Optical Voltage Sensing Nano-Devices using DNA Self-Assembly

使用 DNA 自组装的光学电压传感纳米器件

• 批准号: 319003204
• 负责人: Professor Dr. Philip Tinnefeld
• 金额: --
• 依托单位: Cavendish Laboratory
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/319003204?language=en
• 关键词: Optical; Voltage; Sensing; Nano; Devices

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.

任何活细胞都需要膜电位来实现多种功能，包括能量产生、信息处理和传输。准确了解膜电压对于神经科学，尤其是大脑研究至关重要。现有技术在很大程度上依赖于膜电位的光学测量，而膜电位的光学测量目前受到低灵敏度、低速度和侵入性的限制。基因编码传感器依赖于不稳定的荧光蛋白，并且需要对宿主生物体进行基因改造。在这个项目中，我们提出了一种基于两种均使用 DNA 支架的基本设计的光学电压传感纳米器件 (VSND) 的新方法。 DNA 支架能够对 VSND 的所有重要功能进行空间和化学控制，包括膜定位、电压传感和生物相容性。附着在 DNA 支架上的亲脂性锚将 VSND 靶向膜甚至靶向膜内。附着在支架上并用荧光染料标记的带电柔性元件将通过在电场中移动来对膜电位的变化做出反应。柔性元件的运动将通过单分子荧光-共振-能量-从位于DNA支架上的染料到柔性元件上的染料的转移来检测。我们的两种互补的 VSND 设计将成为该项目的起点。电压传感筏附着在膜上，柔性元件伸入膜内，具有侵入性最小的优点。同时，电压传感孔集成到膜中，并包含受保护在中心孔中的传感器，而不受局部环境的干扰。将使用玻璃纳米移液器在定制装置上对模型膜进行同步电学和光学测量，对 VSND 进行测试和校准。这将使在以定性测量为主的领域中对跨膜电压进行量化成为可能。下一步，我们将应用 VSND 以前所未有的空间和时间分辨率量化活细菌和真核细胞的膜电位。成功实施后，我们将通过对活体斑马鱼的膜电压进行成像来证明 VSND 的体内适用性。这些实验将证明我们的方法具有所需的灵敏度和快速响应能力，可以回答有关细菌膜电位以及真核细胞膜和轴突电位的作用的基本问题。 VSND 的基于 DNA 的模块化设计允许直接优化和适应电压传感问题的通用解决方案，并且可能会找到从神经元功能的视觉测量到离子通道相关药物识别和筛选的应用。

项目成果

Selbstregeneration und Selbstheilung in DNA‐Origami‐Nanostrukturen

• DOI: 10.1002/ange.202012986
• 发表时间: 2021-02
• 期刊: Angewandte Chemie
• 作者: Michael Scheckenbach;T. Schubert;Carsten Forthmann;Viktorija Glembockyte;Philip Tinnefeld

DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity

• DOI: 10.1101/2021.08.18.456762
• 发表时间: 2021-08
• 期刊: bioRxiv
• 作者: Sarah E. Ochmann;Himanshu Joshi;Ece Büber;Henri G. Franquelim;Pierre Stegemann;B. Saccà;U. Keyser;A. Aksimentiev;P. Tinnefeld