Oligonucleotide probes and uses thereof

Abstract

Methods and compositions are provided to identify oligonucleotides that bind targets of interest. The targets include tissues, cells, circulating biomarkers such as microvesicles, including those derived from various diseases. The oligonucleotides can be used in diagnostic and therapeutic applications.

1 - 138 . (canceled) 
     
     
         139 . A method of visualizing a fixed tissue sample comprising:
 (a) permeabilizing the sample;   (b) performing antigen retrieval on the permeabilized sample;   (c) contacting the sample from step (b) with a plurality of oligonucleotides, wherein the plurality of oligonucleotides are in a blocking solution;   (d) removing members of the plurality of oligonucleotides that do not bind the sample; and   (e) visualizing members of the plurality of oligonucleotides that are bound to the sample,.   
     
     
         140 . The method of  claim 139 , wherein the fixed tissue sample comprises formalin fixed paraffin embedded (FFPE) tissue. 
     
     
         141 . The method of  claim 140 , wherein the formalin fixed paraffin embedded (FFPE) tissue sample comprises at least one of an unstained slide, bone marrow core or clot, biopsy sample, surgical sample, core needle biopsy, malignant fluid, and fine needle aspirate (FNA). 
     
     
         142 . The method of  claim 140 , further comprising deparaffinization of the sample prior to antigen retrieval. 
     
     
         143 . The method of  claim 139 , wherein removing the members of the plurality of oligonucleotides that do not bind the sample comprises washing the sample for at least 4 times. 
     
     
         144 . The method of  claim 139 , wherein the permeabilization of the sample is performed by treating the sample with about 0.1% Triton-100 for about 10 minutes. 
     
     
         145 . The method of  claim 139 , wherein the blocking solution comprises about 0.01% Triton-100 and/or protein-blocking components. 
     
     
         146 . The method of  claim 139 , wherein the visualization comprises visualizing a signal linked directly or indirectly to the plurality of oligonucleotides. 
     
     
         147 . The method of  claim 146 , wherein the signal comprises a fluorescent signal or an enzymatic signal. 
     
     
         148 . The method of  claim 147 , wherein the enzymatic signal is produced by at least one of a luciferase, firefly luciferase, bacterial luciferase, luciferin, malate dehydrogenase, urease, peroxidase, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme, a saccharide oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, a heterocyclic oxidase, uricase, xanthine oxidase, lactoperoxidase, and microperoxidase. 
     
     
         149 . The method of  claim 148 , wherein the enzymatic signal is produced by HRP, and wherein the plurality of oligonucleotides are biotinylated and the members of the oligonucleotides that are bound to the sample are visualized using streptavidin-horse radish peroxidase (SA-HRP). 
     
     
         150 . The method of  claim 149 , wherein the streptavidin-horse radish peroxidase (SA-HRP) is contacted with the sample in a blocking solution. 
     
     
         151 . The method of  claim 148 , wherein the enzymatic signal is produced by HRP, and wherein the plurality of oligonucleotides are modified by digoxigenin (DIG) and the members of the oligonucleotides that are bound to the sample are visualized using anti-digoxigenin-horse radish peroxidase (anti-DIG-HRP). 
     
     
         152 . The method of  claim 139 , wherein the visualization comprises use of light microscopy or fluorescent microscopy. 
     
     
         153 . The method of  claim 139 , wherein the target of at least one of the at least one oligonucleotide or plurality of oligonucleotides is known. 
     
     
         154 . The method of  claim 139 , wherein the sample is from a subject suspected of having or being predisposed to a medical condition, disease, or disorder. 
     
     
         155 . The method of  claim 154 , wherein the disease or disorder comprises a cancer, a premalignant condition, an inflammatory disease, an immune disease, an autoimmune disease or disorder, a cardiovascular disease or disorder, neurological disease or disorder, infectious disease or pain. 
     
     
         156 . The method of  claim 155 , wherein the cancer comprises comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS -related cancers; AIDS -related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (including brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non- small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; pleuropulmonary blastema; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor. 
     
     
         157 . The method of  claim 156 , wherein the sample is a tumor sample. 
     
     
         158 . The method of  claim 139 , further comprising visualizing one or more additional sample(s) by repeating steps (a)-(e).