Visualizing modified nucleotide and nucleic acid interactions in single cells

Abstract

A method for visualizing modified nucleotides or specific nucleic acid sequence interactions in a specific nucleic acid sequence in a single cell, wherein the method comprises coupling an in situ hybridization (ISH) reaction with a proximity ligation assay (PLA) reaction.

Description

[0001] field

[0002] The present disclosure relates to methods for visualizing modified nucleotides or specific nucleic acid sequence interactions in specific nucleic acid sequences in single cells. background

[0003] Epigenetics refers to the hereditary changes in gene expression that occur without changes in the DNA sequence. The main epigenetic processes include DNA methylation and covalent modification of histones. There are many techniques that can be used to study epigenetic marks at the molecular level or on a genomic scale. However, what is needed is a technique to visualize epigenetic marks at a single genomic locus in an individual cell.

[0004] Overview

[0005] In various aspects of the present disclosure, a method for visualizing modified nucleotides in a specific nucleic acid sequence in a cell is provided. The method comprises: a) contacting a prepared cell with at least one nucleic acid probe, the at least one nucleic acid probe being labeled with at least one marker and complementary to the specific nucleic acid sequence, wherein the nucleic acid probe hybridizes with the specific nucleic acid sequence to form a hybrid cell; b) contacting the hybrid cell with a first binding agent and a second binding agent, the first binding agent binding to the marker of the nucleic acid probe, the second binding agent binding to the modified nucleotide; and c) detecting the first and second binding agents by a proximity ligation assay to visualize the modified nucleotides in the specific nucleic acid sequence in the cell. In some embodiments, the prepared cell is fixed, permeabilized, and optionally contains denatured chromosomal DNA. In other embodiments, the modified nucleotide is selected from 5-methylcytidine, 3-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 1-methyladenosine, 6-methyladenosine, 7-methylguanosine, xanthine nucleoside, inosine, dihydrouridine or pseudouridine or a modified ribose with 2'-O-methylation. In further embodiments, the specific nucleic acid sequence is selected from promoter DNA, enhancer DNA, CpG islands, coding DNA, intronic DNA, messenger RNA, microRNA, non-coding RNA, long non-coding RNA, ribosomal RNA, transfer RNA, small nuclear RNA, small nucleolar RNA, SmYRNA, YRNA, spliced ​​leader RNA, telomerase RNA component, small interfering RNA, Piwi-interacting RNA or trans-acting RNA. In other embodiments, the marker is a hapten or dye selected from biotin, digoxigenin, dinitrophenyl, fluorescein, diethylaminocoumarin, rhodamine, cyanine 3, cyanine 5 or Texas Red. In still other embodiments, the nucleic acid probe is linear or circular, comprises DNA, RNA, LNA or a combination thereof, and has a length of about 15 nucleotides to about 500 nucleotides. In alternative embodiments, the cell is an individual cell selected from a eukaryotic cell, a mammalian cell, a human cell, a normal cell or a cancer cell, or the cell is within a tissue sample or fluid sample obtained from a eukaryotic organism. In some embodiments, wherein the modified nucleotide is 5-methylcytidine and the specific nucleic acid sequence is Septin 9 promoter.

[0006] Another aspect of the present disclosure includes a method for visualizing the interaction between two nucleic acid sequences in a cell. The method includes: a) contacting a prepared cell with a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe comprises a first marker and has complementarity with a first nucleic acid sequence, and the second nucleic acid probe comprises a second marker and has complementarity with a second nucleic acid sequence, wherein the first and second markers are different, and the first and second nucleic acid probes hybridize with the first and second nucleic acid sequences, respectively; b) contacting the cell with a first binding agent that binds to the first marker and a second binding agent that binds to the second marker; and c) detecting the first and second binding agents by a proximity ligation assay to visualize the interaction between the two nucleic acid sequences in the cell. In some embodiments, the prepared cell is fixed, permeabilized, and optionally contains denatured chromosomal DNA. In other embodiments, the modified nucleotide is selected from 5-methylcytidine, 3-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 1-methyladenosine, 6-methyladenosine, 7-methylguanosine, xanthine nucleoside, inosine, dihydrouridine or pseudouridine or a modified ribose with 2'-O-methylation. In further embodiments, the specific nucleic acid sequence is selected from promoter DNA, enhancer DNA, CpG island, coding DNA, intronic DNA, messenger RNA, microRNA, non-coding RNA, long non-coding RNA, ribosomal RNA, transfer RNA, small nuclear RNA, small nucleolar RNA, SmY RNA, Y RNA, spliced ​​leader RNA, telomerase RNA component, small interfering RNA, Piwi-interacting RNA or trans-acting RNA. In other embodiments, the label is a hapten or dye selected from biotin, digoxigenin, dinitrophenyl, fluorescein, diethylaminocoumarin, rhodamine, cyanine 3, cyanine 5 or Texas red. In still other embodiments, the nucleic acid probe is linear or circular, comprises DNA, RNA, LNA or a combination thereof, and has a length of about 15 nucleotides to about 500 nucleotides. In alternative embodiments, the cell is an individual cell selected from a eukaryotic cell, a mammalian cell, a human cell, a normal cell or a cancer cell, or the cell is within a tissue sample or a fluid sample obtained from a eukaryotic organism.

