High Throughput Cell-Based Screening for Aptamers

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.

1 .- 46 . (canceled) 
     
     
         47 . A method for splitting a randomized aptamer library into smaller aptamer sub-libraries comprising the steps:
 (a) providing a randomized aptamer library, wherein the aptamers in the library comprise multiple 5′ and 3′ constant regions and one or more randomized nucleotides,   (b) performing a two-cycle PCR using the randomized aptamer library as the template and first primers and second primers that are complementary to the 5′ and 3′ constant regions, the primers each including one of a plurality of tag sequences,   (c) isolating the products of the two-cycle PCR, and   (d) PCR amplifying a subset of the isolated products of the two-cycle PCR using primers complementary to a subset of the 5′ and 3′ tag sequences.   
     
     
         48 . The method of  claim 47 , wherein the randomized aptamer library comprises more than about 100,000 aptamers. 
     
     
         49 . The method of  claim 47 , wherein the randomized aptamer library comprises more than about 1,000,000 aptamers. 
     
     
         50 . The method of  claim 47 , wherein the first or second primer in the two-cycle PCR comprises a label selected from the group consisting of biotin, digoxigenin (DIG), bromodeoxyuridine (BrdU), fluorophore, and a chemical group used in click chemistry. 
     
     
         51 . A method for splitting a randomized aptamer library into smaller aptamer sub-libraries comprising the steps:
 (a) providing a randomized aptamer library, wherein the aptamers in the library comprise multiple 5′ and 3′ constant regions and one or more randomized nucleotides and wherein the aptamers in the library are single-stranded,   (b) ligating a plurality of 5′ and 3′ tag sequences to the aptamers in the library using a T4 RNA ligase to produce 5′ and 3′ tagged ligation products, and   (c) PCR amplifying a subset of the 5′ and 3′ tagged ligation products using primers complementary to a subset of the 5′ and 3′ tag sequences.   
     
     
         52 . The method of  claim 51 , wherein the randomized aptamer library comprises aptamers having one or more randomized nucleotides. 
     
     
         53 . The method of  claim 51 , wherein the randomized aptamer library comprises more than about 100,000 aptamers. 
     
     
         54 . The method of  claim 51 , wherein the randomized aptamer library comprises more than about 1,000,000 aptamers. 
     
     
         55 . The method of  claim 51 , wherein the first or second primer in the two-cycle PCR comprises a label selected from the group consisting of biotin, digoxigenin (DIG), bromodeoxyuridine (BrdU), fluorophore, and a chemical group used in click chemistry. 
     
     
         56 . A method for splitting a randomized aptamer library into smaller aptamer sub-libraries comprising the steps:
 (a) providing a randomized aptamer library, wherein the aptamers in the library comprise multiple 5′ and 3′ constant regions and one or more randomized nucleotides and wherein the aptamers in the library are single-stranded,   (b) performing a PCR on the aptamers to generate a randomized double-stranded aptamer library, wherein the aptamers in the library comprise multiple 5′ and 3′ constant regions and one or more randomized nucleotides and wherein the aptamers in the library are double-stranded,   (c) ligating a plurality of 5′ and 3′ tag sequences to the aptamers in the double-stranded aptamer library using a T4 RNA ligase to produce 5′ and 3′ tagged ligation products, and   (d) PCR amplifying a subset of the 5′ and 3′ tagged ligation products using primers complementary to a subset of the 5′ and 3′ tag sequences.   
     
     
         57 . The method of  claim 56 , wherein the randomized aptamer library comprises aptamers having one or more randomized nucleotides. 
     
     
         58 . The method of  claim 56 , wherein the randomized aptamer library comprises more than about 100,000 aptamers. 
     
     
         59 . The method of  claim 56 , wherein the randomized aptamer library comprises more than about 1,000,000 aptamers. 
     
     
         60 . The method of  claim 56 , wherein the first or second primer in the two-cycle PCR comprises a label selected from the group consisting of biotin, digoxigenin (DIG), bromodeoxyuridine (BrdU), fluorophore, and a chemical group used in click chemistry.