Mirna profiling compositions and methods of use

Abstract

Disclosed herein is a nanosensor of miRNA activity in a target cell, and methods of use, for detection and diagnostic applications. The nanosensor comprises a delivery particle comprising an iron oxide crystal coated with a polymer, and a sensor oligonucleotide covalently attached to the polymer. The sensor oligonucleotide comprises a seed region, comprising a nucleic acid sequence that is completely complementary to the target miRNA and comprises a cleavage site which can be engaged and cleaved by the target miRNA. The sensor oligonucleotide also comprises two non-seed regions that each flank the seed region and are each comprised of a nucleic acid sequence that is complementary to the target miRNA to promote hybridization of the sensor oligonucleotide to the target miRNA, and members of a quencher-fluorophore pair. The quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits significant quenching of emitted fluorescent signal.

What is claimed: 
     
         1 . A sensor oligonucleotide for detection of activity of a target miRNA comprising:
 a) a seed region comprising a nucleic acid sequence that is completely complementary to a target miRNA and comprises a cleavage site which can be engaged by the target miRNA and cleaved by the target miRNA in complex with RNA induced silencing complex;   b) two non-seed regions that each flank the seed region and are each comprised of nucleic acid sequences complementary to the target miRNA to promote hybridization of the sensor oligonucleotide to the target miRNA; and   c) members of a quencher-fluorophore pair;   wherein the quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits significant quenching of emitted fluorescent signal.   
     
     
         2 . The sensor oligonucleotide of  claim 1 , that has a nucleic acid sequence that is completely complementary to the target miRNA sequence. 
     
     
         3 . The sensor oligonucleotide of  claim 1 , that comprises RNA, or a combination of RNA and one or more other nucleic acid-like polymers that hybridize with RNA in a sequence dependent manner, wherein at least the entire seed region is RNA. 
     
     
         4 . The sensor oligonucleotide of  claim 3 , wherein the other nucleic acid-like polymer is DNA, LNA or a 2-0-Me/phosphorothioate backbone. 
     
     
         5 . The sensor oligonucleotide of  claim 1 , that is from about 18 to about 30 nucleotides in length. 
     
     
         6 . The sensor oligonucleotide of  claim 1  that is about 20-25 nucleotides in length. 
     
     
         7 . The sensor oligonucleotide of  claim 1 , wherein the quencher-fluorophore pair members are separated by a distance of about 9 to about 30 nucleotides. 
     
     
         8 . The sensor oligonucleotide of  claim 1 , wherein the fluorophore of the quencher-fluorophore pair has an emission maximal over 600 nm. 
     
     
         9 . The sensor oligonucleotide of  claim 1 , wherein the target miRNA is selected from the group consisting of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-10b, miR-340, miR-155, miR-15b, miR-186, miR-222, miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-200c, miR-29c, miR-424, and miR-141. 
     
     
         10 . The sensor oligonucleotide of  claim 4 , further comprising a targeting ligand covalently attached. 
     
     
         11 . The sensor oligonucleotide of  claim 10 , wherein the targeting ligand is a peptide specific for an internalizing receptor located on the exterior plasma membrane of the target cell. 
     
     
         12 . The sensor oligonucleotide of  claim 10 , wherein the targeting ligand is selected from the group consisting of RGD, folic acid, peptide EPPT (SEQ ID NO: 11), polyarginine peptide (MPAP), and chlorotoxin. 
     
     
         13 . The sensor oligonucleotide of  claim 10 , wherein the targeting ligand is covalently attached by thiol crosslinking. 
     
     
         14 . A method for assessing the activity of one or more target miRNA in a target cell comprising,
 a) delivering to the target cell an effective amount of one or more sensor oligonucleotides of  claim 1 ;   b) detecting fluorescence emitted from the one or more sensor oligonucleotides; and   c) comparing the fluorescence detected with that of a normal control cell to thereby assess if the activity of the target miRNA(s) is high, low or normal in the target cell.   
     
     
         15 . The method of  claim 14  further comprising comparing the fluorescence detected for each target miRNA with that of normal control cells to thereby generate a profile of each target miRNA in the target cell or population thereof as deviated from normal. 
     
     
         16 . The method of  claim 15 , wherein the steps are repeated periodically to thereby monitor progression of the miRNA activity in the target cells over time. 
     
     
         17 . The method of  claim 14 , wherein the sensor oligonucleotides are delivered or administered to thereby produce an intracellular concentration in the target cell of about 250 nM. 
     
     
         18 . The method of  claim 14  wherein the target cell is of a primary tumor, of a metastatic tumor, of a biopsy, virally infected, parasitically infected, or non-infected pathology. 
     
     
         19 . The method of  claim 14 , wherein the target cell is in vivo and delivery is by administration to a subject comprising the target cell. 
     
     
         20 . The method of  claim 14 , wherein delivering or administering is by intratumoral injection, transfection, or titration/incubation.