Single-Molecule Fluorescence Meets DNA Origami

Abstract

Recent developments in DNA nanotechnology facilitate a new degree of control to place arbitrary objects. Therefore, DNA is folded into arbitrary 2- and 3-dimensional structures on a nanometer to micrometer scale. In this so-called origami technique, introduced by Paul Rothemund in 2006, one ∼7.3 kbases long single-stranded DNA is hybridized with ∼200 short synthetic DNA “staple” strands to build a desired structure by self-assembly. Objects of interest, e.g. single fluorophores, are attached to individual incorporated DNA strands at specific positions within the structure. Several applications of this approach are shown using single-molecule fluorescence techniques.Revisiting the distance dependence of fluorescence resonance energy transfer (FRET), we used the DNA origami technique to build a spectroscopic ruler. In contrast to double stranded DNA, a commonly used spacer molecule, this technique offers distinct advantages. We designed a rigid DNA origami block, which has a higher persistence length and additionally allows placing the dye molecules all oriented in the same direction on the top surface, limiting static effects of the linker lengths. In contrast to dsDNA, for the origami block the Forster Radius R0 could directly be obtained from the distance dependence of energy transfer based on single-molecule FRET measurements.Guided by the idea to build complex spectroscopic networks by self-assembly, we used rectangular DNA origami as a molecular breadboard to precisely position individual fluorophores. In this artificial system the path of energy transfer can be manipulated on the nanoscale. Fluorophores were incorporated such that the light from the “blue” input dye could either be guided to the “red” or “IR” output dye, by a “green” dye that was placed at two alternative positions. We used a single-molecule four-color FRET approach with alternating laser excitation for analysis of the different energy transfer paths.