Photo-inducible Cell Ablation and Fluorescent Chemosensors for Chemical Biology

我们有兴趣开发化学工具来探测体内生物的复杂性。具体来说，我们提出了两个研究领域：1）光诱导细胞消融。破译整个细胞在生物事件中的作用一直是一个艰巨的挑战，因为细胞在复杂的网络中发挥作用。探测它们的复杂性的一个强有力的方法是关闭细胞，然后监测生物体的发育/行为变化。为了有效地实现这一点，一个有前途的策略是使用小分子光敏剂，它可以在光的存在下产生活性氧，这将杀死或关闭细胞。然而，一旦施用，光敏剂在所有组织中积累，使得难以探测特定位置处的细胞功能。为了解决这个问题，我们建议开发一系列光敏剂，这些光敏剂不仅需要光，还需要一种酶来激活。通过这种化学遗传方法，可以在特定部位有效地破坏细胞（低光剂量），而周围区域的非特异性细胞破坏最小。总的来说，这种策略将使我们能够准确地分配功能的细胞和组织。2）荧光化学传感器。在其天然细胞环境中监测酶将是真正了解其功能的有力方法。能够直接报告其行为的工具可以提供正常和患病状态下活动的准确地图。在这方面，我们建议构建一系列的荧光化学传感器的酶类，甲基转移酶。我们的传感器将是通用的和选择性的，以允许实时感测活细胞中的所有甲基转移酶活性，并将用于回答有关其天然作用的特定问题，否则将难以通过常规生物化学方法获得。反过来，这些新的工具将允许更好地了解甲基转移酶在表观遗传途径的网络。

We are interested in developing chemical tools to probe biological complexity in vivo. Specifically, we are proposing two areas of research:1) Photo-induced Cell Ablation. Deciphering the role of entire cells during biological events has been a difficult challenge since cells function in complex networks. A powerful approach to probing their complexity is to turn “off” cells then monitor developmental/behavioral changes in the organism. To achieve this effectively, a promising strategy is to use small molecule photosensitizers that can produce reactive oxygen species in the presence of light, which will kill or turn “off” cells. However, once administered, photosensitizers accumulate in all tissues making it difficult to probe cellular function at a specific location. To address this issue, we are proposing to develop a series of photosensitizers that will require not only light but also an enzyme for its activation. With this chemical genetic approach, cells can be destroyed efficiently (low light dosage) at a specific site with minimal non-specific cell destruction of the surrounding region. Overall this strategy will enable us to accurately assign functions to cells and tissues.2) Fluorescent Chemosensors. Monitoring enzymes in their native cellular environments would be a powerful approach to truly understand their function. Tools that are capable of directly reporting on their behavior could provide an accurate map of activity in both normal and diseased states. To this regard, we are proposing to construct a series of fluorescent chemosensors for the enzyme class, methyltransferases. Our sensors will be general and selective to permit real-time sensing of all methyltransferase activity in live cells, and will be used to answer specific questions regarding their native roles that would otherwise be difficult to obtain by conventional biochemical methods. In turn, these novel tools will permit a better understanding of the network of methyltransferases in epigenetic pathways.