Identification of micrornas and their targets
OwnerTariq M. Ranainventor
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Abstract
The present invention relates to methods for identifying miRNAs and their targets in vivo and in vitro applicable to situations in which one or both of the sequences of the miRNA and the target nucleic acid is unknown. Further, the method applies to situations in which the miRNA/target interaction is stable or unstable; unstable reactions can be stabilized by the addition of crosslinking agents. The present invention also provides methods of modulating the expression of a target gene in a cell by modulating the activity of an miRNA in the cell that targets the gene.
Claims28
A method for identifying an miRNA and its target RNA, the method comprising
- (a) obtaining an miRNA/target RNA complex;
- (b) optionally crosslinking the complex;
- (c) transcribing target complementary RNA (tcRNA) from the target RNA;
- (d) synthesizing cDNA complementary to the tcRNA;
- and
- (e) sequencing the cDNA,
- thereby identifying the miRNA and its target.
A method for identifying a target RNA of an miRNA, the method comprising
- (a) obtaining a modified miRNA comprising an amino-modified cytosine or amino-modified uracil;
- (b) contacting the miRNA with a target RNA;
- (c) allowing an miRNA/target RNA complex to form;
- (d) labeling the complex with a biotin compound selected from an NHS activated ester of biotin butanoic acid (Compound 2) or photocleavable biotin (Compound 3);
- (e) transcribing target complementary RNA (tcRNA) from the target RNA;
- (f) synthesizing cDNA complementary to the tcRNA;
- and
- (g) sequencing the cDNA,
- thereby identifying the target RNA.
A method for identifying the target RNA of an miRNA, the method comprising
- (a) obtaining an miRNA having a known sequence;
- (b) contacting the miRNA with a target RNA;
- (c) allowing an miRNA/target RNA complex to form;
- (d) labeling the miRNA/target RNA complex with a compound selected from an activated ester of hexanoic acid linked with a biotin and a 4-thio-uracil (Compound 4) or an activated ester of hexanoic acid linked with a biotin and 8-hydroxy-psoralen (Compound 5);
- (e) optionally crosslinking the miRNA/target RNA complex;
- (f) transcribing target complementary RNA (tcRNA) from the target RNA;
- (g) synthesizing cDNA complementary to the tcRNA;
- and
- (h) sequencing the cDNA,
- thereby identifying the target RNA.
A method for identifying the target RNA of an miRNA, the method comprising
- (a) contacting a biotin-labeled miRNA having a known sequence with a target RNA;
- (b) allowing an miRNA/target RNA complex to form;
- (c) crosslinking the miRNA/target RNA complex, e.g. with a psoralen compound or other crosslinking agent;
- (d) immobilizing the complex on avidin-coated beads;
- (e) reversing the crosslink;
- (f) transcribing a complementary strand from the target RNA using reverse transcriptase and a cDNA primer, e.g., a primer having a sequence corresponding to the miRNA;
- (g) synthesizing cDNA complementary to the transcribed strand of (f);
- and
- (h) sequencing the cDNA,
- thereby identifying the target RNA.
A method for identifying a target RNA of an miRNA, the method comprising
- (a) contacting a cell with a psoralen-biotin conjugate such that the conjugate binds to target RNA within the cell;
- (b) allowing the target RNA to form a complex with miRNA within the cell;
- (c) immobilizing the miRNA:target RNA complex on avidin-coated beads;
- (d) reversing the crosslink;
- (e) transcribing a complementary strand from the target RNA using reverse transcriptase and a poly A primer;
- (f) synthesizing cDNA complementary to the transcribed strand of (f);
- and
- (g) sequencing the cDNA,
- thereby identifying the target RNA.
Dependencies are inferred from the claim text.