Optical Voltage Sensing Nano-Devices using DNA Self-Assembly

• Applicant: Professor Dr. Philip Tinnefeld
• Subject Area: Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics ; Biophysics ; Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 319003204

• Final Report Year: 2021

Final Report Abstract

In the project “Optical Voltage Sensing Nano-Devices using DNA Self-Assembly” we aimed to build a DNA origamibased sensor that reacts to changes in the transmembrane potential and translates it into a ratiometric FRET signal. After an in-depth study on the accuracy of FRET as a read-out system and a proof-of-principle study on the suitability of DNA origami as single-molecule voltage sensors, we established various methods in our lab to verify our DNA origami voltage sensors on lipid membranes. Afterwards, we intensively worked on the optimization of our sensor. As the original idea as proposed was challenging, we adopted the sensor design iteratively to meet the requirements. One of the DNA origami designs was the voltage sensing raft. Its design was changed from the original bundle to a plate for a better control of the binding to membranes and for an easier high-throughput screening. For a precise analysis of the sensor, we reduced the number of sensing units from multiple to one per DNA origami. Thereby, we extracted the maximum information content on our system which further helped to identify weaknesses to be optimized. The core task of the project was to develop a suitable sensing mechanism. We therefor tried two different design strategies: one based on a cationic peptide and the other one based on anionic DNA. The sample handling of the strongly hydrophobic and cationic peptide conjugates was very challenging. As the idea was to make a sensor for a broad applicability in biological systems, we changed the strategy to guarantee an easier use of our sensors. Molecular dynamic simulations pin-pointed towards a new direction; Instead of relying on the voltage change inside the membrane core, we placed the sensing unit outside of it and used electrostatic attraction and repulsion act on it. With this approach, we developed two different designs showing sensitivity for either hyper- or depolarized lipid membranes on the level of single DNA origami sensors. Overall, we were able to fulfill most of the objectives proposed; Our DNA origami sensors successfully and very specifically recognize lipid membranes and on the level of single DNA origami structures, we achieved a ratiometic FRET read-out. Also, the sensors show sensitivity even below the range of ±200 mV as intended. With our study on DNA origami self-regeneration, we made attempts towards an application in cellular systems. As an outlook for further development, we have to investigate on the temporal resolution of our system and finally, apply it to cells lines and beyond.

Publications

• Optical Voltage Sensing Using DNA Origami. Nano letters 2018, 18, 1962–1971
  Hemmig, E. A.; Fitzgerald, C.; Maffeo, C.; Hecker, L.; Ochmann, S. E.; Aksimentiev, A.; Tinnefeld, P.; Keyser, U. F.
  
• Precision and accuracy of singlemolecule FRET measurements-a multi-laboratory benchmark study. Nature methods 2018, 15, 669–676
  Hellenkamp, B.; Schmid, S.; Doroshenko, O.; Opanasyuk, O.; Kühnemuth, R.; Rezaei Adariani, S.; Ambrose, B.; Aznauryan, M.; Barth, A.; Birkedal, V.; et al.
  
• DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity, 2021
  Ochmann, S. E.; Joshi, H.; Bueber, E.; Franquelim, H. G.; Stegemann, P.; Sacca, B.; Keyser, U. F.; Aksimentiev, A.; Tinnefeld, P.
  
• Selbstregeneration und Selbstheilung in DNA‐Origami‐Nanostrukturen. Angew. Chem. 2021, 133, 4982–4990
  Scheckenbach, M.; Schubert, T.; Forthmann, C.; Glembockyte, V.; Tinnefeld, P.