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SIMBA Report Summary

Project ID: 261162
Funded under: FP7-IDEAS-ERC
Country: Germany

Final Report Summary - SIMBA (Single-Molecule BioAssays at Elevated Concentrations)

Over the last decade, single-molecule fluorescence spectroscopy has enabled a new view of biomolecular processes and interactions. In order to advance to the next level a quantum leap was required that would ease single-molecule handling and analysis for the non-specialists. In this context, the limited concentration range of optical single-molecule detection was a key limitation that we intended to overcome by nanophotonic approaches.

First, we used a combined top-down and bottom-up strategy of lithography with DNA self-assembly. Small holes in metal films produced by nanolithography (so-called zero-mode waveguides (ZMWs)) reduce the observation volume so that single-molecule detection can be extended from lower nanomolar concentrations to the micromolar range. So far, this technology suffered from the heterogeneity of fluorescence signals that arose from arbitrary positions within the ZMWs and from the limited occupation as only about one third of the ZMWs could be populated with a single fluorescent molecule owing to Poissonian statistics. We introduced DNA origami nanoadapters to place single molecules directly at the center of the ZMWs. This was achieved by labelling the DNA origami nanoadapters with an anchoring group (biotin) placed at the center of the adapter. In this way, we achieved a steric hindrance that allowed binding of the nanoadapters onto the ZMWs coated with the complimentary anchoring group (neutravidin) only in the right orientation flat at the ZMWs surface. In addition, we overcame the Poissonian limit of stochastic single-molecule immobilization. As the nanoadapters are only slightly smaller than the ZMWs, only one nanoadapter can bind per ZMW and doubling of single-molecule occupancy was demonstrated.
Exploiting the blinking of single molecules as well as the fluorescence lifetime and intensity information we obtained a fluorescence map of the nanophotonic environments including the quantum yield of single molecules.

Second, we realized that a complementary nanophotonic approach with nanoantennas could also allow the detection of single-molecule bioassays at elevated concentration. Nanoantennas are able to focus light in ultra-small volumes far beyond the far-field diffraction limit of light. To this end, we developed nanoantennas that consist of two spherical gold nanoparticles (forming a dimer) arranged with the aid of a DNA origami. The DNA origami additionally provides docking strands that enable placing of single dye molecules or biomolecular assays in the fluorescent hot-spots. We demonstrate that the fluorescence of single molecules is enhanced up to 117fold when a dye molecule is placed in the 23-nm gap between two 100-nm diameter gold nanoparticles and up to more than 5000fold for smaller gaps and with low quantum yield dyes. In addition, we demonstrated single-molecule detection at a background of 25 µM of fluorescent molecules. This ground-breaking approach offers several advantages as it does not require any nano-lithography and because the DNA origami offers handles to precisely place the biomolecular assay of interest directly at the nanophotonic hotspot.

To place bioassays in the nanophotonic hotspots we designed DNA origami with specific handles to which the molecules of the biosassay such as proteins could be bound. We first demonstrated biocompatibility of the DNA origami nanoadapters and nanoantennas with biomolecular single-molecule assays. To this end, we showed that the TATA binding protein shows identical binding kinetics in solution as well as on DNA origami. We then established further single-molecule assays that could benefit from the ability to work at higher concentrations. We studied different transcription factors in a single-molecule FRET assay and revealed a two-state mechanism of the nucleic acid processing enzyme argonaute. Additionally, we established a single-molecule assay for RNA polymerase that allowed us to follow the conformation of the polymerase clamp thus revealing new conformational states at different stages of the transcription cycle.

Altogether, the ERC starting grant project SiMBA yielded 29 publications in peer reviewed journals and a granted patent. More manuscripts are in preparation. Our new abilities to improve single-molecule detection with nanoantennas with docking sites have triggered a number of further projects aiming for improved biosensing and other nanobiotechnological applications.

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