High Throughput Cell-Based Screening For Aptamers
AssigneeMeiraGTx Gene Regulation Limited
InventorsXuecui Guo, Alexandria Forbes, Lei Feng
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Abstract
The invention provides eukaryotic cell-based screening methods to identify an aptamer that specifically binds a ligand, or a ligand that specifically binds an aptamer, using a polynucleotide cassette for the regulation of the expression of a reporter gene where the polynucleotide cassette contains a riboswitch in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and an aptamer such that when the aptamer binds a ligand, reporter gene expression occurs.
Claims30
1-46. (canceled)
A method for selecting an aptamer that binds a ligand in eukaryotic cells comprising the steps of:
- (I) providing a library of aptamers;
- (II) introducing members of the library of aptamers into a polynucleotide cassette for the ligand-mediated expression of a reporter gene to create a library of riboswitches, wherein the polynucleotide cassette is present in the coding sequence of the reporter gene;
- (III) introducing the reporter gene comprising the polynucleotide cassette that comprises the library of riboswitches into eukaryotic cells in vitro;
- (IV) contacting the eukaryotic cells with a ligand;
- and
- (V) measuring expression of the reporter gene;
- wherein the polynucleotide cassette comprises sequence encoding from 5′ to 3′:
- (a) a first intron,
- (b) an alternatively-spliced exon, and
- (c) a second intron comprising a riboswitch, wherein the riboswitch comprises from 5′ to 3′:
- (i) a 5′ splice site sequence of the second intron,
- (ii) the aptamer, and
- (iii) a sequence that is complementary to the 5′ splice site sequence of the second intron;
- wherein the 5′ splice site sequence of the second intron and the sequence that is complementary to the 5′ splice site sequence of the second intron form a stem when the ligand binds the aptamer;
- and
- wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the reporter gene coding sequence, when the alternatively-spliced exon is spliced into the target gene mRNA.
A method for selecting a ligand that binds an aptamer in a eukaryotic cell comprising the steps of:
- (I) providing a library of ligands;
- (II) providing a polynucleotide cassette for the ligand-mediated expression of a reporter gene, wherein the polynucleotide cassette is present in the coding sequence of the reporter gene;
- (III) introducing the reporter gene comprising the polynucleotide cassette into the eukaryotic cell in vitro;
- (IV) contacting individual groups of the eukaryotic cells with members of the library of ligands;
- and
- (V) measuring the expression of the reporter gene;
- wherein the polynucleotide cassette comprises sequence encoding from 5′ to 3′:
- (a) a first intron,
- (b) an alternatively-spliced exon, and
- (c) a second intron comprising a riboswitch, wherein the riboswitch comprises from 5′ to 3′:
- (i) a 5′ splice site sequence of the second intron,
- (ii) the aptamer, and
- (iii) a sequence that is complementary to the 5′ splice site sequence of the second intron;
- wherein the 5′ splice site sequence of the second intron and the sequence that is complementary to the 5′ splice site sequence of the second intron form a stem when the ligand binds the aptamer;
- and
- wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the reporter gene coding sequence, when the alternatively-spliced exon is spliced into the target gene mRNA.
Dependencies are inferred from the claim text.