Chemical-selective real-time laser precision control of biomolecules

项目摘要 精确控制活细胞中生物分子行为的能力是一项具有挑战性的任务。当前方法 可以分为基于化学和基于激光的方法。例如，小分子抑制剂或 可将活化剂引入生物系统中以操纵酶活性。但据 不可能以高精度控制交互位置，这造成了脱靶效应。蛋白对照 使用基因沉默或编辑等遗传方法可能会选择性地影响靶蛋白，但需要 转染和孵育，并且不能实时进行。光学技术，如光镊 可以操纵激光焦点处的小目标，但一次只能与少数目标互动。当前激光 操作和消融方法通常需要预先获取的图像以及手动选择 样本上的目标位置。这种方法不仅费时，而且不适用于高度 动态活体生物样本。光遗传学方法可以使用光辐射控制神经元功能， 光敏离子通道，但仅限于单细胞水平。没有现有的技术可以选择 细胞中的分子目标，并仅以亚微米分辨率实时控制这些目标。 在这个应用中，我们开发了一个实时精密光控（RPOC）平台，可以选择性地和 使用激光仅在所需的相互作用位点精确控制生物分子。RPOC基于高速 激光扫描系统首先，在激光扫描期间，在来自像素的特定像素处生成光信号。 靶分子。然后，该光信号将与预设阈值进行比较，并发出电子信号。 用于控制声光调制器的信号，该声光调制器用作快速开关以将另一激光束耦合到 与同一个像素相互作用。光信号检测、处理和激光控制在20 ns内发生， 比像素停留时间快得多。数字逻辑电路也将与比较器电路一起设计， 基于来自多个信号通道的逻辑输出来控制所述相互作用激光束。我们将使用照片- 可转换蛋白质和设计光转换抑制剂和激活剂，以证明精确控制 现场酶活性。此外，我们将使用多个连续波激光器和声光可调谐 滤光片设计一个便携式和可扩展的RPOC，可以在光学实验室外运行。RPOC可以准确地 实时控制和操纵生物分子，而不影响系统中的其他生物分子。极有 化学选择性，因为光信号可以选自荧光、拉曼或吸收信号。它 将允许生物学家在激光扫描期间仅控制和询问感兴趣的生物分子， 以亚微米的精度影响样品的其他部分。RPOC在酶的研究中有着广泛的应用前景 细胞中的活动，了解细胞器的相互作用，改善药物的控释，并执行精确 神经调节

PROJECT SUMMARY The capability to precisely control behaviors of biomolecules in living cells is a challenging task. Current methods can be classified into chemical-based and laser-based approaches. For example, small molecule inhibitors or activators can be introduced into the biological system for manipulating enzyme activities. However, it is impossible to control the interaction locations with high precision, which poses off-target effects. Protein control using genetic methods such as gene silencing or editing might selectively impact a targeted protein, but requires transfection and incubation, and cannot be performed in real-time. Optical techniques such as optical tweezers can manipulate small targets at the laser focus, but can only interact with a few targets at a time. Current laser manipulation and ablation methods usually require a pre-acquired image together with a manual selection of target locations on samples. This method is not only time-consuming but also unsuitable to apply to highly dynamic living biological samples. Optogenetic methods can control neuron functions using light radiation and light-sensitive ion channels, but only at the single-cell level. There’s no existing technology that can select molecular targets in cells and control only these targets at sub-micron resolution in real-time. In this application, we develop a real-time precision opto-control (RPOC) platform that can selectively and precisely control biomolecules only at the desired interaction site using lasers. RPOC is based on a high-speed laser scanning system. First, during the laser scanning, an optical signal is generated at a specific pixel from the target molecule. Then, this optical signal will be compared with a preset threshold and to send out an electronic signal to control an acousto-optic modulator which is used as a fast switch to couple another laser beam to interact with the same pixel. The optical signal detection, processing, and laser control happen within 20 ns, much faster than the pixel dwell time. Digital logic circuits will also be designed with the comparator circuits to control the interaction laser beam based on the logic output from multiple signal channels. We will use photo- switchable proteins and design photo-convertible inhibitors and activators to demonstrate precision control of enzyme activities on site. Furthermore, we will use multiple continuous-wave lasers and acousto-optic tunable filters to design a portable and multicolor RPOC that can operate outside an optical lab. RPOC can accurately control and manipulate biomolecules in real-time without affecting other biomolecules in the system. It is highly chemical selective since the optical signal can be selected from fluorescence, Raman, or absorption signals. It will allow biologists to control and interrogate only the biomolecules of interest during laser scanning without affecting other parts of the sample with sub-micron precision. RPOC would be widely applied to study enzyme activities in cells, understand organelle interactions, improve controlled-release of drugs, and perform precision neuromodulation.