Discovery and development of artificial nucleic acid ligands to probe cellular interactions The regulation followed by measurement of cellular events, such as intercellular communication, receptor- ligand interactions, and inter-receptor interactions using molecular tools, will expand our understanding of cellular decision-making mediated by cell surface receptors. Owing to their synthetic nature and compatibility with a variety of nano-materials, nucleic acid aptamers are well suited as specific recognition probes for incorporation into such tools. Aptamers are single-stranded DNA/RNA/XNA (X= nonstandard nucleic acid base) sequences that bind to a specific target with high affinity and specificity. However, because of their low solubility in aqueous media, purified cell-surface receptors do not make good targets for in vitro screening of aptamer ligands. Additionally, in response to ligand binding, cell surface receptors act in concert, forming transient complexes. Such complexes are hard to constitute in artificial buffer systems while maintaining their native fold. This means that conventional approaches to aptamer-ligand screening may not lead to aptamers that are translatable. Addressing this need, the Mallikaratchy Lab recently pioneered a technology termed Ligand-guided Selection or LIGS to identify functional aptamers from a SELEX (Systematic Evolution of Ligands by EXponential enrichment) library, against known multi-domain cell surface targets in their native functional state. LIGS uses the same combinatorial library but takes advantage of characteristics inherent to SELEX. For example, it is known that the iterative process in conventional SELEX is designed to outcompete low-affinity binders through a competitive process whereby high-affinity binders move through an increasingly selective process. LIGS exploits this competition between weak and strong binders in a combinatorial library by introducing a stronger, known high-affinity secondary ligand, e.g., a monoclonal antibody (mAb), against the target of interest to outcompete and replace highly specific aptamers. These aptamers can recognize their target receptors in cultured and clinical samples specifically. Building on our initial work on LIGS, the first goal of this MIRA is to extend the types of interactions utilized in LIGS to naturally induced conformational switches of membrane proteins of mechanistic/functional interest to discover artificial ligands based on aptamers against them. The second goal explores chemical interventions to understand aptamer folding using bioorthogonal approaches, such as click-chemistry, to facilitate proximity mediated intra-molecular cross-linking. Additionally, we plan to engineer functional aptamer scaffolds to regulate cell-cell and receptor-ligand interactions using aptamers already identified using LIGS, explicitly focusing on the modulation of TCR-CD3ε in T-cells. A successful outcome to these goals will result in aptamers able to probe receptor interactions along with integrated nano-materials enhancing the application of aptamers in biomedicine while providing insights into the modulation of cell receptor biology in cellular decision making.

Project Narrative: Upon the successful completion of this proposal, we expect to make contributions to the scientific community at large by introducing an innovative ligand discovery platform to selectively identify nucleic acid ligands against cell surface targets, followed by developing programmable synthetic ligands study to cell surface receptor proteins.