Optical Voltage Sensing Nano-Devices using DNA Self-Assembly

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Any living cell requires membrane potentials for a wide range of functions including energy production and information processing and transmittance. Exact knowledge of membrane voltages is of paramount importance in neuroscience and especially brain research. The available techniques heavily depend on optical measurements of membrane potentials which are currently limited by low sensitivity, low speed and invasiveness. Genetically encoded sensors rely on unstable fluorescent proteins and require genetic modification of the host organism. In this project, we suggest a new approach for optical voltage sensing nano-devices (VSND) based on two fundamental designs both using a DNA scaffold. The DNA scaffold enables spatial and chemical control over all important functions of the VSNDs including membrane positioning, voltage sensing and biocompatibility. Lipophilic anchors attached to the DNA scaffold will target the VSND to or even into the membrane. A charged, flexible element attached to the scaffold and labeled with a fluorescent dye will react to changes of the membrane potential by moving in the electric field. The movement of the flexible element will be detected by single-molecule Fluorescence-Resonance-Energy-Transfer from a dye located on the DNA scaffold to the dye on the flexible element. Our two complementary designs of VSNDs will be the starting point for this project. A voltage sensing raft that attaches to the membrane with the flexible elements protruding into the membrane offers the advantage of being least invasive. In parallel, a voltage sensing pore integrates into the membrane and contains the sensor protected in a central pore without perturbations from the local environment. The VSNDs will be tested and calibrated using simultaneous electrical and optical measurements on model membranes using glass nanopipettes on a custom-built setup. This will enable quantification of transmembrane voltages in a field that is dominated by qualitative measurements. In the next step, we will apply the VSNDs to quantify membrane potentials in living bacterial and eukaryotic cells with unprecedented spatial and temporal resolution. After successful implementation we will demonstrate the in vivo applicability of the VSNDs by imaging membrane voltages in living zebra fish. These experiments will prove that our approach has the required sensitivity and fast responsiveness to answer fundamental questions with respect to the role of bacterial membrane potentials and eukaryotic membrane and axon potentials. The modular DNA-based design of the VSNDs allows straightforward optimization and adaptation for a generic solution of voltage sensing problems and might find applications ranging from visual measurements of neuronal functions to ion-channel related drug identification and screening.