Engineered DNA/RNA Nano-Assemblies Enable High Throughput Characterization Of Protein Complexes

We will develop tools to automate cryo Electron Microscopy (cryo-EM). Cryo-EM is a unique method to determine the structure of the tiny molecular machines that fill up the body of our cells. In cryo-EM the sample is flash frozen in liquid ethane at 170C below zero. This procedure creates extremely thin layers of glass-like ice in which the sample is embedded. After cryo fixation the particles can be directly visualized using an electron microscope, at ~80.000x magnification. The versatility of cryo-EM has rendered it an indispensable tool for cell biologists to understand the underlying mechanisms inside our cells. Different views of the particles are identified and merged to form a 3D representation of its structure, in a method that is comparable to Computer Tomography (CT). Unfortunately, in cryo-EM this reconstruction step is much more complicated, as the small particles are barely visible and the relative orientation between them has to be computationally determined. In many cases these problems pose significant roadblocks to understand the 3D structure of the molecular machine. Our approach is to use artificially designed, DNA-based structures that will bind to the particles of interest. Using this cutting edge technology it is possible to engineer large, ordered DNA scaffolds that act as molecular markers indicating position and orientation of a particle. We expect that this tool will become tremendously useful and help to automate all steps involved.

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