RNA microarray for detecting interaction between protein and RNA containing a higher-order structure

Patent metadata

Field	Value
Publication number	US-10435738-B2
Application number	US-201515110516-A
Country	US
Kind code	B2
Filing date	Jan 9, 2015
Priority date	Jan 10, 2014
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

Abstract

Interaction with a protein is detected by using an RNA probe containing the following sequences;
(i) a complementary strand sequence to a DNA barcode sequence, (ii) a sequence of a first stem portion, (iii) a sequence of a second stem portion complementary to the first stem portion for hybridizing with the first stem portion to form a double-stranded stem, and (iv) a sequence of a loop portion contained in RNA for linking the first and second stem portions.

First claim

The invention claimed is:

1. A method for detecting RNA which binds to a target protein, comprising the following steps:
(1) contacting an RNA microarray and a DNA microarray with a fluorescence-labeled target protein,
wherein the RNA microarray comprises:
a first multiplicity of DNA barcode sequences attached thereto; and
a multiplicity of RNA probes, each RNA probe comprising:
(i) a complementary strand sequence to each respective DNA barcode sequence in the first multiplicity of DNA barcode sequences;
(ii) a double stranded stem structure comprising a first stem portion complementary to and hybridized with a second stem portion; and
(iii) a sequence of a loop portion linking said first and second stem portions,

wherein each RNA probe is hybridized to each respective complementary DNA barcode sequence in the first multiplicity of DNA barcode sequences; and
wherein a combination of the sequence (i) and (iii) is different in the multiplicity of RNA probes so that each of the sequence (iii) in the multiplicity of RNA probes can be identified by the corresponding sequence (i), and the sequence (ii) has a common sequence in the multiplicity of RNA probes,
wherein the first multiplicity of DNA barcode sequences is selected from a tag, a zip code, an orthonormal sequence or a barcode sequence so as to prevent cross-hybridization reaction;

and
wherein the DNA microarray comprises:
a second multiplicity of DNA barcode sequences attached thereto, the second multiplicity of DNA barcode sequences being identical to the first multiplicity of DNA barcode sequences; and
having no RNA probes hybridized therewith;

(2) identifying RNA probe(s) of said multiplicity of RNA probes of said RNA microarray that bind to the target protein;
(3) identifying DNA barcode sequence(s) of said second multiplicity of DNA barcode sequences of said DNA microarray that bind to the target protein;
(4) identifying RNA probe(s) of step (2) that comprise a sequence complementary to the DNA barcode sequence(s) identified in step (3);
(5) eliminating said RNA probe(s) identified in step (4) from said RNA probe(s) identified in step (2); and
(6) determining the nucleotide sequence of the loop portion of the RNA probe(s) remaining after said elimination step of (5) above, thereby detecting RNA which binds to the target protein,
wherein the RNA probe(s) have a label that is different than the target protein,
wherein said first and second multiplicity of DNA barcode sequences are spotted to the RNA microarray and the DNA microarray so that each spot has a different DNA barcode sequence, and
wherein the RNA probe(s) are identified by positions of the spots on the RNA microarray and the DNA microarray in the identifying step (4).

2. The method according to claim 1 , wherein the sequence of the loop portion is a sequence of a loop portion contained in Pre-miRNA.

3. The method according to claim 1 , wherein each RNA probe of the multiplicity of RNA probes has a labeled 3′ end.

4. The method according to claim 2 , wherein each RNA probe of the multiplicity of RNA probes has a labeled 3′ end.

5. The method according to claim 1 , wherein the RNA probe(s) are labeled with a a radioactive isotope, digoxigenin (DIG), a fluorescent dye, or a molecule (antigen) such as biotin for detection.

6. The method according to claim 2 , wherein the RNA probe(s) are labeled with a a radioactive isotope, digoxigenin (DIG), a fluorescent dye, or a molecule (antigen) such as biotin for detection.

Assignees

• Univ Kyoto

Inventors

• Saito Hirohide
• Taya Toshiki
• Kashida Shunichi

Classifications

• C12Q1/6816 Primary

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

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title