Rotation 1: Aptamers as allosteric modulators of G Protein-Coupled Receptors

BBSRC strategic theme: Transformative technologies

The G protein-coupled receptor (GPCR) superfamily of transmembrane proteins underpin how cells react to extracellular cues. Their extracellular face contains an orthosteric site that may bind agonists, causing the receptor to adopt an active conformation. This superfamily contains many drug targets, with more recent interest having pivoted to allosteric sites that lie away from conserved motifs. As such, allosteric modulators are highly selective for a target receptor. Macromolecules make promising candidates for such drugs, since they have numerous interactions with a target GPCR across a larger surface area. One such example comes in the form of aptamers; oligonucleotides that form complex 3D structures. These may interact with targets over a greater area than antibodies, and diverse libraries are easily generated of both DNA and RNA aptamers. Despite this, very few GPCR-interacting aptamers have been spoken of in the literature. However, the potential for aptamers as GPCR allosteric modulators is already clear, with screens for conformation-selective aptamers already having been performed. This project aims to identify aptamer-based allosteric modulators for the CXCR4 GPCR, with the B1-adrenoceptor as a backup. The receptor will be overexpressed in a HEK cell system, facilitating whole cell SELEX to select for high affinity aptamers for CXCR4. Microfluidic SELEX will be kept as a risk mitigation alternative. The binding method of these aptamers will then be investigated using atomic force and structured illumination microscopies, with CXCR4 being reconstituted in a model supported lipid bilayer system.