Tag: 高速AFMプローブ

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC

Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA.*

In their article “Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC” Alison O. Nwokeoji, Sandip Kumar, Peter M. Kilby, David E. Portwood, Jamie K. Hobbs and Mark J. Dickman have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications.*

The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length.*

The work presented in this study demonstrates the ability of AFM in conjunction with IP RP HPLC to rapidly assess sample heterogeneity and provide important structural information regarding dsRNA.*

For the high resolution images presented in Fig. 1(A, B) and 2(B) in the article NanoWorld Ultra-Short Cantilevers USC-F1.2-k0.15 with a High Density Carbon tip (nominal values: tip radius 10 nm, cantilever length 7 μm, stiffness 0.15 N m−1, resonant frequency 1200 kHz in air) were tuned to 600–650 kHz, oscillated at a free amplitude of <30 mV and scanned at a rate of 0.4–1.0 μm s−1,to visualize the dsRNA and dsDNA grooves.*


Fig. 1 A and B from “Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC” by Alison O. Nwokeoji et al. :
Analysis of dsRNA monomers, multimers and higher order assemblies under non-denaturing conditions. Non-denaturing gel electrophoretograms (A) in vivo synthesised dsRNA (521 bp and 698 bp) (B) in vitro synthesised dsRNA (504 bp). Each dsRNA sample was run in duplicate. The proposed dsRNA multimers or higher order assemblies with reduced electrophoretic mobility are highlighted above the corresponding dsRNA main band.

*Alison O. Nwokeoji, Sandip Kumar,Peter M. Kilby, David E. Portwood, Jamie K. Hobbs and Mark J. Dickman
Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC
Analyst, 2019,144, 4985
DOI: 10.1039/c9an00954j

Please follow this external link for the full article: https://pubs.rsc.org/en/content/articlelanding/2019/an/c9an00954j

Open Access: The article « Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC  by Alison O. Nwokeoji, Sandip Kumar,Peter M. Kilby, David E. Portwood, Jamie K. Hobbs and Mark J. Dickman is licensed under a Creative Commons Attribution 3.0 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 https://creativecommons.org/licenses/by/3.0/.

Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability

DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions.*

In their article “Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability” Charlotte Kielar, Yang Xin, Xiaodan Xu, Siqi Zhu, Nelli Gorin , Guido Grundmeier, Christin Möser, David M. Smith and Adrian Keller investigate the effect of long-term storage of the employed staple strands on DNA origami assembly and stability.*

Atomic force microscopy (AFM) under liquid and dry conditions was employed to characterize the structural integrity of Rothemund triangles assembled from different staple sets that have been stored at −20 °C for up to 43 months.*

NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 were the AFM probes that were used for the AFM measurements under liquid conditions.*

Figure 1. from “Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability” by Charlotte Kielar et al.
(a) Schematic illustration of the Rothemund triangle DNA origami. AFM images of DNA origami triangles assembled from staple sets aged for (b) 2–7 months, (c) 11–16 months, (d) 22–27 months, and (e) 38–43 months. Measurements were performed either in liquid (left column) or dry conditions after gently dipping the sample into water (central column) or after harsh rinsing (right column). Scale bars represent 250 nm. Height scales are given in the individual images. The insets show zooms of individual DNA origami triangles.

*Charlotte Kielar, Yang Xin, Xiaodan Xu, Siqi Zhu, Nelli Gorin , Guido Grundmeier, Christin Möser, David M. Smith and Adrian Keller
Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability
Molecules 2019, 24(14), 2577
doi: https://doi.org/10.3390/molecules24142577

Please follow this external link to the full article: https://www.mdpi.com/1420-3049/24/14/2577/htm

Open Access: The article « Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability » by Charlotte Kielar, Yang Xin, Xiaodan Xu, Siqi Zhu, Nelli Gorin , Guido Grundmeier, Christin Möser, 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/.

Direct Observation of Long-Chain Branches in a Low-Density Polyethylene

The properties of a polymer change significantly depending on the structure of the polymer chain, particularly, with branched structures, depending on the number of branches and the length of the branch.* However, the long-chain branch (LCB) structure of polyethylene was unclear, due particularly to the complex polymer structure and the limitations of its analysis methods.

In their study “Direct Observation of Long-Chain Branches in a Low-Density Polyethylene” Ken-ichi Shinohara, Masahiro Yanagisawa and Yuu Makida measured the chain length of LCBs and the distance between branch points of LDPE by atomic force microscopy.*

The article mentions the use of NanoWorld Ultra-Short Cantilevers (USC) for high speed atomic force microscopy ( AFM probe type USC-F1.2-k0.15 ) for the single-molecule imaging by atomic force microscopy .*

Figure 1 from “Direct Observation of Long-Chain Branches in a Low-Density Polyethylene “ by K. Shinohara et al.: Direct measurement of LCB in a tubular LDPE (F200-0 fractionated). (A) AFM image of a single molecule of LDPE on mica in DMTS at 25 °C. X: 279 nm, Y: 209 nm, Z: 18 nm. (B) Length of each chain of LDPE. (C) A wire model of self-shrinking structure of polymer chain of LDPE. Main chain: red wire. LCB: black wire. The model was created to be one tenth of the length of the extended chain based on AFM observation (B), MD simulation (Fig. S1), and the molecular weight determined by SEC-MALLS-Visc experiments (see Fig. 2).
Figure 1 from “Direct Observation of Long-Chain Branches in a Low-Density Polyethylene “ by K. Shinohara et al.: Direct measurement of LCB in a tubular LDPE (F200-0 fractionated). (A) AFM image of a single molecule of LDPE on mica in DMTS at 25 °C. X: 279 nm, Y: 209 nm, Z: 18 nm. (B) Length of each chain of LDPE. (C) A wire model of self-shrinking structure of polymer chain of LDPE. Main chain: red wire. LCB: black wire. The model was created to be one tenth of the length of the extended chain based on AFM observation (B), MD simulation (Fig. S1), and the molecular weight determined by SEC-MALLS-Visc experiments (see Fig. 2).

*Ken-ichi Shinohara, Masahiro Yanagisawa, Yuu Makida
Direct Observation of Long-Chain Branches in a Low-Density Polyethylene
Nature Scientific Reportsvolume 9, Article number: 9791 (2019)
doi: https://doi.org/10.1038/s41598-019-46035-9

Please follow this external link for the full article: https://rdcu.be/bJY7S

Open Access: The article «Direct Observation of Long-Chain Branches in a Low-Density Polyethylene» by Ken-ichi Shinohara, Masahiro Yanagisawa and Yuu Makida 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/.