A Graphene-based RNA Biosensor to Determine Riboswitch-Ligand Interactions

Abstract

Riboswitches are a class of regulatory structures located in the 5’ untranslated region of many bacterial mRNAs. Validating riboswitch-ligand interactions has historically been costly and low-throughput. Recently, graphene field-effect transistors (G-FETs) have emerged as effective biosensors in detecting interactions of such regulators with charged, high molecular weight analytes. However, a bottleneck still exists in detecting relatively neutral small molecules. The Bacillus subtilis guanine riboswitch (Xpt) within the xpt-pbuX operon contains a purine-responsive aptamer region with affinity for guanine, hypoxanthine, and other purine analogs. The G-FET sensor revealed successful detection of Xpt-hypoxanthine interactions at saturating concentrations. The specificity of Xpt was also demonstrated by a lack of signal detection when incubated with adenine. Therefore, such G-FET devices are effective in detecting aptamer binding to small, electrically-neutral molecules, which will allow for rapid screening of potential therapeutic ligands. Further, different electrical observations of n-doping upon aptamer functionalization and p-doping upon ligand binding reveal unique interactions at the graphene surface. Molecular dynamics simulations were carried out to interpret experimental results and to determine if another well characterized aptamer (FMN) is a suitable candidate for G-FET studies. Trajectory data from the Xpt aptamer domain complexed with hypoxanthine (PDBID: 4FE5) and guanine (PDBID: 1Y27) showed significant differences in root mean square deviation (RMSD) and radius of gyration (Rg) from their respective non-binding mutants. These findings provide evidence that compaction of the RNA phosphodiester backbone is responsible for graphene detection. RMSD and Rg differences from FMN (PDBID: 3F4E) indicate that this aptamer may not show a significant change in G-FET signal. These findings suggest that G-FET biosensors can provide an avenue for the discovery of novel antibiotics for aptamer targets to combat burgeoning antibiotic resistance.