miRNA profiling compositions and methods of use

Patent metadata

Field	Value
Publication number	US-10086093-B2
Application number	US-201414769398-A
Country	US
Kind code	B2
Filing date	Feb 26, 2014
Priority date	Feb 28, 2013
Publication date	Oct 2, 2018
Grant date	Oct 2, 2018

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.

First claim

What is claimed:

1. A nanosensor for detection of miRNA activity in a target cell comprising:
a) a delivery particle comprising an iron oxide crystal coated with a polymer; and
b) a sensor oligonucleotide covalently attached to the polymer, comprising:
i) 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;
ii) 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
iii) 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 quenching of emitted fluorescent signal.

2. The nanosensor of claim 1 , further comprising a targeting ligand covalently attached to the polymer.

3. The nanosensor of claim 2 , wherein the targeting ligand is a peptide specific for an internalizing receptor located on the exterior plasma membrane of the target cell.

4. The nanosensor of claim 2 , wherein the targeting ligand is selected from the group consisting of arginine-glycine-aspartic acid, folic acid, peptide EPPT (SEQ ID NO: 11), polyarginine peptide, and chlorotoxin.

5. The nanosensor of claim 2 , wherein the polymer is selected from the group consisting of polyethylene glycol, dextran, polyvinylpyrrolidone, fatty acids, polypeptides, chitosan and gelatin, chitosan, polyethylenimine, and combinations thereof.

6. The nanosensor of claim 1 , wherein the iron oxide crystal is from about 20-30 nm in diameter and the polymer is dextran.

7. The nanosensor of claim 2 , wherein the sensor oligonucleotide and/or to the targeting ligand are covalently attached to the polymer by thiol crosslinking.

8. The nanosensor of claim 1 , wherein the entire sensor oligonucleotide nucleic acid sequence is completely complementary to the target miRNA sequence.

9. The nanosensor of claim 1 , wherein the sensor oligonucleotide is RNA, or a combination of RNA and one or more other modified nucleotides that hybridize with RNA in a sequence dependent manner, wherein at least the entire seed region is RNA.

10. The nanosensor of claim 1 , wherein the sensor oligonucleotide is from about 18 to about 30 nucleotides in length or from about 20-25 nucleotides in length.

11. The nanosensor of claim 1 , wherein the members are separated by a distance of about 9 to about 30 nucleotides.

12. The nanosensor of claim 1 , wherein the fluorophore of the quencher-fluorophore pair has an emission maximal over 600 nm.

13. The nanosensor of claim 1 , wherein the delivery particle is functionalized with amines to thereby facilitate endosomal swelling and rupture upon cellular uptake.

14. The nanosensor 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.

15. The nanosensor of claim 1 , wherein the delivery particle is covalently attached to the sensor oligonucleotide at a ratio selected from the group consisting of about 1:1, 1:10, about 1:20, about 1:30, about 1:40, and about 1:50.

16. The nanosensor of claim 1 , wherein quenching is with an efficiency of greater than 95%.

17. The nanosensor of claim 1 , wherein quenching is with an efficiency of about 99%.

18. A method for assessing the activity of one or more target miRNA in a target cell comprising,
a) delivering an effective amount of one or more nanosensors of any of claims 1 - 17 to the target cell;
b) detecting fluorescence emitted from the one or more nanosensors; 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 higher than, lower than, or the same as in the target cell.

Assignees

• Massachusetts Gen Hospital

Inventors

• Moore Anna V
• Medarova Zdravka

Classifications

• C12N2320/10

  in screening processes · CPC title

• C12Q2600/178

  miRNA, siRNA or ncRNA · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title

• C12Q2525/207

  siRNA, miRNA · CPC title

• A61K49/10 Primary

  Organic compounds · CPC title