[go: up one dir, main page]

WO2025184998A1 - Procédé d'hybridation in situ par fluorescence basé sur l'obtention d'une imagerie à super-résolution - Google Patents

Procédé d'hybridation in situ par fluorescence basé sur l'obtention d'une imagerie à super-résolution

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
abnormal
situ hybridization
sample
fluorescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/097317
Other languages
English (en)
Chinese (zh)
Other versions
WO2025184998A8 (fr
Inventor
高军涛
李亚书
田丰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Original Assignee
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University filed Critical Tsinghua University
Publication of WO2025184998A1 publication Critical patent/WO2025184998A1/fr
Publication of WO2025184998A8 publication Critical patent/WO2025184998A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12Q1/6806—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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé d'hybridation in situ par fluorescence basé sur l'obtention d'une imagerie à super-résolution. Le procédé comprend les étapes d'un traitement d'expansion d'un échantillon à tester, et d'hybridation in situ de l'échantillon à tester à l'aide d'une sonde dirigée vers une cible d'intérêt. Le traitement d'expansion de l'échantillon à tester comprend les étapes suivantes : a1) et/ou a2) : a1) réaliser un traitement d'expansion sur l'échantillon à tester avant l'hybridation in situ ; et a2) réaliser une expansion secondaire sur l'échantillon après l'hybridation in situ. La sonde dirigée vers une cible d'intérêt est combinée à une technologie de microscopie à expansion. Après l'expansion cellulaire, le volume augmente d'environ 60 fois par rapport au volume d'origine, de telle sorte que l'effet de résolution de la microscopie à super-résolution peut être réalisé, créant ainsi un site de détection plus précis. Par ailleurs, le problème selon lequel un meilleur résultat de détection de fluorescence ne peut pas être obtenu en raison de faibles signaux de fluorescence de sondes commerciales existantes est évité.
PCT/CN2024/097317 2024-03-06 2024-06-04 Procédé d'hybridation in situ par fluorescence basé sur l'obtention d'une imagerie à super-résolution Pending WO2025184998A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202410254373.5 2024-03-06
CN202410254373.5A CN120608130A (zh) 2024-03-06 2024-03-06 一种基于实现超分辨成像的荧光原位杂交方法

Publications (2)

Publication Number Publication Date
WO2025184998A1 true WO2025184998A1 (fr) 2025-09-12
WO2025184998A8 WO2025184998A8 (fr) 2025-10-02

Family

ID=96924610

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/097317 Pending WO2025184998A1 (fr) 2024-03-06 2024-06-04 Procédé d'hybridation in situ par fluorescence basé sur l'obtention d'une imagerie à super-résolution

Country Status (2)

Country Link
CN (1) CN120608130A (fr)
WO (1) WO2025184998A1 (fr)

Also Published As

Publication number Publication date
WO2025184998A8 (fr) 2025-10-02
CN120608130A (zh) 2025-09-09

Similar Documents

Publication Publication Date Title
US20240425908A1 (en) Methods of detecting nucleic acid sequences with high specificity
US20230151413A1 (en) Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations
US20210032689A1 (en) Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations
EP2529030B1 (fr) Methode de detection d'acides nucleiques in situ
ES2562821T3 (es) Método ultrasensible para la detección in situ de ácidos nucleicos
JP4435259B2 (ja) 微量胃癌細胞の検出法
ES2659798T3 (es) Detección de la restricción de cadena ligera de inmunoglobulina por hibridación in situ de ARN
US20200199687A1 (en) Materials and methods for assessing progression of prostate cancer
US20220380837A1 (en) Method for preparing probe targeting target nucleic acid target
CN115094063A (zh) 一种肺癌早期智能诊断的多价可激活适配体探针及其制备与应用
JP6284487B2 (ja) 膀胱癌の診断及びその再発のモニタリングのための材料及び方法
US20130171622A1 (en) Compositions and methods for detecting viral infection using direct-label fluorescence in situ hybridization
WO2025184998A1 (fr) Procédé d'hybridation in situ par fluorescence basé sur l'obtention d'une imagerie à super-résolution
JP6313713B2 (ja) 膵胆管癌の診断、予後、再発のモニターおよび治療的／予防的治療の評価のための物質および方法
US20070111960A1 (en) High affinity probes for analysis of human papillomavirus expression
JP2015504665A5 (fr)
WO2017114009A1 (fr) Sonde de détection du gène egfr, son procédé de préparation et trousse d'essai
Xiong et al. A single-molecule biosensor based on multiple fluorophore nucleic acid probe and STORM imaging for detection of Hantaan virus
WO2005090608A2 (fr) Sondes haute affinite pour analyse de l'expression du papillomavirus humain

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24927933

Country of ref document: EP

Kind code of ref document: A1