Production of artificial micrornas using synthetic microrna precursors
AssigneeThe Rockefeller University
InventorsNam-Hai CHUA, Qi-Wen NIU
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Abstract
The invention provides methods and compositions useful in target sequence suppression, target sequence validation and target sequence down regulation. The invention provides polynucleotide constructs useful for producing artificial microRNA (amiRNA) using synthetic amiRNA precursors.
Claims57
A nucleic acid construct comprising a promoter operatively linked to a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary sequence are replaced by an amiRNA sequence and its complementary sequence, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA and wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor.
A cell comprising a host cell promoter operatively linked to a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary (miRNA* strand) sequence are replaced by an amiRNA sequence and its fully complementary sequence, respectively, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA and wherein the host cell promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor.
A transgenic plant comprising a host cell promoter operatively linked to a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary (miRNA* strand) sequence are replaced by an amiRNA sequence and its fully complementary sequence, respectively, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA and wherein the host cell promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor.
A transgenic seed comprising a host cell promoter operatively linked to a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary (miRNA* strand) sequence are replaced by an amiRNA sequence and its fully complementary sequence, respectively, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA and wherein the host cell promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor.
A method for down regulating a target sequence in a cell comprising:
- (a) introducing into a cell a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary (miRNA* strand) sequence are replaced by an amiRNA sequence and its fully complementary sequence, respectively, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA, wherein the polynucleotide is operably linked to a promoter, and wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor and
- (b) growing the cell of (a) under conditions wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor, the amiRNA is produced and the RNA containing the target sequence is cleaved.
A method for producing artificial miRNA (amiRNA) in a cell comprising:
- (a) introducing into a cell a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary (miRNA* strand) sequence are replaced by an amiRNA sequence and its fully complementary sequence, respectively, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA, wherein the polynucleotide is operably linked to a promoter, and wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor and
- (b) growing the cell of (a) under conditions wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor and the amiRNA is produced.
A method to assay for production of an artificial miRNA comprising the steps of:
- (a) identifying a precursor miRNA containing a miRNA sequence and a complementary miRNA sequence (a miRNA* strand sequence),
- (b) introducing into a cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding an artificial miRNA (amiRNA) precursor wherein the amiRNA precursor comprises the following:
- (1) an artificial miRNA sequence replacing the endogenous miRNA sequence;
- (2) a fully complementary sequence of the artificial miRNA replacing the endogenous miRNA* strand sequence;
- and
- wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor;
- (c) growing the cell of (b) under conditions wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor;
- and
- (d) assaying for production of the artificial miRNA.
A method for down regulating an RNA containing a target sequence in a plant comprising:
- (a) introducing into a plant cell a polynucleotide which encodes an artificial miRNA (amiRNA) precursor capable of forming a double-stranded RNA or a hairpin, wherein the amiRNA precursor comprises a modified miRNA precursor in which the miRNA sequence and its complementary (miRNA* strand) sequence are replaced by an amiRNA sequence and its fully complementary sequence, respectively, wherein the amiRNA sequence is (i) fully complementary to a target sequence, (ii) fully complementary to the target sequence except for GU base pairing or (iii) fully complementary to the target sequence in the first ten nucleotides counting from the 5′ end of the amiRNA, wherein the polynucleotide is operably linked to a promoter, and wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor,
- (b) regenerating a plant from the plant cell of (a) wherein the plant comprises in its genome the nucleic acid construct,
- (c) growing the plant of (b) under conditions wherein the promoter initiates and mediates transcription of the antisense strand of the amiRNA precursor, the amiRNA is produced and the RNA containing the target sequence is cleaved.
Use of a synthetic pre-miRNA to generate one or more mature miRNAs in transformants to down regulate one or more target genes.
Dependencies are inferred from the claim text.