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High resolution optical tweezers for single molecule studies of hierarchical folding in the pbuE riboswitch aptamer.

机译:高分辨率光学镊子,用于pbuE核糖开关适体中的分层折叠的单分子研究。

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摘要

Riboswitches are gene regulatory elements found in messenger RNA that function by changing structure upon the binding of a ligand to an aptamer domain. Single adenine-binding pbuE riboswitch aptamer RNAs were unfolded and refolded co-transcriptionally using optical tweezers for single molecule force spectroscopy. The kinetic and energetic properties of distinct folding intermediates were characterised with and without the binding of adenine. These observed intermediates were related to structural elements of the aptamer, which were found to fold sequentially, in a transcriptionally independent manner. The mechanical switch underlying the regulatory action of the riboswitch was observed directly (adenine stabilisation of the weakest helix), and the energy landscape for the folding was reconstructed.The construction of a dual-beam optical trap with separate detection and trapping laser beams manipulated and focused into a rigid, modified inverted microscope is also described. This instrument aims to achieve angstrom-level resolution through careful design to reduce noise.
机译:核糖开关是在信使RNA中发现的基因调控元件,其通过在配体与适体结构域结合后改变结构而起作用。使用光学镊子对单个腺嘌呤结合的pbuE核糖开关适体RNA进行解折叠和共转录重新折叠,以用于单分子力光谱法。在有和没有腺嘌呤结合的情况下,表征了不同折叠中间体的动力学和能量特性。这些观察到的中间体与适体的结构元件有关,发现它们以转录独立的方式顺序折叠。直接观察到核糖开关调节作用背后的机械开关(最弱螺旋的腺嘌呤稳定化),并重建折叠的能量分布图。双束光阱的构造,其具有分别检测和捕获激光束的操作和还介绍了聚焦到刚性,改进型倒置显微镜中的方法。该仪器旨在通过精心设计来降低噪音,从而达到埃级分辨率。

著录项

  • 作者

    Foster, Daniel.;

  • 作者单位

    University of Alberta (Canada).;

  • 授予单位 University of Alberta (Canada).;
  • 学科 Physics Molecular.Biophysics General.Biophysics Biomechanics.
  • 学位 M.Sc.
  • 年度 2010
  • 页码 144 p.
  • 总页数 144
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 R592;
  • 关键词

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