Nanopore Force Spectroscopy on Nucleic Acid Structures & their Target Complexes using Biological and Synthetic Ion Channels

Abstract

Nanopore force spectroscopy (NFS) is a versatile tool for the investigation of single molecule interactions with high throughput and sensitivity. We parallelize this method using a 16-channel chip-based bilayer setup (Nanion-Technologies), making it suitable for screening assays.In our experiments, we study the binding of the ATP aptamer to its target in terms of complex stability as well as binding affinity. Aptamer-target complexes show a stability that is considerably higher compared to unbound aptamer structures. This allows us to determine the binding affinity of the ATP aptamer to its target molecules by comparing populations in the stability distributions for varying target concentrations. Due to the high force resolution of NFS we observe that the stability of bound aptamer structures splits up into several sub-populations that reveal more detailed information about the binding process.We extend the use of NFS from DNA to RNA-based molecular structures, which play important roles in biological processes e.g. in the form of aptamers or in gene regulation. In particular RNA hairpin structures are investigated, which are found to display an increased stability compared to their DNA analogues, as their predicted thermodynamic stability suggested. Furthermore, first experiments on RNA aptamers have been performed.Previous NFS studies were based on naturally occurring membrane channels like α-hemolysin or solid-state nanopores. Tailoring these systems to specific applications remains a challenging task, when altering the channels' geometry or chemical modification becomes necessary. We report on the use of a novel synthetic membrane channel constructed entirely from DNA to perform nanopore-sensing experiments. Scaffolded DNA origami was used to create the channel, allowing for easy modifications in terms of geometry and chemical features. We demonstrate its functionality by studying DNA hairpin unzipping and G-quadruplex unfolding.