An In Vitro Selection Platform to Identify Multiple Aptamers against Multiple Cell-Surface Markers Using Ligand-Guided Selection

Aptamer ligand discovery against multiple mole-cules expressed on whole cells is an essential component in molecular tool development. However, owing to their intrinsic structural characteristics, cell-surface receptors have proven to be challenging targets in ligand discovery. Several variants to systematic evolution of ligands by exponential enrichment (SELEX) have been introduced to address the & DPRIME;target problem & DPRIME; for aptamer screening. To this end, we introduced a variant of SELEX, termed ligand-guided selection (LIGS), to identify highly specific aptamers against complex cell-surface markers in their native state. So far, the application of LIGS has been aimed at identifying aptamers against the most dominant receptors on the cell surface. Here, we report that LIGS can be expanded to identify two receptors on the same cell surface, paving the way to generate a multiplexed ligand discovery platform based on SELEX-targeting membrane receptors in their native functional state. Using CD19 and CD20 expressed on Toledo cells as a model system, multiple aptamer families were evolved against Toledo cells. We then utilized two monoclonal antibodies (mAbs) against CD20 and CD19 to selectively partition specific aptamers against CD19 and CD20. Following biochemical characterization, we introduce two specific aptamers against CD19 and two specific aptamers against CD20 with high affinity. Multi-target LIGS, as reported here, demonstrates a successful combinatorial approach for nucleic acid library screening to generate multiple artificial nucleic acid ligands against multiple receptors expressed on a single cell.