A Single‐Molecule Förster Resonance Energy Transfer Analysis of Fluorescent DNA–Protein Conjugates for Nanobiotechnology

Abstract The development of nanobiotechnological devices requires the ability to build various components with nanometer accuracy. DNA is a well‐established nanoscale building block that self assembles due to specific interactions that are encoded in its sequence. Recently, it has become possible to couple proteins to DNA, thereby expanding the capabilities of DNA for use with molecular photonics and bioelectronics. Here, we present the design and characterization of a supramolecular Förster resonance energy transfer (FRET) system by using a fluorescent protein bound to single‐stranded DNA (ssDNA), a fluorophore attached to a second ssDNA molecule, and a complementary strand for hybridizing the two fluorophores together. The FRET efficiency was studied by using both ensemble and single‐pair FRET measurements. The distance between the two fluorophores was determined from the single‐pair FRET efficiency and could be described by a simple cylindrical model for the DNA. Hence, DNA can be used as a scaffold for positioning fluorescent proteins, as well as traditional fluorophores, with nanometer accuracy and shows great potential for use in the future of nanobiotechnology.