Compositions and methods for the detection of small RNAs

Patent metadata

Field	Value
Publication number	US-9714446-B2
Application number	US-201113025458-A
Country	US
Kind code	B2
Filing date	Feb 11, 2011
Priority date	Feb 11, 2010
Publication date	Jul 25, 2017
Grant date	Jul 25, 2017

Abstract

The invention provides compositions and methods for the detection of small RNA molecules in a multiplexed reaction. The assays and kits described herein are applicable for the identification, diagnosing, and monitoring of disorders including, but not limited to cancer, developmental and degenerative disease, neurological disorders, and stem cell disorders.

First claim

What is claimed is:

1. A composition comprising at least two tripartite complexes, each tripartite complex comprising:
(a) a tag molecule, wherein the tag molecule comprises a first DNA sequence and a plurality of reporter attachment regions and wherein the first DNA sequence comprises a DNA sequence having no greater than 85% identity across 35-50 bases with a maximum continuous homology of 15 bases or less to any known genomic DNA or RNA transcribed from a genome;
(b) one or more reporter molecules each non-covalently attached to one of the plurality of reporter attachment regions of the tag molecule, wherein the one or more reporter molecules together produces a code comprising an ordered series of colored fluorescent spots that identifies a target RNA molecule; and
(c) a bridge molecule, wherein the bridge molecule comprises a second DNA sequence that is complementary to the target RNA molecule and a third DNA sequence that is complementary to the first DNA sequence of the tag molecule;
wherein a first tripartite complex identifies a first target RNA molecule and comprises a bridge molecule that is complementary to the first target RNA molecule;
wherein an at least second tripartite complex identifies an at least second target RNA molecule and comprises a bridge molecule that is complementary to the at least second target RNA molecule; and
wherein the first and the at least second target RNA molecules are different.

2. The composition of claim 1 , wherein the second DNA sequence and the third DNA sequences of each bridge molecule are contiguous.

3. The composition of claim 1 , wherein the first target RNA molecule or the at least second target RNA molecules comprises a non-coding RNA.

4. The composition of claim 3 , wherein the non-coding RNA is a transfer RNA (tRNA), a small nucleolar RNA (snoRNA), a microRNA (miRNA), a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a repeat associated small interfering RNA (rasiRNA), or a piwi-interacting RNA (piRNA).

5. The composition of claim 3 , wherein the non-coding RNA is a miRNA.

6. The composition of claim 1 , wherein Bathe second DNA sequence of each bridge molecule and the target RNA molecule of each tripartite complex form a DNA/RNA heteroduplex having a melting temperature of between 37-95° C.

7. The composition of claim 1 , wherein the second DNA sequence of each bridge molecule and the target RNA molecule of each tripartite complex form a DNA/RNA heteroduplex having a melting temperature of between 44-53° C.

8. The composition of claim 1 , wherein the third DNA sequence of the each bridge molecule and the first DNA sequence of each tag molecule form a DNA/DNA duplex having a melting temperature of between 37-95° C.

9. The composition of claim 1 , wherein the third DNA sequence of each bridge molecule and the first DNA sequence of each tag molecule form a DNA/DNA duplex having a melting temperature of between 44-53° C.

10. The composition of claim 1 , wherein the second DNA sequence and the third DNA sequence of each bridge molecule form nucleic acid duplexes having substantially the same melting temperature.

11. The composition of claim 1 , wherein the third DNA sequence of each bridge molecule comprises a DNA sequence having no greater than 85% identity across 35-50 bases with a maximum continuous homology of 15 bases or less to any known genomic DNA.

12. The composition of claim 1 , wherein the target RNA molecule or the tag molecule of each tripartite complex contains a sequence that specifically hybridizes to the bridge molecule of the tripartite complex with complete complementarity.

13. The composition of claim 1 , wherein the target RNA molecule or the tag molecule of each tripartite complex contains a sequence that specifically hybridizes to the bridge molecule of the tripartite complex with partial complementarity.

14. The composition of claim 1 , wherein when each of said tripartite complex in the composition is complexed with its target RNA molecule, said target RNA molecule can be ligated to said tag molecule.

15. The composition of claim 1 , wherein at least one of said tripartite complexes comprises or is complementary to one or more of SEQ ID NO: 1 to SEQ ID NO: 35.

Assignees

• Webster Philippa J
• Dennis Lucas M
• Boykin Richard K
• Nanostring Technologies Inc

Inventors

• Webster Philippa J
• Dennis Lucas M
• Boykin Richard K

Classifications

• C12Q2525/207

  siRNA, miRNA · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q2561/125

  Ligase Detection Reaction [LDR] · CPC title