[0007] Other aspects and features of the disclosure are described in detail below. Sequence Listing <110> sigma-aldrich llc <120> Visualizing modified nucleotide and nucleic acid interactions in single cells <130> 047497-509514 <150> US 62 / 095,660 <151> 2014-12-22 <150> US 62109,979 <151> 2015-01-30 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1819 <212> DNA <213> Homo sapiens <400> 1 gcgtcgcccg tccctggctt ctctgacagc cgtgttccat ccccgccctg tgccccttct 60 cccggacagt gccttctcca gggctcaccc aggagggtgc agcggtggcc cccggggcgg 120 tggtcgtggt gggggtgtta gctgcagggg tgccctcggt gggtgggagt tggtggcctc 180 tcgctggtgc catgggactc gcatgttcgc cctgcgcccc tcggctcttg agcccacagg 240 ccgggatcct gcctgccagc cgcgtgcgct gccgtttaac ccttgcaggc gcagagcgcg 300 cggcggcggt gacagagaac tttgtttggc tgcccaaata cagcctcctg cagaaggacc 360 ctgcgcccgg ggaaggggag gaatctcttc ccctctgggc gcccgccctc ctcgccatgg 420 cccggcctcc acatccgccc acatctggcc gcagcggggc gcccgggggg aggggctgag 480 gccgcgtctc tcgccgtccc ctgggcgcgg gccaggcggg gaggaggggg gcgctccggt 540 cgtgtgccca ggactgtccc ccagcggcca ctcgggcccc agccccccag gcctggcctt 600 gacaggcggg cggagcagcc agtgcgagac agggaggccg gtgcgggtgc gggaacctga 660 tccgcccggg aggcgggggc ggggcggggg cgcagcgcgc ggggaggggc cggcgcccgc 720 cttcctcccc cattcattca gctgagccag ggggcctagg ggctcctccg gcggctagct 780 ctgcactgca ggagcgcggg cgcggcgccc cagccagcgc gcagggcccg ggccccgccg 840 ggggcgcttc ctcgccgctg ccctccgcgc gacccgctgc ccaccagcca tcatgtcgga 900 ccccgcggtc aacgcgcagc tggatgggat catttcggac ttcgaaggtg ggtgctgggc 960 tggctgctgc ggccgcggac gtgctggaga ggaccctgcg ggtgggcctg gcgcgggacg 1020 ggggtgcgct gaggggagac gggagtgcgc tgaggggaga cgggacccct aatccaggcg 1080 ccctcccgct gagagcgccg cgcgcccccg gccccgtgcc cgcgccgcct acgtggggga 1140 ccctgttagg ggcacccgcg tagaccctgc gcgccctcac aggaccctgt gctcgttctg 1200 cgcactgccg cctgggtttc cttcctttta ttgttgtttg tgtttgccaa gcgacagcga 1260 cctcctcgag ggctcgcgag gctgcctcgg aactctccag gacgcacagt ttcactctgg 1320 gaaatccatc ggtcccctcc ctttggctct ccccggcggc tctcgggccc cgcttggacc 1380 cggcaacggg atagggaggt cgttcctcac ctccgactga gtggacagcc gcgtcctgct 1440 cgggtggaca gccctcccct cccccacgcc agtttcgggg ccgccaagtt gtgcagcccg 1500 tgggccggga gcaccgaacg gacacagccc aggtcgtggc agggtctaga gtgggatgtc 1560 ccatggcccc catccaggcc tggggatatc ctcatccgcc tcccagaatc gggccgtggg 1620 ggacagaagg ggcctgcgtg cgggcaggga gagtattttg gctctctcct gtcttcgggg 1680 tttacaaagt gtgttgggac ttgcggggct gctctgtcca agcctgggtc tggcgtccgc 1740 gtctctgagc ctgtgagtgc gtgcgctttc ctgcgtcctc ttgactgccg gtgctggggc 1800 tctgcgtcct gcgtccgcg 1819 BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication in color drawing will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0009] Figure 1 An overview of the in situ hybridization–proximity ligation assay (ISH-PLA) protocol is presented.

[0010] Figure 2Representative sequence of the SEPTIN 9 promoter and the positions (underlined) of 18 oligonucleotide probes designed to target the promoter region surrounding the CpG island (shown in italics) of the SEPTIN 9 promoter.

[0011] Figure 3A , Figure 3B and Figure 3C Illustration of visualization of SEPTIN 9 promoter methylation in prostate cancer cells (DU145). Figure 3A Control prostate cancer (DU145) cells incubated with LacZ probe (no PLA signal) stained with DAPI are shown. Figure 3B and Figure 3C Cells incubated with a SEPTIN 9 oligonucleotide probe are shown. PLA signals are shown in red. Two red dots per nucleus represent the diploid number of chromosome 17 in DU145 cells where SEPTIN 9 is localized. Figure 3B Overlay of PLA signal and DAPI staining is presented. Figure 3C An overlay of PLA signal and differential interference contrast (DIC) is presented.

[0012] Figure 4A and 4B Shows SEPTIN 9 promoter methylation in colon cancer cells (SW480). PLA signal is shown in red. Three red dots per nucleus represent the triploid number of chromosome 17 in SW480 cells where SEPTIN 9 is localized. Figure 4A PLA signal and DAPI overlay are shown. Figure 4B Overlay of DIC and PLA signals is presented.

[0013] Figure 5 Figure 3 shows reduced SEPTIN 9 promoter methylation after 5-AzaC treatment of SW480 cells. Several drug-treated cells showed cytotoxic effects in terms of altered cell shape, which led to capture of the PLA probe and was responsible for the diffuse nonspecific red fluorescence observed in several cells.

