Porous Biomimetric Nanocontainers

描述（由申请人提供）：我们建议开发一种强大的方法将生物大分子封装在多孔纳米级（直径30-100纳米）磷脂囊泡中。葡萄球菌毒素-溶血素会形成毛孔。这些超小的、生物相容的容器将允许小分子（如ATP和镁离子）通过，同时限制了大分子在zeptollil体积内的扩散运动，从而在单分子水平上实现新型的生物物理分析。虽然这种方法使分子基本上没有表面伪影，但囊泡可以被拴在一个支持的双层上，这样单分子反应可以被观察几秒钟甚至几分钟。因此，这项技术有可能改变世界上许多实验室进行单分子荧光测量的方式。我们还将追求光活化孔，以便生物化学反应可以在局部触发，这应该能够实现高通量，高时间分辨率的单分子分析。如果孔隙活化的时间尺度很快，这种方法也可以被证明在系综动力学研究中很有用，因为它比停止流动方法或传统的松开方法有许多优点。我们将使用表征良好的系统，如发夹核酶和Holliday结来探测孔隙形成过程。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.