Compositions for selection of aptamers

Abstract

The present disclosure describes compositions and methods for rapid selection of both binding and functional oligonucleotides (DNA, RNA, or any natural or synthetic analog of these). In certain embodiments, provided herein are flow cells (e.g., flow cells for an Illumina sequencing instrument or a Polonator sequencing instrument) comprising within its flow chamber a plurality of immobilized aptamer clusters (e.g., from an aptamer library described herein) and, optionally, one or more target cells (e.g., cancer cells, immune cells, etc.) and/or a detectable indicator of cellular function (e.g., a fluorescent indicator of apoptosis, cell proliferation, gene or protein expression, etc.). In certain embodiments, provided herein are methods of using such an aptamer cluster-containing flow cell to identify functional aptamers from an aptamer library (e.g., in a sequencing instrument, such as an Illumina sequencing instrument).

What is claimed is: 
     
         1 . A method for identifying one or more aptamers that mediate a cell function in a target cell, the method comprising: (i) contacting a plurality of surface-immobilized aptamer clusters with the target cell; and (ii) identifying immobilized aptamer clusters that mediate the cell function in the target cell. 
     
     
         2 . The method of  claim 1 , wherein the surface-immobilized aptamer clusters are immobilized on beads. 
     
     
         3 . The method of  claim 2 , wherein the beads are paramagnetic bead. 
     
     
         4 . The method of  claim 2 , wherein the beads are detectably labeled. 
     
     
         5 . The method of  claim 1 , further comprising the steps of:
 (a) immobilizing a plurality of aptamers from an aptamer library on surfaces; and   (b) amplifying the plurality of immobilized aptamers locally on the surfaces to form the plurality of immobilized aptamer clusters.   
     
     
         6 . The method of  claim 5 , wherein the amplification is conducted via bridge PCR amplification or rolling circle amplification. 
     
     
         7 . The method of  claim 6 , wherein the method further comprises removing the complementary strands from the immobilized aptamer clusters to provide single stranded immobilized aptamer clusters. 
     
     
         8 . The method of  claim 1 , further comprising an aptamer folding step, wherein the aptamer folding step comprises raising and then lowering the temperature of the surface. 
     
     
         9 . The method of  claim 1 , further comprising an aptamer folding step, wherein the aptamer folding step comprises adding a denaturing agent to the surfaces and then removing the denaturing agent from the surfaces. 
     
     
         10 . The method of  claim 1 , further comprising the step of sequencing the aptamer clusters identified in step (ii). 
     
     
         11 . The method of  claim 10 , wherein the sequencing is conducted via Illunmia sequencing or Polonator sequencing. 
     
     
         12 . The method of  claim 1 , wherein at least 10 8  distinct aptamers are immobilized on the one or more surfaces and each aptamer cluster comprises 10 3  to 10 6  copies of an aptamer. 
     
     
         13 . The method of  claim 1 , wherein the target cell is detectably labeled. 
     
     
         14 . The method of  claim 1 , wherein the target cell is a mammalian cell. 
     
     
         15 . The method of  claim 14 , wherein the mammalian cell is a human cell. 
     
     
         16 . The method of  claim 15 , wherein the human cell in an immune cell. 
     
     
         17 . The method of  claim 15 , wherein the human cell is a cancer cell. 
     
     
         18 . The method of  claim 15 , wherein the cell function is cell death, caspase-3/7 activity, proliferation, gene expression, or cytokine expression. 
     
     
         19 . The method of  claim 18 , wherein the cell function is cell death. 
     
     
         20 . The method of  claim 19 , wherein the target cell is labeled with a fluorescent reporter of cell death.