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Temperature-Independent Porous Nanocontainers for Single-Molecule Fluorescence Studies

机译:与温度无关的多孔纳米容器,用于单分子荧光研究

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In this work, we demonstrate the capability of using lipidnvesicles biofunctionalized with protein channels to performnsingle-molecule fluorescence measurements over anbiologically relevant temperature range. Lipid vesicles cannserve as an ideal nanocontainer for single-molecule fluorescencenmeasurements of biomacromolecules. One seriousnlimitation of the vesicle encapsulation method hasnbeen that the lipid membrane is practically impermeablento most ions and small molecules, limiting its applicationnto observing reactions in equilibrium with the initial bufferncondition. To permeabilize the barrier, Staphylococcusnaureus toxin r-hemolysin (aHL) channels have beennincorporated into the membrane. These aHL channelsnhave been characterized using single-molecule fluorescencenresonance energy transfer signals from vesicleencapsulatednguanine-rich DNA that folds in a G-quadruplexnmotif as well as from the Rep helicase-DNA system.nWe show that these aHL channels are permeable tonmonovalent ions and small molecules, such as ATP, overnthe biologically relevant temperature range (17-37 °C).nIons can efficiently pass through preformed aHL channelsnto initiate DNA folding without any detectable delay. Withnaddition of the cholesterol to the membrane, we alsonreport a 35-fold improvement in the aHL channel formationnefficiency, making this approach more practical fornwider applications. Finally, the temperature-dependentnsingle-molecule enzymatic study inside these nanocontainersnis demonstrated by measuring the Rep helicasenrepetitive shuttling dynamics along a single-stranded DNAnat various temperatures. The permeability of the biofriendlynnanocontainer over a wide range of temperaturenwould be effectively applied to other surface-based highthroughputnmeasurements and sensors beyond the singlemoleculenfluorescence measurements.
机译:在这项工作中,我们证明了使用与蛋白质通道生物功能化的脂质小囊在生物相关温度范围内进行单分子荧光测量的能力。脂质囊泡可以作为生物大分子单分子荧光测量的理想纳米容器。囊泡包囊方法的一个严重局限性是脂质膜实际上不能渗透大多数离子和小分子,从而限制了其在观察与初始缓冲条件平衡的反应中的应用。为了透化屏障,已将葡萄球菌毒素r-溶血素(aHL)通道并入膜中。这些aHL通道已使用来自囊泡封装的富含鸟嘌呤的DNA的单分子荧光共振能量转移信号进行了表征,该信号在G-四链体基元中以及在Rep解旋酶-DNA系统中折叠.n我们证明了这些aHL通道是可渗透的单价离子和小分子,例如ATP,在生物学上相关的温度范围(17-37°C)范围内,离子可以有效地通过预先形成的aHL通道,从而启动DNA折叠而没有任何可察觉的延迟。除了在膜上添加胆固醇外,我们还没有报告aHL通道形成效率提高了35倍,使这种方法在更广泛的应用中更加实用。最后,通过测量沿着不同温度的单链DNAnat的Rep螺旋重复穿梭动力学,证明了这些纳米容器内部的温度依赖性单分子酶学研究。生物友好型纳米容器在宽温度范围内的渗透性将有效地应用于单分子荧光测量以外的其他基于表面的高通量测量和传感器。

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  • 来源
    《Analytical Chemistry》 |2010年第23期|p.9694-9701|共8页
  • 作者单位

    Department of Physics, Center for the Physics of Living Cells, and Department of Chemical and BiomolecularEngineering, University of Illinois at Urbana-Champaign, and Howard Hughes Medical Institute, Urbana,Illinois 61801, United States;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 入库时间 2022-08-17 13:36:50

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