A novel microfluidic aptamer selection approach named “1D bead-array SELEX” with the advantages of library-in-droplet assembly, automation of repeated binding/washes, simultaneous counter-selection and cross-check of selected sequences is developed and studied for STAT3-targeted aptamer selection. The approach uses a single-channel PDMS microfluidic device embedded with an 1D array of functionalized micro-glass beads that immobilize STAT3 (for selection) and HSA (for counter-selection) in turn (see Fig. 1(a)). For binding, an ssDNA or dsDNA library in a droplet is driven to flow through the bead array in a back-and-forth format at a programmed shuttling rate, and then non-specific binding events are washed off by repeated flowing through buffers. After washes, the device is opened and each bead is transferred to qPCR amplification of bound DNA ligands. It is found that the difference between the DNA binding amounts on STAT3 and HSA-coated beads can be tuned by the shuttling rate (see Fig. 1(b)). The amplified DNA ligands from all STAT3 beads are collected together and processed as a new ligand pool in a droplet for the next microfluidic selection round. After three rounds of selection, the DNA ligands bound on each bead are cloned and sequenced, separately. The sequences appear on every STAT3 bead but not on both HSA beads are considered as the STAT3-targeted aptamers, which are further investigated for both diagnostic and therapeutic applications. In addition to single-protein SELEX, 1D bead-array SELEX can be extended for multiplex aptamer selection against a family of protein analogues, e.g., STAT family proteins.

1D Bead-Array SELEX for STAT3-targeted aptamer selection

254th National Meeting (2017)

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