DNA-Origami-Based Tethered Particle Motion for the Single-Molecule Study of Bimolecular Interactions

Abstract

Tethered Particle Motion (TPM) is a single-molecule method based on capturing the confined Brownian motion of a solid particle attached to a surface via a polymer tether. Its applications include studies on the mechanical properties of nucleic acids and protein-DNA interactions. In order to reduce the particle's effect on the dynamics of the measurement, small particle sizes compared to tether length are desirable. Tethered fluorophore motion (TFM) reduces the particle to a single fluorescent dye, but the maximum observation time of individual molecules before bleaching remains limited. We introduce an approach that combines fluorescence and DNA Origami to realize a TPM setup with small particle size and observation times surpassing 1 hour while maintaining a sub-second temporal resolution of positional fluctuations. Exploiting the versatility and positional addressability offered by state-of-the-art DNA nanotechnology, we can easily produce tether-particle systems of different, well-defined geometries and with an inherent 1:1 stoichiometry of particle to tether. Our system offers attachment points for interacting molecules of interest. We demonstrate its use as a platform for a multiplexed bimolecular binding/unbinding assay. We report on the dynamical behavior of DNA tethers with lengths between 250 and 3000 base pairs, as well as kinetic data of short complementary DNA motifs. Moreover, we present means to adjust the local concentration of the observed interaction partners and perform a calibration necessary for quantitative kinetic measurements.