[0014] Details

[0015] The present disclosure provides a method for the interaction between the modified nucleotides or specific nucleic acid sequences and proteins or other specific nucleic acid sequences in the specific nucleic acid sequences in the visualization individual cells. The method includes coupling the in situ hybridization (ISH) reaction with the proximity ligation assay (PLA) reaction, and can be used to visualize epigenetic markers at the single genomic locus in the individual cells, such as DNA methylation. Many diseases, such as cancer, are related to abnormal promoter hypermethylation. Therefore, the method disclosed herein can be used to detect cancer cells and / or as a diagnostic tool for screening cancer biopsy tissue samples under the background of normal cells.

[0016] I. Methods for Visualizing Modified Nucleotides in Cells

[0017] One aspect of the present disclosure provides a method for visualizing modified nucleotides in a target nucleic acid sequence in a cell. The method includes contacting a prepared cell with at least one nucleic acid probe, which is labeled with at least one marker and is complementary to a target-specific nucleic acid sequence, wherein the nucleic acid probe hybridizes with a specific nucleic acid sequence. The method also includes contacting the cell with a first binding agent that binds to the marker of the nucleic acid probe and a second binding agent that binds to the target modified nucleotide. Finally, the method includes detecting the first and second binding agents by a proximity ligation assay to visualize the modified nucleotides in the target-specific nucleic acid sequence in the cell.

[0018] (a) Step A - Hybridizing the labeled probe to the specific nucleic acid

[0019] The first step of the method comprises an in situ hybridization reaction. This step comprises contacting the prepared cell with at least one nucleic acid probe, which is labeled with at least one marker and has complementarity with a specific nucleic acid sequence, so that the nucleic acid probe hybridizes with a specific nucleic acid sequence comprising the modified nucleotides.

[0020] (i) Modified nucleotides

[0021] The methods disclosed herein can be used to detect and visualize various modified nucleotides. The modified nucleotides can be in DNA or RNA, and the nucleotides can be modified by methylation, hydroxylation, acetylation, formylation, acylation, carboxylation, thiolation, alkylation, amination, esterification, phosphorylation, or a combination thereof. Specific examples of modified nucleotides include 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 3-methylcytidine, N 4 -methylcytidine, N 4 -acetylcytidine, 2-thiocytidine, 1-methyladenosine, 2-methyladenosine, N 6 -methyladenosine, N 6 ,N6 -Dimethyladenosine, N 6 ,N 6 ,N 6 -trimethyladenosine, N 6 -Isopenyl adenosine, 2-methylthio-N 6 -Isopentyl adenosine, 1-methylguanosine, N 2 -methylguanosine, N 2 ,N 2 -Dimethylguanosine, N 2 ,N 2 ,N 2 -trimethylguanosine, 7-methylguanosine, xanthine riboside, inosine, 1-methylinosine, dihydrouridine, pseudouridine, 1-methylpseudouridine, 3-methyluridine, 5-methyluridine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, 5-carboxymethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, wybutoxosine, wybutosine, queuosine, ribosylthymine, pyrimidine dimers and 2'-O-methyl derivatives of standard or modified ribonucleotides. In specific embodiments, the modified nucleotide is 5-methylcytidine.

[0022] (ii) Specific nucleic acid sequence

[0023] The specific nucleic acid sequence comprising the modified nucleotides can and will vary. Non-limiting examples of suitable nucleic acid sequences include chromosomal DNA, transcriptional control regions of DNA, promoter DNA, CpG, enhancer DNA, silencer DNA, locus control regions of DNA, protein-coding DNA, intronic DNA, RNA-coding DNA, episomal DNA, viral RNA, messenger RNA (mRNA), microRNA (miRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, Y RNA, spliced ​​leader RNA (SL RNA), telomerase RNA component (TERC), small interfering RNA (siRNA), Piwi interacting RNA (piRNA) or trans-acting RNA (rasiRNA).

[0024] The feature of the specific nucleic acid sequence comprising the modified nucleotide can and will change, provided that the sequence of all or part of the specific nucleic acid sequence is known.In some embodiments, the specific nucleic acid sequence can be the region of the chromosome sequence (that is, gene) encoding the protein related to the disease or illness, and the disease or illness is related to abnormal gene expression.Abnormal gene expression can be related to hypermethylation or hypomethylation promoter control region.Non-limiting examples of the disease or illness related to abnormal gene expression include cancer (such as colon cancer, gastric cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, rectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, brain cancer, glioma, leukemia, melanoma, prostate cancer and head and neck cancer), autoimmune diseases (such as type 1 diabetes, inflammatory bowel disease), inflammatory diseases (such as asthma) and metabolic diseases. For example, non-limiting examples of genes associated with cancer include Septin 9, ATM, APC, BRCA1, BRCA2, CDH1, E-Cad, CDKN2B, DAPK, FANCB, FANCF, GATA-4, GATA-5, GSTP1, HER2, HIC1, MGMT, MLH1, MSH2, MSH4, NEIL1, PITX2, p14ARF, p15INK4B, p16INK4A, p53, p73, RAD51C, RASSF1, RB, TIMP3, VHL, and WRN. Examples of genes associated with an autoimmune or inflammatory disease or disorder include, but are not limited to, AAA1, ABCB1, ARTS-1, ATG16L1, BSN, CLSTN3, CTLA4, ERBB3, FCER1A, GSTP1, GSDML, HLA, HLA-DQA1, IFIH1, IL2Ra, IL23R, IBD5, IRGM, JAZF1, KIQQ1109, LNPEP, LPP, MYO9B, MST, NKKX2-3, NELL1, NOD2, NOTCH2, PLA2G7, PPATG, PTPN2, RGS1, SH2B3, TAGAP, THADA, TNF, and WSF1. In other embodiments, the modified nucleotides can be in specific mRNA sequences (e.g., transcripts of any of the genes listed above), in specific miRNAs (e.g., miRNAs associated with colon cancer include miR-551a, miR-552, miR-138, miR-451, miR-144, miR548h, miR-658, miR-595, miR-338-3p, etc.), or in specific lncRNAs (e.g., RepA, HOTAIR, Airnm, Kcnq1ot1, Evf-2, HSR1, SRA, NRON, MALAT1, NEAT2, etc.).

