Porous Biomimetric Nanocontainers

描述（由申请人提供）：我们建议开发一种稳健的方法，将生物大分子包封在多孔纳米级（直径30-100 nm）磷脂囊泡内。葡萄球菌毒素α-溶血素将形成孔。这些超小型生物相容性容器将允许ATP和镁离子等小分子通过，同时限制zeptoliter体积内大分子的扩散运动，从而实现单分子水平的新型生物物理分析。虽然该方法保持分子基本上没有表面伪影，但囊泡可以被拴系到支持的双层上，使得可以观察单分子反应数秒甚至数分钟。因此，这项技术有可能改变世界各地许多实验室进行单分子荧光测量的方式。我们还将追求光激活孔，使生化反应可以被局部触发，这应该能够实现高通量，高时间分辨率的单分子分析。如果孔活化的时间尺度是快的，这种方法也可以证明是有用的系综动力学研究，因为它具有许多优势，停止流动的方法或传统的uncaging方法。我们将使用良好的表征系统，如发夹核酶和霍利迪连接，以探测孔形成过程。此外，我们还将利用这些技术对RNA酶和解旋酶的活性进行新的生物学发现。

DESCRIPTION (provided by applicant): We propose to develop a robust method to encapsulate biological macromolecules inside porous nano-scale (30-100 nm diameter) phospholipid vesicles. The pores will be formed by Staphylococcal toxin alpha-hemolysin. These ultrasmall, biocompatible containers will allow the passage of small molecules such as ATP and magnesium ions, while limiting the diffusional motion of macromolecules inside the zeptoliter volume, thereby enabling new types of biophysical analysis at the single-molecule level. While the method keeps the molecules essentially free of surface artifacts, the vesicles can be tethered to a supported bilayer so that single molecule reactions can be observed for seconds or even minutes. Therefore, this technique has the potential of transforming the way single-molecule fluorescence measurements are performed in many laboratories around the world. We will also pursue light-activatable pores so that biochemical reactions can be triggered locally, which should enable high-throughput, high time resolution single molecule analysis. If the time scale of pore activation is fast, this approach could also prove useful in ensemble kinetic studies since it has a number of advantages over stopped flow methods or conventional uncaging methods. We will use well-characterized systems such as the hairpin ribozyme and the Holliday junction to probe the pore formation process. Furthermore we will use these techniques to make new biological discoveries on the activities of RNA enzymes and helicases.