Compositions for rna-chromatin interaction analysis and uses thereof

Patent metadata

Field	Value
Publication number	US-2016177380-A1
Application number	US-201615059605-A
Country	US
Kind code	A1
Filing date	Mar 3, 2016
Priority date	Sep 5, 2013
Publication date	Jun 23, 2016
Grant date	—

Abstract

The invention described herein provides reagents (e.g., kits), compositions, and methods for carrying out an unbiased genome-wide strategy to identify the functional targets for all ncRNAs.

First claim

We claim:

1 . A kit comprising:
(1) an RNA linker comprising:
(i) a first polynucleotide, and,
(ii) a second polynucleotide,
wherein the first and the second polynucleotides form a first double stranded region flanked by a first ligation compatible end, and a 3′-overhang at the 3′-end of the first polynucleotide, wherein the 3′-overhang comprises a random-sequence primer; and,
(2) a DNA linker comprising:
(iii) a third polynucleotide, and,
(iv) a fourth polynucleotide,
wherein the third and the fourth polynucleotides form a second double stranded region flanked by a blunt end and a second ligation compatible end,
wherein the first and the second ligation compatible ends ligate to each other, or are adaptable to ligate to each other.

2 . The kit of claim 1 , wherein the first ligation compatible end is a 3′-overhang at the 3′-end of the second polynucleotide, and the second ligation compatible end is a 3′-overhang at the 3′-end of the third polynucleotide, wherein both 3′-overhangs anneal to each other for ligation.

3 . The kit of claim 1 , wherein the first double stranded region comprises a first recognition site for a first restriction enzyme (RE) that cleaves 3′ to the random-sequence primer.

4 . The kit of claim 1 , wherein the second double stranded region comprises a second recognition site for a second restriction enzyme (RE) that cleaves 5′ to the third polynucleotide.

5 . The kit of claim 1 , wherein one or more of said first, second, third, and fourth polynucleotides are DNA.

6 . The kit of claim 1 , wherein one or more of said first, second, third, and fourth polynucleotides comprise a modified nucleotide.

7 . The kit of claim 6 , wherein said modified nucleotide is a biotinylated T (Thymidine).

8 . The kit of claim 1 , wherein said first polynucleotide comprises a plurality of polynucleotides, each differing only at the random-sequence primer region.

9 . The kit of claim 1 , wherein said first polynucleotide comprises a homogeneous population of polynucleotides having identical random-sequence primer.

10 . The kit of claim 1 , wherein said random-sequence primer comprises 4, 5, 6, 7, 8, or more nucleotides.

11 . The kit of claim 1 , wherein the first double stranded region comprises a unique sequence that distinguishes the RNA linker from the DNA linker.

12 . The kit of claim 1 , wherein the second double stranded region comprises a unique sequence that distinguishes the RNA linker from the DNA linker.

13 . The kit of claim 1 , wherein the last nucleotide of the first recognition site is the last base-paired nucleotide 5′ to the random-sequence primer.

14 . The kit of claim 1 , wherein the last nucleotide of the second recognition site is a base-paired nucleotide at the blunt end.

15 . The kit of claim 1 , wherein the first and the second restriction enzymes are the same.

16 . The kit of claim 1 , wherein the first or the second restriction enzyme is independently selected from: AarI, AceIII, Alol, BaeI, Bbr7I, BbvI, BbvII, BccI, Bce83I, BceAI, BcefI, BcgI, BciVI, BfiI, BinI, BplI, BsaXI, BscAI, BseMII, BseRI, BsgI, BsmI, BsmAI, BsmFI, Bsp24I, BspCNI, BspMI, BsrI, BsrDI, BstF5I, BtgZI, BtsI, CjeI, CjePI, EciI, Eco31I, Eco57I, Eco57MI, EcoP15I, Esp3I, FalI, FauI, FokI, GsuI, HaeIV, HgaI, Hin4I, HphI, HpyAV, Ksp632I, MboII, MlyI, MmeI, Mn1I, PleI, PpiI, PsrI, RleAI, SapI, SfaNI, SspD5I, Sth132I, StsI, TaqII, TspDTI, TspGWI, TspRI or Tth111II.

