Porous Biomimetric Nanocontainers

描述(由申请人提供)：我们建议开发一种稳健的方法，将生物大分子包裹在多孔纳米级(直径30-100 nm)磷脂微囊中。毛孔将由葡萄球菌毒素α-溶血素形成。这些超小的、生物兼容的容器将允许小分子，如三磷酸腺苷和镁离子通过，同时限制大分子在泽普托体积内的扩散运动，从而实现单分子水平的新型生物物理分析。虽然这种方法使分子基本上没有表面假象，但可以将小泡拴在支撑的双层上，以便可以观察到几秒钟甚至几分钟的单分子反应。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。我们还将追求可光激活的毛孔，以便可以在局部触发生化反应，这将使高通量、高时间分辨率的单分子分析成为可能。如果孔道活化的时间尺度很快，这种方法也可以在系综动力学研究中被证明是有用的，因为它比停流法或传统的去孔化方法有许多优点。我们将使用特征良好的系统，如发夹核酶和Holliday连接来探索孔形成过程。此外，我们将利用这些技术在RNA酶和解旋酶的活性方面取得新的生物学发现。

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.