[0025] (iii) Nucleic acid probe

[0026] One or more nucleic acid probes used in the method are labeled with at least one marker and have complementarity with a specific nucleic acid sequence comprising the modified nucleotides. The nucleic acid probe may be DNA, RNA, LNA, PNA, or a combination thereof; may comprise standard nucleotides or nucleotide analogs; and may comprise a standard sugar-phosphate backbone or a modified backbone (e.g., phosphorothioate). The nucleic acid probe may be single-stranded, or the nucleic acid probe may be double-stranded (and denatured before use). The nucleic acid probe may be linear or cyclic and may comprise a secondary structure (e.g., hairpin, loop, stem, protrusion, etc.).

[0027] The length of the nucleic acid probe can vary. For example, the nucleic acid probe can be a length ranging from about 10 nucleotides to thousands of nucleotides. In some embodiments, the nucleic acid probe can be a length of about 10 to 30 nucleotides, a length of about 15 to 25 nucleotides, or a length of about 20 nucleotides. In other embodiments, the length of the nucleic acid probe can be about 15 to 500 nucleotides, about 30 to 100 nucleotides, about 100 to 300 nucleotides, about 300 to about 1000 nucleotides, or about 1000 to about 10000 nucleotides. Thus, in various embodiments, the nucleic acid probe can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 1750, 2000, 2250, 2500, 27500, 3000, 3250, 3500, 375000 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 nucleotides, or any integer falling between given adjacent sets of the above integers (e.g., 11, 12, 13 or 14, 16, 17, 18 or 19; 21, 22, 23 or 24, etc.).

[0028] The nucleic acid probe is labeled with at least one marker. The marker may be a hapten or a dye. Non-limiting examples of suitable haptens and dyes include biotin, digoxigenin, dinitrophenyl, fluorescein, diethylaminocoumarin, rhodamine, cyanine 3, cyanine 5, and Texas Red. In some embodiments, the marker may be located at the 5'-end or the 3'-end of the nucleic acid probe. For example, the marker may be attached to either end of the nucleic acid probe via a linker (e.g., a tetraethylene glycol (TEG) spacer, a polyethylene glycol (PEG) spacer, a C6 linker, or another linker known in the art). In other embodiments, the marker may be located throughout the nucleic acid probe, i.e., the nucleic acid probe may contain labeled nucleotides.

[0029] The nucleic acid probe can be synthesized chemically or enzymatically. In one embodiment, the nucleic acid probe can be synthesized using standard phosphoramidite solid phase synthesis techniques. The resulting probe can be end-labeled with a marker using standard procedures. In another embodiment, the DNA probe labeled can be synthesized by nick translation in the presence of labeled nucleotides (e.g., labeled dATP or labeled dCTP). In yet another embodiment, the RNA probe labeled can be synthesized by in vitro transcription in the presence of labeled ribonucleotides (e.g., labeled UTP).

[0030] The nucleic acid probe is designed to have complementarity with the target-specific nucleic acid (i.e., can be paired with the target-specific nucleic acid base). In various embodiments, the nucleic acid probe can have about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity with the target-specific nucleic acid. In a specific embodiment, the nucleic acid probe has at least about 90% or 95% sequence complementarity with the target-specific nucleic acid.

[0031] In some embodiments, the nucleic acid probe comprises a population of short nucleic acid sequences, each of which is generally complementary to an adjacent region of a target-specific nucleic acid, such that after hybridization, the nucleic acid probe "tiles" on the target-specific nucleic acid. In other embodiments, the nucleic acid probe is a longer nucleic acid sequence that includes labeled nucleotides throughout the probe length.

[0032] (iv) cell

[0033] Various cells can be used in the methods disclosed herein. Generally, the cell is a eukaryotic cell. In various aspects, the cell can be a human cell, a non-human mammal cell, a non-mammalian vertebrate cell, an invertebrate cell, an insect cell, a plant cell, a yeast cell or a unicellular eukaryotic organism. The cell can be a normal cell, an abnormal cell or a cancer cell. The cell can be a primary cell or a cell line cell. The cell can be an adult cell or an embryonic cell (e.g., an embryo). In other aspects, the cell can be a stem cell. Suitable stem cells include, but are not limited to, embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, pluripotent stem cells, oligopotent stem cells, unipotent stem cells, etc. In exemplary aspects, the cell is a mammalian cell.

