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US 2014/0213460 A1Published application
RNA-YY1 Interactions
AssigneeThe General Hospital Corporation
InventorsJeannie T. Lee, Yesu Jeon
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Abstract
Methods relating to obtaining libraries of YY1-binding long non-coding RNAs, libraries obtained thereby, and methods of use thereof.
Claims19
A method of preparing a library of nuclear ribonucleic acids (nRNAs) that specifically bind YY1, the method comprising:
- (a) contacting a sample containing nRNAs, e.g. at least 104 different nRNAs, with (i) YY1 protein and (ii) a YY1 binding agent, under conditions sufficient to form complexes between the nRNA, YY1 protein and the YY1 binding agent, and
- (b) isolating the complexes.
A method of preparing a plurality of cDNAs complementary to a pool of nuclear ribonucleic acids (nRNAs), the method comprising:
- (a) providing a sample comprising nuclear ribonucleic acids, e.g., a sample comprising nuclear lysate, e.g., comprising nRNAs bound to nuclear proteins;
- (b) contacting the sample with an agent, e.g., an antibody, that binds specifically to YY1 protein, under conditions sufficient to form complexes between the agent and YY1 proteins, e.g., such that the nRNAs remain bound to the YY1 proteins;
- (c) isolating the complexes;
- (d) synthesizing DNA complementary to the nRNAs to provide an initial population of cDNAs;
- (e) optionally PCR-amplifying the cDNAs using strand-specific primers;
- purifying the initial population of cDNAs to obtain a purified population of cDNAs that are at least about 20 nucleotides (nt) in length, e.g., at least 25, 50, 100, 150 or 200 nt in length;
- (f) sequencing at least part of substantially all of the purified population of cDNAs;
- comparing the high-confidence sequences to a reference genome, and selecting those sequences that have a high degree of identity to sequences in the reference genome, e.g., at least 95%, 98%, or 99% identity, or that have fewer than it 0, 5, 2, or 1 mismatches;
- and
- (g) selecting those cDNAs that have (i) reads per kilobase per million reads (RPKM) above a desired threshold, and (ii) are enriched as compared to a control library (e.g., a protein-null library or library made from an IgG pulldown done in parallel);
- thereby preparing the library of cDNAs.
8 more elements
A method of identifying a compound that disrupts binding of one or more long non-coding RNAs (lncRNAs) to YY1 protein, the method comprising:
- (a) providing a sample comprising a lncRNA and YY1, wherein the lncRNA can bind to the YY1 and form lncRNA-YY1 complexes;
- (b) contacting the sample with a test compound;
- and
- (c) detecting the formation of lncRNA-YY1 complexes in the presence and the absence of the test compound,
- wherein a decrease in formation of lncRNA-YY1 complexes in the presence of the test compound as compared to formation of lncRNA-YY1 complexes in the absence of the test compound indicates that the test compound disrupts binding of the lncRNA to YY1.
2 more elements
A method of identifying an RNA target for the treatment of cancer, the method comprising:
- (a) comparing (i) a library of nRNAs that specifically bind YY1 prepared from a normal cell with (ii) a library of nRNAs that specifically bind YY I prepared from a cancerous cell, wherein the normal cell and cancerous cell are of the same tissue type;
- and
- (b) identifying an nRNA that is differentially expressed between the libraries of (a)(i) and (a)(ii) as an RNA target for treatment of cancer.
A method of identifying a therapeutic target for the treatment of cancer, the method comprising:
- (a) providing a population of nRNAs from a first cell type, by:
- (1) providing a sample comprising nuclear ribonucleic acids, e.g., a sample comprising nuclear lysate, e.g., comprising nRNAs bound to nuclear proteins, from the first cell type;
- contacting the sample with an agent, e.g., an antibody, that binds specifically to YY1 protein, under conditions sufficient to form complexes between the agent and YY1 proteins, e.g., such that the nRNAs remain bound to the YY1 proteins;
- isolating the complexes;
- and
- thereby providing a population of nRNAs from the first cell type;
- (b) providing a population of nRNAs from a second cell type, by:
- (1) providing a sample comprising nuclear ribonucleic acids, e.g., a sample comprising nuclear lysate, e.g., comprising nRNAs bound to nuclear proteins, from the second cell type;
- contacting the sample h an agent, e.g., an antibody, that binds specifically to YY1 protein, under conditions sufficient to form complexes between the agent and YY1 proteins, e.g., such that the nRNAs remain bound to the YY1 proteins;
- isolating the complexes;
- synthesizing DNA complementary to the nRNAs to provide an initial population of cDNAs;
- (2) thereby providing a population of cDNAs from the second cell type;
- wherein the first and second cell types are from the same type of tissue, and the first or second cell type is a tumor cell;
- (c) contacting the population of nRNAs from the first cell type with the cDNAs from the second cell type, tinder conditions sufficient for the nRNAs to bind to complementary cDNAs;
- and
- (d) identifying an nRNA that is differentially expressed in the first or second cell type as a therapeutic target for the treatment of cancer.
13 more elements
Dependencies are inferred from the claim text.