Nanofabricated quartz cylinders for angular trapping: DNA supercoiling torque detection

Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853, USA.
Nature Methods (Impact Factor: 32.07). 04/2007; 4(3):223-5. DOI: 10.1038/nmeth1013
Source: PubMed


We designed and created nanofabricated quartz cylinders well suited for torque application and detection in an angular optical trap. We made the cylinder axis perpendicular to the extraordinary axis of the quartz crystal and chemically functionalized only one end of each cylinder for attachment to a DNA molecule. We directly measured the torque on a single DNA molecule as it underwent a phase transition from B-form to supercoiled P-form.

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Available from: Christopher Deufel, Aug 18, 2014
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    • "One focus has been on modelling DNA. For example, in experiments [1] [2] [3] single molecules of twist-storing polymers such as double stranded DNA can be held torsionally constrained and under constant stretching force. These experiments show abrupt phase transitions known as buckling, and the formation of conformational structures known as plectonemes. "
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    ABSTRACT: We consider a simple lattice model of a topological phase transition in open polymers. To be precise, we study a model of self-avoiding walks on the simple cubic lattice tethered to a surface and weighted by an appropriately defined writhe. We also consider the effect of pulling the untethered end of the polymer from the surface. Regardless of the force we find a first-order phase transition which we argue is a consequence of increased knotting in the lattice polymer, rather than due to other effects such as the formation of plectonemes.
    Preview · Article · Oct 2014 · Journal of Physics A Mathematical and Theoretical
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    • "In order to probe nucleosome stability under torsion, we twisted a nucleosomal DNA molecule at low force to generate a desired degree of supercoiling, and then stretched the DNA to disrupt the nucleosome (Fig.1a). The range of forces (1–30 pN) and torques (−10 to +40 pN·nm) used were chosen so as to maintain DNA in its native B-form conformation37–40 and are comparable to those measured or estimated for molecular motors, such as RNA polymerase32,33,35. In order to ensure precise and efficient nucleosome positioning, we have utilized the high affinity 601 nucleosome positioning element41. "
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    ABSTRACT: The nucleosome, the fundamental packing unit of chromatin, has a distinct chirality: 147 bp of DNA are wrapped around the core histones in a left-handed, negative superhelix. It has been suggested that this chirality has functional significance, particularly in the context of the cellular processes that generate DNA supercoiling, such as transcription and replication. However, the impact of torsion on nucleosome structure and stability is largely unknown. Here we perform a detailed investigation of single nucleosome behaviour on the high-affinity 601-positioning sequence under tension and torque using the angular optical trapping technique. We find that torque has only a moderate effect on nucleosome unwrapping. In contrast, we observe a dramatic loss of H2A/H2B dimers on nucleosome disruption under positive torque, whereas (H3/H4)2 tetramers are efficiently retained irrespective of torsion. These data indicate that torque could regulate histone exchange during transcription and replication.
    Full-text · Article · Oct 2013 · Nature Communications
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    • "Solid-state synthesis of DNA can yield 100–150-bp primers of this design, but such primers are expensive (∼$200 per label). A variation of this more expensive method has been successfully implemented by ligating on a short (62 bp) dsDNA segment containing six biotin tags (38). However, we did not pursue this option but rather focused on a more general approach that did not require costly primers for each new DNA construct. "
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    ABSTRACT: Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two ‘megaprimers’, 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (∼500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.
    Preview · Article · Aug 2013 · Nucleic Acids Research
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