Tag: biology

Cryopreservation of DNA Origami Nanostructures

Although DNA origami nanostructures have found their way into numerous fields of fundamental and applied research, they often suffer from rather limited stability when subjected to environments that differ from the employed assembly conditions, that is, suspended in Mg2+-containing buffer at moderate temperatures.*

In the article “Cryopreservation of DNA Origami Nanostructures” Yang Xin, Charlotte Kielar, Siqi Zhu, Christoph Sikeler, Xiaodan Xu, Christin Möser, Guido Grundmeier, Tim Liedl, Amelie Heuer-Jungemann, David M. Smith and Adrian Keller investigate means for efficient cryopreservation of 2D and 3D DNA origami nanostructures and, in particular, the effect of repeated freezing and thawing. It is found that, while the 2D DNA origami nanostructures maintain their structural integrity over at least 32 freeze–thaw cycles, ice crystal formation makes the DNA origami gradually more sensitive toward harsh sample treatment conditions. *

The cryoprotectants glycerol and trehalose are found to efficiently protect the DNA origami nanostructures against freeze damage at concentrations between 0.2 × 10−3and 200 × 10−3m and without any negative effects on DNA origami shape. This work thus provides a basis for the long-term storage of DNA origami nanostructures, which is an important prerequisite for various technological and medical applications. *

NanoWorld Ultra-Short Cantilevers for High-Speed AFM USC-F0.3-k0.3 were used for the AFM imaging in liquid of the DNA  origami sample described in this article.

Figure 2 from “Cryopreservation of DNA Origami Nanostructures” by Yang Xin et al.: AFM images of triangular DNA origami nanostructures after 32 freeze–thaw cycles measured a) in air and b) in liquid. AFM images of triangular DNA origami nanostructures assembled from scaffold and staple strands that were subjected to 32 freeze–thaw cycles measured c) in air and d) in liquid. Images have a size of 1.5 × 1.5 μm2 and height scales are 2.3 nm.
Figure 2 from “Cryopreservation of DNA Origami Nanostructures” by Yang Xin et al.:

AFM images of triangular DNA origami nanostructures after 32 freeze–thaw cycles measured a) in air and b) in liquid. AFM images of triangular DNA origami nanostructures assembled from scaffold and staple strands that were subjected to 32 freeze–thaw cycles measured c) in air and d) in liquid. Images have a size of 1.5 × 1.5 μm2 and height scales are 2.3 nm.

*Yang Xin, Charlotte Kielar, Siqi Zhu, Christoph Sikeler, Xiaodan Xu, Christin Möser, Guido Grundmeier, Tim Liedl, Amelie Heuer-Jungemann, David M. Smith and Adrian Keller
Cryopreservation of DNA Origami Nanostructures
Small 2020, 16, 1905959
DOI: 10.1002/smll.20190595

Please follow this external link to read the full article: https://onlinelibrary.wiley.com/doi/pdf/10.1002/smll.201905959

Open Access The article “ Cryopreservation of DNA Origami Nanostructures “ by Yang Xin, Charlotte Kielar, Siqi Zhu, Christoph Sikeler, Xiaodan Xu, Christin Möser, Guido Grundmeier, Tim Liedl, Amelie Heuer-Jungemann, David M. Smith and Adrian Keller is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision.*

In the article “Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides” Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis and James A Frank report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells.*

They combine atomic force microscopy on model bilayers with metabolic tracing studies in cells, and demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. The findings described in the article disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light.*

The High-speed AFM in AC mode described in the article was done with NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 with a typical stiffness of 0.3 N/m. The AFM cantilever oscillation was tuned to a frequency of 100–150 kHz and the amplitude kept below 10 nm. The scan rate was set to 25–150 Hz. Images were acquired at 256 × 256 pixel resolution. All measurements were performed at room temperature. The force applied on the sample was minimized by continuously adjusting the set point and gain during imaging. Height, error, deflection and phase-shift signals were recorded and images were line-fitted as required.*

Figure 2 b from “ Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides “ by Matthijs Kol et al. :
Photo-isomerization of caCers affects membrane fluidity and lipid domain structure in supported lipid bilayers.
(b) Atomic force microscopy of supported lipid bilayers ( SLBs )prepared as in (a). Isomerization of caCer-3 (top) and caCer-4 (bottom) to cis with UV-A light (365 nm) resulted in a fluidification inside the Lo domains, as indicated by the appearance of small fluid Ld lakes and an increased Ld/Lo area ratio. This effect was reversed on isomerization back to trans with blue light (470 nm), marked by a drop in the Ld/Lo area ratio. Scale bars, 2 μm. (c) Time-course plotting the normalized Lo area over multiple 365/470 nm irradiation cycles for caCer-3 (top) and caCer-4 (bottom).
Please refer to https://doi.org/10.7554/eLife.43230.007 for the full figure.

*Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis, James A Frank
Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides
eLife 2019;8:e43230
DOI: 10.7554/eLife.43230
https://doi.org/10.7554/eLife.43230.001
https://doi.org/10.7554/eLife.43230.007

Please follow this external link to read the full article: https://elifesciences.org/articles/43230

Open Access The article “Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides “ by Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis and  James A Frank is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

The free energy landscape of retroviral integration

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved.*

In the article “The free energy landscape of retroviral integration” published in Nature Communications Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser and Jan Lipfert use biochemical assays, atomic force microscopy (AFM), and multiplexed single-molecule magnetic tweezers (MT) to study tetrameric prototype foamy virus (PFV) strand-transfer dynamics.*

Their finding that PFV intasomes employ auxiliary-binding sites for modulating the barriers to integration raises the question how the topology of higher-order intasomes governs integration of pathogenic retroviruses, most notably HIV. The single-molecule assays developed in this work are expected to be particularly useful to further unravel the complexity of this important class of molecular machines.*

The AFM images were recorded in amplitude modulation mode under ambient conditions and by using NanoWorld high resolution SuperSharpSilicon™ SSS-NCH cantilevers ( resonance frequency ≈300 kHz; typical end-radius 2 nm; half-cone angle <10 deg). Typical scans were recorded at 1–3 Hz line frequency, with optimized feedback parameters and at 512 × 512 pixels.*

Figure 2 e, f and g from “The free energy landscape of retroviral integration” by Willem Vanderlinden et al. (please refer to the full article for the complete figure 2 https://rdcu.be/b0R63 ) : e Atomic Force Microscopy image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes. f Atomic Force Microscopy image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation. g Atomic Force Microscopy image of a gel-purified STC
Figure 2 e, f and g from “The free energy landscape of retroviral integration” by Willem Vanderlinden et al.
(please refer to the full article for the complete figure 2 https://rdcu.be/b0R63 ) :
 e AFM image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes.
 f AFM image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation.
g AFM image of a gel-purified STC

*Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser, Jan Lipfert
The free energy landscape of retroviral integration
Nature Communications volume 10, Article number: 4738 (2019)
DOI: https://doi.org/10.1038/s41467-019-12649-w

Please follow this external link to read the full article: https://rdcu.be/b0R63

Open Access The article “The free energy landscape of retroviral integration“ by Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser and Jan Lipfert is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.