[0034] In some embodiments, the cell may be a human cell line cell. Non-limiting examples of suitable cell lines include DU145 (metastatic carcinoma), SW490 (colon cancer), DLD-1 (colon cancer), KM20L2 (colon cancer), COLO 205 (colon cancer), HCC-2998 (colon cancer), HCT-116 (colon cancer), HCT-15 (colon cancer), HT29 (colon cancer), KM12 (colon cancer), SW-620 (colon cancer), SF-268 (CNS), SF-295 (CNS), SF-539 (CNS), SNB-19 (CNS), SNB-75 (CNS), U251 (CNS), CCRF-CEM (leukemia), HL-60 (TB) (leukemia), K-562 (leukemia), MOLT-4 (leukemia), RPMI-8226 (leukemia), SR (leukemia), A549 (non-small cell lung cancer), EKVX (non-small cell lung cancer), HOP-62 (non-small cell lung cancer), HOP-92 (non-small cell lung cancer), NCI-H226 (non-small cell lung cancer), NCI -H23 (non-small cell lung cancer), NCI-H322M (non-small cell lung cancer), NCI-H460 (non-small cell lung cancer), NCI-H522 (non-small cell lung cancer), LOXIMVI (melanoma), MALME-3M (melanoma), M14 (melanoma), MDA-MB-435 (melanoma), SK-MEL-2 (melanoma), SK-MEL-28 (melanoma), SK-MEL-5U (melanoma), ACC-257 (melanoma), UACC-62 (melanoma), IGR-OV1 (ovarian), OVCAR-3 (ovarian), OVCAR-4OVCAR-5 (ovarian), OVCAR-8 (ovarian), SK-OV-3 (ovarian), 786-0 (kidney), A498 (kidney), ACHN (kidney), CAKI-1 (kidney), RXF 393 (kidney), SN12C (kidney), TK-10 (kidney), UO-31 (kidney), PC-3 (prostate), DU-145 (prostate), MCF7 (breast), MDA-MB-231 (breast), MDA-MB-468 (breast), HS578T (breast), BT-549 (breast), and T-47D (breast).

[0035] In other embodiments, the cell can be a mammalian cell line cell. Non-limiting examples of suitable mammalian cell lines include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells; mouse myeloma NS0 cells, mouse embryonic fibroblast 3T3 cells (NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2 / 0 cells; mouse embryonic mesenchymal C3H-10T1 / 2 cells; mouse cancer CT26 cells, mouse prostate DuCuP cells; Mouse mammary gland EMT6 cells; mouse hepatoma Hepa1c1c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mouse myocardial MyEnd cells; mouse kidney RenCa cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; rat glioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35 cells; rat hepatoma cell carcinoma (HTC); buffalo rat liver BRL 3A cells; canine kidney cells (MDCK); canine mammary gland (CMT) cells; rat osteosarcoma D17 cells; rat monocyte / macrophage DH82 cells; monkey kidney SV-40 transformed fibroblasts (COS7) cells; monkey kidney CVI-76 cells; African green monkey kidney (VERO-76) cells; and human embryonic kidney cells (HEK293, HEK293T). An extensive listing of mammalian cell lines can be found in the catalog of the American Type Culture Collection (ATCC, Manassas, VA).

[0036] In still other embodiments, the cell can be in a tissue sample or a fluid sample obtained from a subject. For example, a tissue sample or a fluid sample can be removed by surgical excision, excisional biopsy, incisional biopsy, core biopsy or needle aspiration biopsy. The subject can be a person, a non-human mammal (for example a rodent, a cat, a dog, livestock animals, etc.) or a non-mammalian vertebrate (for example a fish, a bird, etc.). The tissue sample can be frozen or fixed with a fixative, as described in detail below. The fixed tissue sample can be embedded in an embedding medium such as paraffin, plasticized paraffin (paraplast) or a similar embedding medium.

[0037] (v) Preparation of cells

[0038] Cells are prepared for in situ hybridization by fixing cells, permeabilizing cells and optionally denaturing the chromosome DNA of cells. Various fixatives can be used for fixing or cross-linking cells. Examples of suitable fixatives include acetone, acetic acid, ethanol, formaldehyde (or formalin, a 37% aqueous solution of formaldehyde), glutaraldehyde, iodoform, lactic acid, methanol, paraformaldehyde, picric acid and combinations thereof. In a specific embodiment, the fixative can be paraformaldehyde. The concentration of the fixative and the duration of the fixation process will vary according to the type of cell (e.g., cells in a cell line cell or tissue).

[0039] Permeabilize fixed cells by incubating with a solution comprising at least one surfactant and / or protease. The non-limiting examples of suitable surfactants include Tween-20, Tween-80, Triton X-100, cetyl alcohol, decyl glucoside, digitonin, dodecyl glucoside, IGEPAL CA-630, leucoperm, NP-40, nonoxynol-9, octaethylene glycol monododecyl ether, n-octyl β-D-thiopyranoglucoside, oleyl alcohol, octyl glucoside, polysorbate 20, polysorbate 80, saponin, stearyl alcohol or its combination. Suitable protease includes but is not limited to proteinase K, caspase (caspase), chymotrypsin, papain, pepsin and trypsin. In some embodiments, cells are incubated with a solution comprising Tween-20 and / or TritonX-100. The concentration of surfactant or protease and the duration of the incubation period can and will vary according to the type of cell, tissue or fluid.

[0040] In the embodiment where the specific nucleic acid sequence of the nucleotides comprising the modification is double-stranded, the cell is contacted with a denaturing solution to convert the double-stranded nucleic acid into a single-stranded nucleic acid. The denaturing solution can be acidic or it can be alkaline. Acidic solutions include acids such as hydrochloric acid, and alkaline solutions include alkalis such as alkali metal hydroxides (e.g., sodium hydroxide or potassium hydroxide). The concentration of acid or alkali in the denaturing solution and the duration of the denaturing step can and will vary according to the type of cell, tissue or fluid.

