Driving biomolecular nanodevices with synthetic in vitro gene circuits: One of the major challenges for synthetic biology is the realization of large biochemical circuits composed of smaller, well-defined functional "modules". Due to the large number of interacting components and potentially detrimental, unintended "feedback loops", connecting several modules together can lead to a severe deterioration of systems performance. We here addressed this problem by studying an in vitro model system. We utilized a synthetic biochemical clock based on in vitro transcription reactions to periodically drive the motion of a DNA nanodevice (the "DNA tweezers"). Using a variety of different "coupling modes" between oscillator circuit and nanodevice, it was indeed possible to control the motion of the tweezers. However, the dynamics of the oscillator were strongly affected, when we increased the "load". We therefore developed an insulator circuit (corresponding to a "buffer amplifier" in electronics) that effectively uncoupled the load from the driving circuit, and therefore reduced undesirable biochemical "back-action".

This work was conducted in collaboration with the Winfree and Murray labs at Caltech.

E. Franco, E. Friedrichs, J. Kim, R. Jungmann, R. Murray, E. Winfree, F. C. Simmel, Timing molecular production and motion with a synthetic transcriptional clock, PNAS 108, E784-E793 (2011). DOI: 10.1073/pnas.1100060108

DNA-PAINT: DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.

The DNA-PAINT software is available in the software section.

Nanopore translocation experiments are performed using a novel setup based on microemulsion droplets. This scales down the required analyte volume to only a few picoliters and provides very stable membranes for single-molecule nanopore experiments. Using this setup, the stability of hairpin DNA and G-quadruplex DNA is studied by nanopore force spectroscopy.

Resolving the distances: Rectangular DNA origami labeled with fluorophores at specific positions has been used as a nanoscopic ruler. Super-resolution microscopy based on the subsequent localization of single molecules enables two fluorophores at a distance of about 90 nm to be optically resolved. This combination of subdiffraction imaging and DNA nanotechnology opens up new avenues for studying nanostructures and their dynamics.