Abstract
G protein-coupled receptors (GPCRs) play diverse roles in physiological processes, and hence the ligands to modulate GPCRs have served as important molecules in biological and pharmacological approaches. However, the exploration of novel ligands for GPCR still remains an arduous challenge. In this study, we report a method for the discovery of nucleic acid ligands against GPCRs by an advanced RNA aptamer screening technology that employs a virus-like particle (VLP), exposing the GPCR of interest. An array of biochemical analyses coupled with a cell-based assay revealed that one of the aptamers raised against purinergic receptor P2Y2 (P2RY2), a GPCR, exhibits an activation potency to unliganded receptor and prohibits a further receptor activation by endogenous ligand, behaving like a partial agonist. However, the aptamer enhances the activity of intrinsic ligand-binding P2RY2, thereby acting as a positive allosteric modulator (PAM) to liganded receptor. Our findings demonstrate that the nucleic acid aptamer conditionally exerts PAM and agonist effects on GPCRs, depending on their intrinsic ligand binding state. These results indicate the validity of our VLP-based aptamer screening targeting GPCR and reemphasize the great potential of nucleic acid ligands for exploring the GPCR activation mechanism and therapeutic applications.
Highlights
Gprotein–coupled receptors (GPCRs) are involved in varied human physiological functions and are targets for one-third of currently marketed drugs [1]
We used a library composed of modified nucleotides with 2′-fluoro-pyrimidine, 2′-deoxy-adenosine and 2′-hydroxy-guanosine for tolerance to nucleases because virus-like particle (VLP) targeted in systematic evolution of ligands by exponential enrichment (SELEX) are relatively crude material
During the SELEX cycle (SI Appendix, Table S1), we used an immobilization-free separation system with an ultrafiltration column capable of separating aptamers complexed with VLPs from free RNA sequences to avoid any conformational distortion of GPCR induced by immobilization (SI Appendix, Fig. S2)
Summary
A scatter plot based on the above-mentioned clustering analysis with FASTAptamer indicated eight enriched sequences with the highest RPM in each of the top eight clusters (see Materials and Methods and Fig. 2B). These eight sequences were subjected to motif and cluster analyses (SI Appendix, Fig. S3). Pretreatment of P2RY2-overexpressing HEK293 cells with each aptamer inhibited the calcium signaling induced by the specific chemical agonist PSB1114, thereby indicating the inhibitory effect of the aptamers, in c11 and c37, on the molecular interaction between the receptor and chemical agonist PSB1114 (Fig. 2C and SI Appendix, Fig. S4). Antagonist assay (vs. PSB1114) Cells Aptamer PSB Measure c37_8-40 c37_8-40 (2 ́OH) AR-C118925XX c37_8-40 (2 ́OMe) 160
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