[0041] After nucleic acid denaturation, the cell can be contacted with a protease to remove protein from the nucleic acid, thereby making the specific nucleic acid more accessible to the nucleic acid probe. Non-limiting examples of suitable proteases include caspase, chymotrypsin, papain, pepsin, proteinase K, trypsin and combinations thereof. The concentration of the protease and the duration of the incubation period can and will vary according to the type of cell, tissue or fluid.

[0042] In some embodiments, the cell can be in a tissue sample or in a section of a tissue sample. The sample can be a frozen sample or a formalin-fixed paraffin-embedded (FFPE) sample. When the sample is a frozen sample, the cell can be fixed and permeabilized, the chromosomal DNA can be denatured, and the cell can be contacted with a protease as described above. When the sample is a FFPE sample, dimethylbenzene or other organic solvents can be used to dewax the sample, the cell can be permeabilized, the chromosomal DNA can be denatured, and the cell can be contacted with a protease as described above.

[0043] (vi) Hybridization

[0044] Described method comprises making the cell of preparation contact with the nucleic acid probe of labeling to form the cell of hybridization, and wherein the nucleic acid probe of labeling carries out base pairing with the specific nucleic acid sequence of the nucleotide comprising modification.For this reason, the cell of preparation and the nucleic acid probe of labeling are hatched under the condition of intracellular (in situ) hybridization between the nucleic acid probe of labeling and the target nucleic acid sequence.In situ hybridization method and in situ hybridization solution are known in the art.In specific embodiments, described hybridization solution can contain 50% formamide, 10%Ficoll 400, 0.1%SDS and 2X SSC (sodium chloride-sodium citrate).The temperature and duration of hybridization reaction can change according to the type of cell or tissue.For example, in the embodiment that wherein cell is cultured cell, described hybridization can carry out about 16 hours at about 37 ℃.

[0045] (b) Step B—Binding the First and Second Binding Agents

[0046] The method also includes contacting a hybrid cell containing a hybridized nucleic acid probe-specific nucleic acid sequence complex with a first binding agent that binds to a marker of the nucleic acid probe and a second binding agent that binds to a target modified nucleotide. The first and second binding agents can each independently be a protein, an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment (e.g., an Fc fragment or a Fab fragment), a nucleic acid, or an aptamer. In a specific embodiment, each of the first and second binding agents is an antibody, wherein each antibody is prepared in a different species. For example, the first binding agent can be a rabbit polyclonal antibody that recognizes a marker in the labeled nucleic acid probe, and the second binding agent can be a mouse monoclonal antibody that recognizes the modified nucleotide. Methods for incubating cells with antibodies are well known to those skilled in the art, as are suitable solutions (e.g., blocking solutions, rinse or wash solutions, incubation solutions, etc.).

[0047] (c) Step C—Detection of the First and Second Binding Agents

[0048] The method also includes detecting first and second binding agents in close proximity. The first binding agent binds to a marker of the hybridized nucleic acid probe, and the second binding agent binds to each and every modified nucleotide in the cell. In order to detect the first and second binding agents bound to the hybridized nucleic acid probe-specific nucleic acid sequence complex (and thus detect the modified nucleotides in the target specific nucleic acid sequence), a proximity ligation assay (PLA) is used to detect the first and second binding agents in close proximity (i.e., within about 40 nm, Gomez et al., 2013, Nature Methods, 10(2): 171-177). PLA kits are commercially available (e.g., Sigma-Aldrich). PLA signals can be detected and analyzed using standard fluorescence microscopy and image analysis software programs.

[0049] (d) Specific implementation plan

[0050] In one embodiment, the modified nucleotide can be 5-methylcytidine, the specific nucleic acid sequence can be Septin 9 promoter DNA, and the cell can be a human cancer cell (e.g., a colon cancer cell). The specific nucleic acid sequence can be labeled with biotin, and the first binding agent can be an anti-biotin polyclonal antibody produced in rabbits. The second binding agent can be an anti-5-methyl-cytosine monoclonal antibody produced in mice.

[0051] II. Methods for Visualizing Nucleic Acid Interactions in Cells

[0052] Another aspect of the present disclosure provides a method for visualizing the interaction between specific nucleic acids in a cell. The method includes contacting a prepared cell with a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe comprises a first marker and has complementarity with a first nucleic acid sequence, and the second nucleic acid probe comprises a second marker and has complementarity with a second nucleic acid sequence, wherein the first and second markers are different, and the first and second nucleic acid probes hybridize with the first and second nucleic acid sequences, respectively. The method also includes contacting the cell with a first binding agent that binds to the first marker and a second binding agent that binds to the second marker, and then detecting the first and second binding agents by a proximity ligation assay to visualize the interaction between the two nucleic acids in the cell.

[0053] Various nucleic acid interactions can be detected using disclosed methods. Non-limiting examples include interactions between RNA and DNA (e.g., non-coding RNA or functional RNA interacts with specific chromosome sequences during transcription, replication, recombination, repair or another nuclear process), interactions between different classes of RNA (e.g., during RNA processing, splicing, translation, nuclear translocation and other cell events). Examples of suitable nucleic acids are described in detail in Part I (a) (ii) as above.

[0054] The first and second nucleic acid probes are each substantially as described above in section 1(a)(iii), however, the first and second nucleic acid probes are each labeled with different markers. In one embodiment, the first nucleic acid probe can be labeled with biotin and the second nucleic acid probe can be labeled with digoxigenin. In another embodiment, the first nucleic acid probe can be labeled with biotin and the second nucleic acid probe can be labeled with fluorescein (e.g., fluorescein isothiocyanate, FITC).

