Intracellular Biophotonic Nanoswitches

最近对特定细胞中蛋白质之间所有物理相互作用的图谱证实了这样一个概念，即蛋白质之间的相互作用受到高度调控，并支撑着所有细胞过程。研究人员和技术人员面临着一个重大挑战--如何对这种复杂的系统提出具体问题，特别是当给定细胞中的蛋白质相互作用随时间变化并导致不同的末端状态时。例如，当人类细胞对压力做出反应时，主调控蛋白之间的特定相互作用开始驱动恢复过程或启动对细胞死亡的受控承诺。该项目旨在产生一种解决这一问题的通用技术--将合成开关引入活细胞，可以通过远程控制‘微调’蛋白质相互作用。这一令人兴奋的方法基于非常有希望的前期工作，将允许研究人员通过引入小干扰分子来规划已定义的蛋白质-蛋白质相互作用的变化，这些小干扰分子可以通过精心选择的波长的光来开启和关闭。小分子结构的变化是由外部光脉冲诱导肽骨架的构象重排，从而改变细胞内生物光子纳米开关(IBN)的生物学性质而引发的。IBN是一种光敏的基于纳米颗粒的分子结构，连接到短肽序列，识别蛋白质表面的特征，成为切换的目标。将IBN输送到活细胞的传统和新方法将允许将开关形成细胞群体的图案。用光操作这些IBN将使研究人员能够在如此复杂的细胞群体中对开关的激活进行模式识别，或者在单个细胞中对开关过程进行编程--这是操纵离散细胞内通路的主调节器技术的一大进步。我们的建议的冒险和风险涉及IBN设计的问题及其在活细胞中自我报告的可能性。IBN将允许研究人员通过生物物理手段切换或编程活细胞中主调节器的状态，并探索对整个系统的影响，以揭示不同途径的内部联系。此外，我们的建议解决了如何跟踪选择性切换的下游后果，即使在不同的谱系中，以揭示细胞在发展其反应时如何对不同的信号(幅度或频率)做出反应，即使这些信号在不同的细胞世代中快速或缓慢地发展。由于我们的愿景是为生命科学界提供基于强大的化学系统的新颖、健壮和易于实施的技术，该建议包含了与IBN技术的潜在用户和下游需求的接触，重点关注在复杂系统水平上理解细胞生物学的迅速增长的需求。令人兴奋的前景是，获得可编程的光子控制正常生理(指导干细胞分化、操纵伤口愈合和延缓细胞衰老)、肿瘤(细胞周期检查点功能障碍的癌症生物学和光控治疗)、构建细胞群落(光定向组织工程)和分子靶标识别(寻找新药和产品)。

Recent mapping of all physical interactions between proteins in a given cell has confirmed the notion that interactions between proteins are highly regulated and underpin all cellular processes. Researchers and technologists have been presented with a major challenge - how to ask specific questions of such complex systems especially when protein interactions change with time in a given cell and result in different end states. For example when a human cell responds to stress, specific interactions between master regulatory proteins start to drive a recovery process or initiate a controlled commitment to cell death. This project aims to generate a generic technology for solving this problem - introducing synthetic switches into live cells that can 'fine-tune' protein interactions by remote control. This exciting approach, based on highly promising preliminary work, would allow the investigator to programme changes in defined protein-protein interactions by the introduction of small interfering molecules engineered to be switched on and off by light of carefully selected wavelengths. Changes in the structure of a small molecule are triggered by external light pulses inducing conformational rearrangements in the peptide backbone and hence alterations of the biological properties of the Intracellular Biophotonic Nanoswitch (IBN). IBNs are light-sensitive nanoparticle-based molecular structures linked to the short peptide sequences that recognize features on the surface of a protein that has been targeted for switching. Conventional and novel methods for IBN delivery into live cells will allow patterning of the swiches into populations of cells. Operating these IBNs by light will allow the researcher to pattern the activation of switches in such complex cell populations or to 'programme' the switching process in single cells - a step-forward in the technology of manipulating master regulators of discrete intracellular pathways. Our proposal's adventure and risk relates to the problems of IBN design and their potential for self-reporting in live cells.IBNs will allow a researcher to switch or programme the state of a master regulator in a live cell by biophysical means and explore the consequences on the whole system to reveal the internal linking of different pathways. Furthermore, our proposal addresses how to track the downstream consequences of selective switching, even in different lineages, to reveal how cells respond to different signals (amplitude or frequency) in developing their responses even if these arise quickly or indeed develop slowly through different cell generations.Since our vision is to provide the life sciences community with novel, robust and readily implemented technologies based on robust chemical systems, the proposal encompasses engagement with potential user & downstream demands of IBN technology with a focus on the burgeoning demand to understand cellular biology at the complex 'systems' level. The exciting prospect looms of gaining programmable photonic control over normal physiology (directing stem cell differentiation, manipulating wound healing and delaying cell senescence), neoplasia (cancer biology of cell cycle checkpoint dysfunction and photonically-controlled therapeutics), constructed cell communities (light-directed tissue engineering) and molecular target identification (the search for new medicines and products).