Single Molecule Snapshots of Riboswitch Conformational Change and RNA Switch Based Biosensing on a Nanopore Maglet Device

Abstract

Riboswitches are a class of biosensory-regulatory segments of messenger-RNAs. Their binding with specific cellular ligands e.g. metabolic intermediates, can induce a conformational change in riboswitch's tertiary structure, ultimately programming the production of the proteins encoded by the mRNA. Precision tools to explore ligand-programmed riboswitch folding/unfolding mechanism are important in both developing riboswitch-targeted antibiotic intervention and RNA switch-based biosensors. Our nanopore weak-interaction spectroscopy can snapshot a single RNA riboswitch's folding/unfolding pathway at single base-pair resolution. Different from thermodynamic approaches and force microscopies (e.g. AFM and optic tweeze), the unique feature of nanopore is unfolding along the backbone of RNA-Vectorial Unfolding. This tool enables us to discriminate RNA unfolding pathway in both 3’>5’ and 5’>3’ directions, dissect the non-canonical triple-strand conformations and interactions that control the RNA stability, and many interconnected folding intermediates. Based on these findings, we further develop a nanopore maglet automatic machine that can “lock” a single riboswitch (preQ1) molecule in the nanopore. The locked single RNA can be electrically driven to repeatedly move back and forth to transform among different conformations: partial folding→ligand binding →fully folding→Unfolding. We can probe the folding change of riboswitch upon ligand (preQ1) binding, explore riboswitch's induced fit and/or conformational selection folding mechanism, and study how designed drug compounds regulate the riboswitch folding pathway. Finally, this nanopore molecular machine can be utilized as RNA switch-based biosensor for detection a variety of disease-derived ligands and proteins.