10 research outputs found

    Fluorescence and Scattering based Single Molecule Microscopy with DNA Origami Nanostructures

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    Seit der Einführung der DNA-Origami-Technik im Jahr 2006 durch P. Rothemund können dreidimensionale Nanostrukturen durch eine einfache Bottom-up-Synthese hergestellt werden. Diese ermöglichen eine exakte Positionierung von Bio- und Farbstoffmolekülen im einstelligen Nanometerbereich. In Kooperation mit der Arbeitsgruppe von T. Liedl (LMU München) wurde eine molekulare Kraftklammer entwickelt, bei der über Einzelmolekül-Förster-Resonanz-Energie-Transfer (smFRET) die zwischen Biomolekülen wirkenden Kräfte ausgelesen werden. Die Untersuchung der Holliday Junction und des TATA-Box-bindenden Proteins auf ihr Verhalten unter kontrollierter Krafteinwirkung hin zeigte, dass die molekulare Kraftklammer aufgrund ihrer Robustheit ein geeignetes System ist, Kräfte im niedrigen Pikonewton Bereich zu messen und jegliche Systeme zu untersuchen, die mit DNA wechselwirken oder selbst durch DNA modifizierbar sind. Dass FRET nicht nur ein Werkzeug, sondern auch ein in der Natur auftretender Effekt ist, zeigt sich in Lichtsammelkomplexen (LSK). Dort sammeln mehrere Donormoleküle Sonnenlicht ein und übertragen die Energie via FRET auf einen Akzeptor. Um die Effizienz eines solchen Systems zu bestimmen, wurden in Kooperation mit der Arbeitsgruppe von U. Keyser (University of Cambridge) synthetische LSKs basierend auf der DNA-Origami-Technik entwickelt und mittels smFRET-Messungen auf ihren Antenneneffekt hin untersucht. Dabei konnte gezeigt werden, dass sich DNA-Nanostrukturen aufgrund ihrer Homogenität und Robustheit als Gerüst für die Entwicklung von LSKs eignen. Die notwendigen hohen Laserleistungen (bis zu 1 kW/cm²) bei der Einzelmolekülmikroskopie schränken die möglichen Anwendungen jedoch stark ein. Daher wurden neuartige Nanoantennen auf DNA-Basis entwickelt, die durch die räumliche Nähe von metallischen Nanopartikeln zu Farbstoffmolekülen eine Verstärkung der Anregungs- und Emissionsrate erreichen und so Messungen bei deutlich niedrigeren Laserleistungen ermöglichen. Um diese für die molekulare Diagnostik weiterentwickeln zu können, müssen die auftretenden Effekte verstanden werden. Im Mittelpunkt dieser Arbeit steht die Entwicklung einer Methode um die optischen Eigenschaften des metallischen Nanopartikels mit denen des Farbstoffmoleküls auf Einzelmolekülebene zu korrelieren und so das Verständnis über plasmonische Effekte in Nanoantennen zu erweitern.Since the DNA origami technique was introduced in 2006 by P. Rothemund, three dimensional nanostructures can be assembled via bottom-up synthesis. These enable the exact positioning of biomolecules and dyes in the range of a few nanometres. Together with the working group of T. Liedl from the LMU München, we developed a molecular force clamp which is used for the readout of forces that exist between biomolecules via single-molecule Förster resonance energy transfer (smFRET). By studying the properties of the Holliday Junction and the TATA-binding protein with respect to the amount of applied force, it was demonstrated that, due to its robustness, the molecular force clamp is a system which is suitable for the measurement of forces of a few piconewton of every system that interacts with DNA or can be modified with DNA. However, FRET is not only a tool to study distance dependent interactions but it is also present in natural light harvesting complexes (LHC). These consist of multiple donor molecules that collect sun light and transfer the energy to an acceptor via FRET. To determine the efficiency of such a system, the antenna effect of synthetic LHCs based on the DNA origami technique were studied together with the working group of U. Keyser from the University of Cambridge. With single-molecule FRET measurements, it was possible to show that the synthesized samples are very homogenous and further that the DNA nanostructures are suitable as a platform for the development of LHCs. The high laser powers (up to 1 kW/cm²) required for single-molecule microscopy limit the range of applications. Thus, new nanoantennas based on the DNA origami technique were developed that lead to an enhanced excitation and emission rate due to the positioning of metallic nanoparticles next to a dye and enable measurements at low laser powers. For further development, which will lead to their use in diagnostic experiments, it is necessary to understand the occurring effects. The main part of this work focuses on the development of a method to correlate the fluorescence properties of dyes with the scattering spectra of adjacent metallic nanoparticles at the single-molecule level and thus to give an insight into the plasmonic effects occurring in these nanoantennas

    Programming Light-Harvesting Efficiency Using DNA Origami.

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    The remarkable performance and quantum efficiency of biological light-harvesting complexes has prompted a multidisciplinary interest in engineering biologically inspired antenna systems as a possible route to novel solar cell technologies. Key to the effectiveness of biological "nanomachines" in light capture and energy transport is their highly ordered nanoscale architecture of photoactive molecules. Recently, DNA origami has emerged as a powerful tool for organizing multiple chromophores with base-pair accuracy and full geometric freedom. Here, we present a programmable antenna array on a DNA origami platform that enables the implementation of rationally designed antenna structures. We systematically analyze the light-harvesting efficiency with respect to number of donors and interdye distances of a ring-like antenna using ensemble and single-molecule fluorescence spectroscopy and detailed Förster modeling. This comprehensive study demonstrates exquisite and reliable structural control over multichromophoric geometries and points to DNA origami as highly versatile platform for testing design concepts in artificial light-harvesting networks.A. W. C. acknowledges support from the Winton Programme for the Physics of Sustainability. U. F. K. was partly supported by an ERC starting grant (PassMembrane, EY 261101). E. A.H. acknowledges support from Janggen-Pöhn Stiftung and the Schweizerischer Nationalfonds (SNF). P. T. acknowledges support by a starting grant (SiMBA, EU 261162) of the European Research Council (ERC). B. W. gratefully acknowledges support by the Braunschweig International Graduate School of Metrology B-IGSM and the DFG Research Training Group GrK1952/1 ‘Metrology for Complex Nanosystems’. P. M. thankfully acknowledges the support of the EPSRC Centre for Doctoral Training in Sensor Technologies and Applications EP/L015889/1.This is the final version of the article. It first appeared from ACS via https://doi.org/10.1021/acs.nanolett.5b0513

    Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

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    Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

    Molecular force spectroscopy with a DNA origami–based nanoscopic force clamp

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    Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein-induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation
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