Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Abstract Fluorescence resonance energy transfer (FRET) has become a work‐horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single‐molecule spectroscopy to compare the origami‐based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor–acceptor distance is significantly reduced. Based on a simple geometric model for the inter‐dye distances on the origami block, the Förster radius R 0 could directly be determined from the distance dependence of energy transfer yielding R 0 =5.3±0.3 nm for the Cy3–Cy5 pair.