Porous Biomimetric Nanocontainers

多孔仿生纳米容器

• 批准号: 7228913
• 负责人: Taekjip Ha
• 金额: $ 15.87万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228913
• 关键词: ATP Hydrolysis; Bacterial Toxins; Biochemical; Biochemical Reaction; Biocompatible; Biological; Biological Assay; Buffers; Caliber; Chemicals; Collaborations; Condition; Cruciform DNA; Cysteine; Cytolysis; DNA; Dissociation; Encapsulated; Enzymes; Erythrocytes; Fluorescence; Gel; Genus staphylococcus; Goals; Hemolysin; Image; Ions; Kinetics; Laboratories; Ligation; Measurement; Measures; Methods; Morphologic artifacts; Motion; Numbers; Oryctolagus cuniculus; Permeability; Phospholipids; Process; RNA; Range; Reaction; Resolution; Sampling; Staphylococcus alpha toxin; Surface; System; Techniques; Temperature; Time; Ultraviolet Rays; Universities; Vesicle; desire; hairpin ribozyme; helicase; improved; macromolecule; magnesium ion; mutant; nano container; nanoscale; professor; research study; single molecule; small molecule; stop flow technique

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.

描述（由申请人提供）：我们建议开发一种稳健的方法，将生物大分子封装在多孔纳米级（直径 30-100 nm）磷脂囊泡内。毛孔将由葡萄球菌毒素α-溶血素形成。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上的新型生物物理分析。虽然该方法使分子基本上没有表面伪影，但囊泡可以束缚在支撑的双层上，以便可以观察单分子反应几秒钟甚至几分钟。因此，该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，以便可以局部触发生化反应，从而实现高通量、高时间分辨率的单分子分析。如果孔隙激活的时间尺度很快，这种方法也可以在系综动力学研究中发挥作用，因为它比停流方法或传统的解笼方法具有许多优点。我们将使用发夹核酶和霍利迪连接体等充分表征的系统来探测孔形成过程。此外，我们将利用这些技术对 RNA 酶和解旋酶的活性做出新的生物学发现。

项目成果

A single-vesicle content mixing assay for SNARE-mediated membrane fusion.

• DOI: 10.1038/ncomms1054
• 发表时间: 2010-08-10
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Diao, Jiajie;Su, Zengliu;Ishitsuka, Yuji;Lu, Bin;Lee, Kyung Suk;Lai, Ying;Shin, Yeon-Kyun;Ha, Taekjip
• 通讯作者: Ha, Taekjip

Single-molecule FRET study of SNARE-mediated membrane fusion.

• DOI: 10.1042/bsr20110011
• 发表时间: 2011-12
• 期刊: Bioscience reports
• 影响因子: 4
• 作者: Diao J;Ishitsuka Y;Bae WR
• 通讯作者: Bae WR