Empirical evidence generated over five decades has demonstrated that cells undergo structural changes at the molecular level in response to environmental cues. As membrane receptor proteins are most attuned to such cues, they remain the most attractive therapeutic targets. In order to discover specific DNA ligands against cell-surface receptors, our group recently introduced a unique variant of SELEX, termed Ligand-guided Selection (LIGS). LIGS exploits the inherent evolutionary step of competition between weak and strong binders in a SELEX library. The iterative process in conventional SELEX is designed to outcompete low-affinity binders through a competitive process whereby high affinity binders move on through the selection process. By introducing a naturally occurring stronger, highly specific binder, for example, an antibody (Ab) interacting with its cognate epitope, LIGS out-competes specific aptamers from a partially enriched SELEX pool against cells expressing the same epitope. Here, we show that LIGS can be expanded to evolve functional nucleic acid (NA) ligands against the multi-component cell-surface receptor T-cell receptor-CD3ε complex expressed by cultured and primary cells using two monoclonal Abs, OKT3 and UCHT1, as competing ligands. Bioinformatics analysis of the sequences obtained from Illumina sequencing, a total of five aptamer candidates were identified. These aptamers show affinities from 3.06±0.485 nM to 325±62.7 nM towards TCR-CD3ε. The aptamer family was validated utilizing multiple strategies, including competitive binding analysis with respective antibodies used in LIGS, and a double-knockout Jurkat cell line generated by CRISPR technology. Collectively this report shows that, by exploiting the inherent competition between weak and strong binders in a combinatorial library with existing ligand receptor interactions, multiple NA ligands against a multi-component receptor protein can be identified without changing the cell-surface landscape.

Integrating ligand receptor interactions and in vitro evolution for streamlined discovery of artificial nucleic acid ligands against T-cell receptor-CD3 complex in human T-cells

2019 Fall National Meeting

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