Janissen Richard, Berghuis Bojk A, Dulin David, Wink Max, van Laar Theo, Dekker Nynke H
Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
Nucleic Acids Res. 2014 Oct;42(18):e137. doi: 10.1093/nar/gku677. Epub 2014 Aug 19.
Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.
磁镊是一种强大的单分子技术，可在施加力的情况下对生物分子过程进行实时定量研究。例如，为了探测积极转录或包装基因组的蛋白质的力范围，可能需要超过数十皮牛顿的高拉力。然而，频繁施加这样的力会缩短样品寿命，阻碍数据采集。为了在高拉力下提供实验上可行的样品寿命，我们设计了一种用于磁镊中DNA的新型固定策略。我们的方法利用基于异双功能聚乙二醇交联剂的共价功能化，使我们能够牢固地束缚DNA，同时抑制不期望的非特异性粘附。对这些共价固定的DNA系链进行的完整力和寿命表征表明，与更常用的固定策略相比，它们能承受高三倍的拉力（高达150 pN），并且在150 pN的恒定力下寿命延长高达200倍（超过24小时）。这一进展使得将生物相关的全范围力尺度应用于生物分子过程成为可能，并且其简单的实施方式应会将其应用范围扩展到单分子力谱领域的众多应用中。
