A Genetically-Targeted Molecular Wire Fluorescent Sensor for Monitoring Voltage

描述(由申请人提供)：监测神经元中的电活动对于了解脑细胞交流的动态方式至关重要。目前，电生理学可以提供对单个细胞内电压变化和动作电位的精确测量，但电生理学测量所获得的空间信息有限。光学成像为同时监测多个神经元的电压变化提供了一个有吸引力的解决方案。不幸的是，没有单一的电压光学探头能够提供适合于动作电位和其他亚阈值事件的灵敏检测的探头的必要特性。在这项研究中，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)、细胞上可忽略的电容负载、遗传靶向性和合成可控性。在外加电场的作用下，苯胺供体和黄杂环基荧光团受体之间的光致电子转移效率将发生改变。变化的幅度将与激发到单重态时形成的场的强度和偶极子的长度成正比。考虑到神经元环境中的电场在大约105V/cm处相对恒定，施加电场时荧光的较大变化可以通过增加偶极或从供体到受体的距离来实现。为了克服电子传递的指数距离依赖性，这将显著削弱基于PET的传感器对电压的响应能力，将使用分子导线连接供体和受体。分子线通过改变电子转移的机制，从近距离的超交换转变为很远距离的电子跳跃，从而降低了电子转移的距离依赖性。分子导线将允许有效的电子转移，同时在引入电场时最大化荧光变化。通过使用化学反应手柄，将探针定位于遗传定义的神经元亚群，可以进一步改进传感器。膜结合的融合蛋白的表达将使传感器能够针对特定的神经元亚型，提高探针的信噪比，从而能够在不同的样本中研究神经元的遗传亚型。开发一种电压荧光探针，在保持良好的时间保真度和避免电容负载的同时，能够响应电压的变化而提供大量的荧光增加，将在广泛的神经系统研究中引起广泛的兴趣。 与公共健康相关：对神经细胞中电势变化的光学成像为研究神经元通讯的动力学提供了一种有吸引力的方法。传统的监测神经元活动的光学方法受到低信噪比的影响，以响应细胞膜上电压的变化。这项研究将开发新的分子线基荧光探针，用于以高空间和时间分辨率光学监测活细胞的电压。

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.