Abstract
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.
Highlights
Single-molecule techniques have become increasingly important in the last two decades, as they have permitted detailed insights to biological processes that are not readily apparent in ensemble measurements
We introduce the application of ethanolamine and heterobifunctional poly(ethylene glycol) (PEG) linkers
Toward higher averaging times up to 3 s, the bead stability decreases globally, with excursions up to 13 Athat result from the increasing influence of instrumental drift. These results demonstrate that both approaches are suitable for highresolution measurements [41,46,49] and, more importantly, that we have provided a protocol for reference bead fixation that is compatible with covalent tethering of DNA tethers
Summary
Single-molecule techniques have become increasingly important in the last two decades, as they have permitted detailed insights to biological processes that are not readily apparent in ensemble measurements. They provide access to novel parameters such as force, which plays a fundamental role in a variety of biological processes ranging from cellular motility to the replication, repair and segregation of DNA [1,2]. The magnetic and optical tweezers methods have garnered particular attention as they provide high temporal, spatial and force sensitivity in a range from hundreds of femtoNewtons to tens of picoNewtons (pN), and, in contrast to AFM, they are capable of measuring and applying torque [4,5,6,7]. To allow measurements over longer timeframes and broader force spectra, the anchoring of biomolecules in tweezers experiments should employ strong chemical bonding
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