A Genetically-Targeted Molecular Wire Fluorescent Sensor for Monitoring Voltage

描述（由申请人提供）：监测神经元的电活动对于理解脑细胞交流的动态方式至关重要。目前，电生理学可以精确测量单个细胞的电压变化和动作电位，但电生理学测量得到的空间信息是有限的。光学成像为同时监测多个神经元的电压变化提供了一种有吸引力的解决方案。不幸的是，没有一种单独的电压光学探头能够提供适合灵敏检测动作电位和其他亚阈值事件的探头所必需的特性。在本研究中，设计了一种检测神经元细胞电压变化的方法，该方法结合了大的荧光变化（1-2%/mV），快速动力学（亚微秒），细胞可忽略的电容负荷，遗传靶向性和合成可追溯性。在外加电场的作用下，苯胺供体和杂蒽基荧光基团受体之间的光诱导电子转移（PeT）的效率将发生改变。变化的大小将与场的强度和激发到单重态激发态时形成的偶极子的长度成正比。考虑到神经元环境中的电场相对恒定，约为105 V/cm，通过增加偶极子或从供体到受体的距离，可以实现电场施加后荧光的大变化。为了克服电子转移的指数距离依赖性，这将大大削弱基于pet的传感器对电压的响应能力，将使用分子线连接供体和受体。分子线通过改变电子从近距离的超交换到极远距离的电子跳跃的转移机制，降低了电子转移的距离依赖性。分子线将允许有效的电子转移，同时在引入电场时最大化荧光的变化。通过使用化学反应手柄将探针定位到基因定义的神经元亚群，传感器可以进一步改进。膜结合融合蛋白的表达，共价自连接正交化学手柄到自己，将允许传感器靶向特定的神经元亚型，增加探头响应电压变化的信噪比，并允许在异质样品中研究神经元的遗传亚型。开发一种电压荧光探针，可以在响应电压变化的同时提供大量荧光增加，同时保持良好的时间保真度并避免电容性负载，这将对研究广泛的神经系统具有广泛的兴趣。

DESCRIPTION (provided by applicant): Monitoring electrical activity in neurons is of critical importance for understanding the dynamic manner in which brain cells communicate. Currently, electrophysiology can provide exquisitely precise measurements of voltage changes and action potentials in a single cell, but the spatial information derived from electrophysiological measurement is limited. Optical imaging provides an attractive solution for monitoring voltage changes in multiple neurons simultaneously. Unfortunately, no single optical probe for voltage has been able to supply the requisite characteristics of a probe suitable for sensitive detection of action potentials and other sub-threshold events. In this research, a method is devised for detecting changes in voltage in neuronal cells which incorporates large fluorescence changes (1-2%/mV), fast kinetics (submicrosecond), negligible capacitative load on the cell, genetic targetability, and synthetic tractability. The efficiency of photo- induced electron transfer (PeT) between an aniline donor and a xanthene-based fluorophore acceptor will be altered in the presence of an applied electric field. The magnitude of the change will be proportional to the strength of the field and the length of the dipole formed upon excitation to the singlet excited state. Given that the electric field in a neuronal context will be relatively constant at approximately 105 V/cm, large changes in fluorescence upon application of an electric field can be realized by increasing the dipole, or the distance from donor to acceptor. To combat the exponential distance dependence of electron transfer, which will significantly impair the ability of a PeT-based sensor to respond to voltage, a molecular wire will be used to connect donor to acceptor. Molecular wires decrease the distance dependence of electron transfer, by changing the mechanism of electron transfer from superexchange at close distances to electron hopping at very large distances. A molecular wire will allow effective electron transfer while maximizing the change in fluorescence upon introduction of an electric field. The sensor can be further improved by targeting the probe, through the use of a chemically reactive handle, to genetically defined neuronal sub-populations. Expression of membrane- bound fusion proteins that covalently self-ligate orthogonal chemical handles to themselves will allow for targeting of the sensor to specific sub-types of neurons, increasing the signal-to-noise ratios of the probe in response to voltage changes and allowing for investigation of genetic sub-types of neurons within heterogeneous samples. Development of a fluorescent probe for voltage which can deliver large fluorescence increases in response to changes in voltage while maintaining good temporal fidelity and avoiding capacitative loading will be of broad interest for studying a wide range of neurological systems. PUBLIC HEALTH RELEVANCE: Optical imaging of changes in electrical potential in neuronal cells offers an attractive method for studying the dynamics of neuronal communication. Traditional optical methods for monitoring neuronal activity suffer from low signal-to-noise ratios in response to changes in voltage across the cellular membrane. This research will develop new molecular wire-based fluorescent probes for optically monitoring voltage in living cells with high spatial and temporal resolution.