表达异常引起的疾病的检测方法，其特征在于，包括权利要求1-10任一所述的基于实现超分辨成像的荧光原位杂交方法进行RNA的信号定位的步骤。
  13. 根据权利要求12所述的RNA表达异常引起的疾病的检测方法，其特征在于，所述RNA表达异常引起的疾病包括：MYC mRNA表达异常的疾病、TP53 mRNA表达异常的疾病、EGFR mRNA表达异常的疾病、BRAF mRNA表达异常的疾病、HER2 mRNA表达异常的疾病和/或SRC mRNA表达异常的疾病。
  14. 根据权利要求13所述的RNA表达异常引起的疾病的检测方法，其特征在于，所述MYC mRNA表达异常的疾病包括淋巴瘤、肺癌、乳腺癌、胃癌、结直肠癌、宫颈癌、白血病、神经母细胞瘤、视网膜母细胞瘤、肉瘤；
    TP53 mRNA表达异常的疾病包括乳腺癌、肺癌、胰腺癌、结直肠癌、肝癌、胃癌、卵巢癌、血液系统恶性肿瘤、神经胶质瘤；
    EGFR mRNA表达异常的疾病包括非小细胞肺癌、头颈部癌、结直肠癌、脑癌、胰腺癌；
    BRAF mRNA表达异常的疾病包括恶性黑色素瘤、甲状腺癌、结直肠癌、非小细胞肺癌、胰腺癌、试管癌、食管癌、乳腺癌、卵巢癌；
    HER2 mRNA表达异常的疾病包括乳腺癌、胃癌；
    SRC mRNA表达异常的疾病包括乳腺癌、结直肠癌、肺癌、前列腺癌、胃癌、骨肉瘤、软组织肉瘤、肾癌、家族性巨颌症、心血管疾病。
PCT/CN2024/097317 2024-03-06 2024-06-04 一种基于实现超分辨成像的荧光原位杂交方法 Pending WO2025184998A1 (zh)

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