[0055] As various changes could be made in the above methods without departing from the scope of the invention, it is intended that all matter contained in the above description and examples given below shall be interpreted as illustrative and not in a limiting sense.

[0056] definition

[0057] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Edition, R. Rieger et al. (ed.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, as used herein, the following terms have the meanings deemed to belong to them.

[0058] When introducing elements of the present disclosure or preferred aspects thereof, the articles "a", "an", "the" and "said" mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements besides the listed elements.

[0059] As used herein, the term "complementary" or "complementarity" refers to the association of double-stranded nucleic acids by base pairing via specific hydrogen bonds. Base pairing can be standard Watson-Crick base pairing (e.g., 5'-AGT C-3' pairs with the complementary sequence 3'-TCA G-5'). Base pairing can also be Hoogsteen or reverse Hoogsteen hydrogen bonding. Complementarity is usually measured relative to the duplex region, so that, for example, overhangs are excluded. If only some base pairs are complementary, the complementarity between the two chains of the duplex region can be partial and expressed as a percentage (e.g., 70%). Non-complementary bases are "mismatched". If all base pairs in the duplex region are complementary, complementarity can also be complete (i.e., 100%).

[0060] The term "CpG island" refers to a cluster of CpG sites, where a CpG site refers to a region of DNA where cytosine nucleotides are present adjacent to guanine nucleotides along its length in a linear base sequence, where "CpG" is an abbreviation for a "-C-phosphate-G-" bond, i.e., cytosine and guanine separated by a single phosphate.

[0061] As used herein, "gene" refers to a DNA region encoding a gene product (including exons and introns) and all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0062] The terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotide or ribonucleotide polymers in linear or circular configurations and in single-stranded or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. The term can include known analogs of natural nucleotides, as well as nucleotides modified in base, sugar and / or phosphate moieties (e.g., phosphorothioate backbones). Typically, analogs of a particular nucleotide have the same base pairing specificity; i.e., an analog of A will base pair with T.

[0063] The term "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. The nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or a nucleotide analog. Nucleotide analogs refer to nucleotides with modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine) or non-naturally occurring nucleotides. Non-limiting examples of modifications on the sugar or base moieties of nucleotides include adding (or removing) acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, and replacing the carbon and nitrogen atoms of the bases (e.g., 7-deazapurine) with other atoms. Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.

[0064] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0065] The technology for determining nucleic acid and amino acid sequence identity is known in the art. Generally, such technology includes determining the nucleotide sequence of the mRNA of the gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. It is also possible to determine and compare genomic sequences in this way. Generally, identity refers to the exact nucleotide and nucleotide or amino acid and amino acid correspondence of two polynucleotides or polypeptide sequences respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percentage identity. The percentage identity of two sequences (whether nucleic acid or amino acid sequence) is the number of the exact matches between the two comparison sequences divided by the length of the shorter sequence and multiplied by 100. The approximate comparison of nucleotide sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). The algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff, 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and standardized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986). An exemplary implementation of the algorithm to determine the percent identity of a sequence is provided by Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code = standard; filter = none; chain = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs can be found on the GenBank website. Example

[0066] The following examples illustrate certain aspects of the invention.

[0067] Example 1: Visualizing DNA methylation on the SEPTIN9 promoter in individual cancer cells

[0068] The purpose of the following examples is to develop a highly sensitive detection method to detect DNA methylation at a single genomic locus in individual cells. To this end, a method was developed to perform in situ hybridization followed by a proximity ligation assay (PLA) (also known as Sigma-Aldrich) and cell imaging to visualize DNA methylation (5meC) on the SEPTIN 9 promoter. SEPTIN 9 promoter methylation is a known biomarker for colon cancer (Wasserkort et al., 2013, BMC Cancer, 13: 398). After optimizing cell cross-linking (i.e., fixation), cell permeabilization, and chromatin accessibility, genomic specificity was verified by hybridization with a pool of biotinylated oligonucleotide probes targeting CpG islands in the human SEPTIN 9 promoter. PLA assays were performed using anti-biotin and anti-5meC antibodies, corresponding (+ and -) PLA probes, and Far Red detection reagents. Figure 1 The strategy is outlined.

[0069] Oligonucleotide (20-mer) probes were designed using free online software (ie, Stellaris FISH Probe Designer) against the sequence of human SEPTIN 9. Probes with 3' TEG-biotin modification were synthesized by Sigma Custom Products and purified by HPLC. Figure 2 The SEPTIN 9 promoter sequence is presented and the positions of the 18 oligonucleotide probes are indicated.

[0070] Human cancer cells, DU145 (metastatic carcinoma; derived from the prostate, migrated to the brain) or SW480 (colon carcinoma), were grown in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS and Pen / Strep antibiotics and seeded (~10 cells / well) per well of 8-well chamber cover slip plates (with silicone gaskets). 4Cells were washed twice with HBSS and then fixed with 4% paraformaldehyde solution (40 μL / well, 16% paraformaldehyde stock diluted 1:4 in 1X PBS) for 30 minutes at room temperature (RT). Cross-linking was terminated by adding 5 μL / well of 1.25 M glycine and incubating at room temperature for 5 minutes. Cells were permeabilized by incubation with permeabilization buffer (0.75% Tween 20 and 0.75% Triton X-100 in 1X PBS) for 1 hour at room temperature. Cells were rinsed with 1X PBS. For detection of 5-meC DNA, cells were treated with 4N HCl for 10 minutes at room temperature. Cells were washed with 1X PBS for 30 minutes at room temperature. Cells were treated with pre-warmed (37°C) 0.25% trypsin-EDTA solution for 1 minute, and trypsin was inactivated by incubation in pre-warmed (37°C) quenching solution (i.e., DMEM supplemented with 10% FBS and 3 mg / mL BSA) for 2 minutes. The cells were washed with 1X PBS and blocked with in situ hybridization blocking solution for 1 hour at room temperature.

