Expanding the Scope of Single Molecule FRET with DNA Origami

Abstract

Typical dimensions of cellular components are on the order of a few nanometers and their diverse cellular functions often involve conformational changes that require movements ranging from fractions of a nanometer up to tens of nanometers. The current state of the art tool for studying conformational dynamics is single molecule Fluorescence Resonance Energy Transfer (FRET), whereby energy transfer between two fluorescent dyes is correlated to their spatial separation; however, distance predictions made by FRET theory are only accurate in the range of ∼ 3-7 nm, and the working range is limited to below 10 nm. Furthermore, quantitative distance predictions require case-specific calibrations.Here we present a nanoscale device constructed by DNA origami that improves the quantitative accuracy of FRET distance predictions and expands its potential working range. The device integrates 3d structures built from self-assembled DNA, attachment sites for molecules of interest, sites for surface immobilization, and fluorescent markers for the direct visualization of biomolecular dimensions and dynamics by FRET. The device takes advantage of a distance calibration that can be generally applied to any molecule of interest. We have demonstrated that the device can easily achieve 1 nm distance resolution. For the purpose of proof-of-concept studies we specifically integrated a piece of double stranded DNA (dsDNA) containing a recognition sequence for Catabolite Activator Protein (CAP), which is know to bend dsDNA upon binding, between the arms of this device. The bending angle will be evaluated with single particle electron microscopy (EM), and FRET microscopy will be employed in solution to resolve real-time kinetics and deformations of CAP-DNA binding.