Methods and kits used in identifying microrna targets

Patent metadata

Field	Value
Publication number	US-2017009293-A1
Application number	US-201615275628-A
Country	US
Kind code	A1
Filing date	Sep 26, 2016
Priority date	Sep 16, 2011
Publication date	Jan 12, 2017
Grant date	—

Abstract

Described herein are methods and kits used to identify an endogenously expressed target mRNA of a microRNA of interest. The method involves the use of a dominant negative GW182 polypeptide that forms a stable complex with the target mRNA. The method further involves purifying the complex and identifying the target mRNA.

First claim

1 . A method of identifying a target mRNA of a microRNA of interest, the method comprising:
a. associating the microRNA of interest with a protein complex comprising a dominant negative GW182 polypeptide comprising at least 90% sequence identity with SEQ ID NO: 9 within a cell; b. purifying the complex comprising the dominant negative GW182 polypeptide and an endogenously expressed target mRNA of the microRNA of interest; and c. identifying the endogenously expressed target mRNA.

2 .- 3 . (canceled)

4 . The method of claim 1 , wherein the dominant negative GW182 comprises a mutation in its RRM domain.

5 . The method of claim 1 , wherein the dominant negative GW182 comprises a mutation in its silencing domain.

6 . The method of claim 1 , wherein the dominant negative GW182 comprises a deletion in its silencing domain.

7 . The method of claim 6 , wherein the deletion is a:
deletion of less than 550 amino acids; deletion of less than 100 amino acids; deletion of the entire RRM domain; or deletion of the entire silencing domain.

8 . (canceled)

9 . The method of claim 1 , wherein contacting the microRNA of interest with the dominant negative GW182 polypeptide comprises introducing into the cell a first nucleic acid construct, the first nucleic acid construct comprising a first polynucleotide sequence, the first polynucleotide sequence comprising the sequence of the microRNA of interest.

10 . The method of claim 9 , wherein the first polynucleotide sequence is
a pre-microRNA sequence of the microRNA of interest; or a mature sequence of the microRNA of interest.

11 . (canceled)

12 . The method of claim 1 , wherein contacting the microRNA of interest with the dominant negative GW182 polypeptide further comprises transfecting a cell with a second nucleic acid construct, the second nucleic acid construct comprising a second polynucleotide sequence that encodes the dominant negative GW182 polypeptide and third polynucleotide sequence that is a promoter operably linked to second polynucleotide sequence.

13 . The method of claim 12 , wherein the second nucleic acid construct is stably transfected.

14 . (canceled)

15 . The method of claim 13 , wherein the second nucleic acid construct further comprises a fourth polynucleotide sequence that is a sequence derived from a virus.

16 . The method of claim 15 , wherein the virus is selected from adenovirus and lentivirus.

17 . (canceled)

18 . The method of claim 15 wherein the second nucleic acid construct comprises a SEQ ID NO: 16.

19 . The method of claim 1 , wherein purifying the complex comprises contacting the complex with a first reagent that specifically binds to a component of the complex.

20 . The method of claim 19 , wherein the first reagent comprises an antibody.

21 . The method of claim 19 , wherein the first reagent specifically binds to the dominant negative GW182 polypeptide.

22 . The method of claim 21 , wherein the dominant negative GW182 polypeptide comprises a label and wherein the first reagent specifically binds to the label.

23 . The method of claim 22 , wherein the label is a myc tag, a FLAG® tag, or a His tag.

24 . The method of claim 23 , wherein
the label is biotin and the dominant negative GW182 polypeptide is encoded by SEQ ID NO: 19; the label is a myc tag and the dominant negative GW182 polypeptide is encoded by SEQ ID NO: 23; or the label is a His tag or FLAG® tag and wherein the dominant negative GW182 polypeptide is encoded by SEQ ID NO: 22.

25 . The method of claim 1 , wherein identifying the endogenously expressed target mRNA comprises a method selected from polymerase chain reaction, microarray analysis, and nucleic acid sequencing.

26 . The method of claim 25 , wherein identifying the endogenously expressed target mRNA comprises nucleic acid sequencing and wherein sequences that are enriched at least two-fold relative to a mean value of all sequences detected in the screen are identified as target mRNA.

27 . The method of claim 1 , wherein the microRNA of interest is a mutant form of microRNA relative to its native sequence.

28 . The method of claim 1 , further comprising confirming the regulation of the target mRNA by the microRNA of interest by transfecting the microRNA of interest into a cell and assessing the expression of a protein encoded by the target mRNA.

29 .- 36 . (canceled)

37 . The method of claim 16 , wherein the virus is a lentivirus and wherein the second nucleic acid construct comprises a sequence selected from SEQ ID NO: 26.

Assignees

• Univ Oregon Health & Science

Inventors

• Cambronne Xiaolu Lim Ang

Classifications

• C12Q1/702 Primary

  for retroviruses · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/178

  miRNA, siRNA or ncRNA · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q2600/136

  Screening for pharmacological compounds · CPC title