17 . The kit of claim 1 , wherein the cleavage site of the first or the second restriction enzyme is at least about 10, 12, 14, 16, 18, 20, or more nucleotides 3′ to the last nucleotide of the recognition site.

18 . The kit of claim 1 , wherein the first and the fourth polynucleotides are dephosphorylated.

19 . The kit of claim 1 , further comprising a reagent that cross-links protein and polynucleotide.

20 . The kit of claim 19 , wherein the reagent comprises formaldehyde.

21 . The kit of claim 1 , further comprising an affinity reagent (e.g., an antibody, or a monoclonal antibody) that specifically or selectively bind a component of chromatin (e.g., histone).

22 . The kit of claim 1 , further comprising an end-repairing mixture that converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA.

23 . The kit of claim 1 , further comprising a DNA ligase (e.g., T4 ligase).

24 . The kit of claim 1 , further comprising a reagent that reverses cross-linking of protein and polynucleotide (e.g., Proteinase K).

25 . The kit of claim 1 , further comprising the first and/or the second restriction enzyme(s).

26 . The kit of claim 1 , further comprising a pair of concatenating adapters for PCR amplification of blunt-ended double stranded DNA.

27 . The kit of claim 1 , further comprising a Taq DNA polymerase.

28 . The kit of claim 1 , further comprising a reverse transcriptase.

29 . A paired-end tag (PET) polynucleotide comprising a central region comprising the first and second double stranded regions of claim 1 , said central region being flanked by:
(1) at a site proximal to said first double stranded region, a sequence tag of a non-coding RNA (ncRNA); and (2) at a site proximal to said second double stranded region, a sequence tag of a genomic DNA.

30 . The PET polynucleotide of claim 29 , wherein the sequence tag of the non-coding RNA (ncRNA) has a free end resulting from digestion by said first restriction enzyme.

31 . The PET polynucleotide of claim 29 , wherein the sequence tag of the non-coding RNA (ncRNA) uniquely identifies a genomic region from which the ncRNA is transcribed.

32 . The PET polynucleotide of claim 29 , wherein said sequence tag of the non-coding RNA (ncRNA) is about 8-30 base pairs in length.

33 . The PET polynucleotide of claim 29 , wherein the sequence tag of the genomic DNA has a free end resulting from digestion by said second restriction enzyme.

34 . The PET polynucleotide of claim 29 , wherein the sequence tag of the genomic DNA uniquely identifies a genomic region at which the genomic DNA is located.

35 . The PET polynucleotide of claim 29 , wherein said sequence tag of the genomic DNA is about 8-30 base pairs in length.

36 . A paired-end tag (PET) library comprising two or more members of the PET polynucleotide of claim 29 , wherein each member of the PET library comprises the same said central region, and different said sequence tag of the non-coding RNA (ncRNA) of claim 29 or different said sequence tag of the genomic DNA of claim 29 or both.

37 . A vector comprising a PET polynucleotide of claim 29 .

38 . The vector of claim 37 , comprising a plurality of concatenated PET polynucleotide of claim 29 .

39 . A concatemer of two or more PET polynucleotides of claim 29 .

40 . A method of identifying functional interaction loci within a genome for non-coding RNAs (ncRNAs) of the genome, the method comprising:
(1) providing chromatin fragments comprising cross-linked genomic DNA fragments and cross-linked ncRNAs; (2) using the RNA linker and the DNA linker of claim 1 , ligating an end of a cross-link

Assignees

• Jackson Lab

Inventors

• Ruan Yijun
• Zheng Meizhen
• Luo Junhong Oscar

Classifications

• C12Q2565/113

  based on agglutination/precipitation · CPC title

• C12Q2565/133

  conformational analysis · CPC title

• C12Q2525/207

  siRNA, miRNA · CPC title

• C12Q1/6806 Primary

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

• C12Q1/6809 Primary

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