[0071] Cells were incubated with hybridization buffer (50% formamide, 10% Ficoll 400, 0.1% SDS, 2X SSC) prepared in water at 37°C using a slide hybridizer (Abbott Molecular) for 30 minutes. Oligonucleotide probes were denatured by adding hybridization buffer, and probes (i.e., (i) all 18 SEPTIN 9 probes, (ii) 5 SEPTIN 9 probes closest to the CpG island, or (iii) LacZ control probe; 100 μM each) were added to each well, which was sealed with a hybridization cover, and the slide hybridizer denaturation / hybridization program was run (i.e., denaturation at 80°C for 5 minutes, followed by hybridization at 37°C for 16 hours). Cells were washed three times with wash buffer (2X SSC, 0.1% NP-40) and then subjected to the PLA method, starting with the blocking step. The antibodies used were anti-biotin (Abcam 53494, polyclonal antibody raised in rabbit, diluted 1:200 in PLA antibody diluent) and anti-5-methylcytosine (Eurogentec BI-MECY-0100, monoclonal antibody raised in mouse, diluted 1:200 in PLA antibody diluent).

[0072] The SEPTIN 9 gene is located on human chromosome 17. Therefore, in a cancer cell line that is diploid for chromosome 17 and in which both copies of the SEPTIN 9 promoter are methylated, one would expect to see two punctate PLA (red) dots in the nucleus. This was observed in several DU145 cells incubated with a Septin 9 promoter-specific probe (i.e., probe set (ii) oligonucleotides #5-9 surrounding the methylated CpG island) ( Figure 3B and3C Similar data were obtained with probe set (i) (data not shown), but not with the nonspecific LacZ probe ( Figure 3A No PLA signal was observed in normal foreskin fibroblasts (BJ) or pancreatic cancer cells (BxPC3), data not shown.

[0073] The specificity of methylated SEPTIN 9 promoter detection was further confirmed by performing the same ISH-PLA assay in the colon cancer cell line SW480, which is triploid for chromosome 17. Indeed, when hybridized with SEPTIN9 probe set (ii), three PLA dots per nucleus were observed in several SW480 cells ( Figure 4A and 4B ), but not when hybridized to the LacZ probe (data not shown). This nuclear ISH-PLA signal was lost in SW480 cells treated with 5-azacytidine (5-AzaC, 500 nM, 24 h), a drug known to block DNA methylation in vivo, confirming the 5-meC epitope-dependent nature of the PLA signal ( Figure 5 ). Several drug-treated cells showed cytotoxic effects in terms of altered cell shape, which resulted in capture of the PLA probe and was responsible for the diffuse nonspecific red fluorescence observed in several cells. 5-AzaC is a pyrimidine nucleoside analog of cytidine with antitumor activity. Azacytidine is incorporated into DNA where it reversibly inhibits DNA methyltransferases, thereby blocking DNA methylation.

Claims

1. A non-disease diagnostic method for visualizing the interaction between two nucleic acid sequences in a cell, the method comprising: a) contacting the prepared cells with a first nucleic acid probe and a second nucleic acid probe, wherein the first nucleic acid probe comprises a first label and has complementarity with a first nucleic acid sequence, and the second nucleic acid probe comprises a second label and has complementarity with a second nucleic acid sequence, wherein the first and second labels are different, and the first and second nucleic acid probes hybridize with the first and second nucleic acid sequences, respectively, wherein the prepared cells are fixed and permeabilized; b) contacting the cells from step a) with a first binding agent that binds to said first marker and a second binding agent that binds to said second marker; c) detecting the first and second binding agents by a proximity ligation assay to visualize the interaction between the two nucleic acid sequences in the cell.

2. The method of claim 1, wherein the first and second nucleic acid sequences are each selected from promoter DNA, enhancer DNA, CpG island DNA, coding DNA, intronic DNA, messenger RNA, microRNA, noncoding RNA, long noncoding RNA, ribosomal RNA, transfer RNA, small nuclear RNA, small nucleolar RNA, SmY RNA, Y RNA, spliced ​​leader RNA, telomerase RNA component, small interfering RNA, Piwi-interacting RNA or trans-acting RNA.

3. The method of claim 1 or 2, wherein the first and second labels are each selected from biotin, digoxigenin, dinitrophenyl, fluorescein, diethylaminocoumarin, rhodamine, cyanine 3, cyanine 5, or Texas Red.

4. The method of claim 1 or 2, wherein the first and second nucleic acid probes are each linear or circular, comprise DNA, RNA, LNA, or a combination thereof, and have a length of 15 nucleotides to 500 nucleotides.

5. The method of claim 1 or 2, wherein the cell is an individual cell that is a eukaryotic cell, or the cell is within a tissue sample or a fluid sample obtained from a eukaryotic organism.

6. The method of claim 1 or 2, wherein the cell is an individual cell that is a mammalian cell.

7. The method of claim 1 or 2, wherein the cell is an individual cell that is a human cell.

8. The method of claim 1 or 2, wherein the cell is an individual cell that is a normal cell.

9. The method of claim 1 or 2, wherein the cell is an individual cell that is a cancer cell.

10. The method of claim 1 or 2, wherein the prepared cells contain denatured chromosomal DNA.

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