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/.

Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study

“Motile cells require reversible adhesion to solid surfaces to accomplish force transmission upon locomotion. In contrast to mammalian cells, Dictyostelium discoideum ( a soil dwelling amoeba) cells do not express integrins forming focal adhesions but are believed to rely on more generic interaction forces that guarantee a larger flexibility; even the ability to swim has been described for Dictyostelium discoideum (D.d.).”* In order to understand the origin of D.d. adhesion, Nadine Kamprad, Hannes Witt, Marcel Schröder, Christian Titus Kreis, Oliver Bäumchen, Andreas Janshoff and Marco Tarantola  describe in their publication “Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study”* how they realized and modified a variety of conditions for the amoeba comprising the absence and presence of the specific adhesion protein Substrate Adhesion A (sadA), glycolytic degradation, ionic strength, surface hydrophobicity and strength of van der Waals interactions by generating tailored model substrates. By employing AFM-based single cell force spectroscopy (using NanoWorld Arrow-TL2 tipless cantilevers) they could show that experimental force curves upon retraction exhibit two regimes described in detail in the article cited above. The study describes a versatile mechanism that allows the cells to adhere to a large variety of natural surfaces under various conditions.
Fig. 2 A from "Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study": Cell parametrization: β, angle between the normal on the cell membrane and the cell axis; R1, contact radius between the cell and substrate; R0, equatorial cell radius; R2, contact radius between the cell and cantilever, ϕ1 contact angle towards the substrate; ϕ2, contact angle between the cell and cantilever, in the background is a section of the confocal image in B. B: morphology of the carA-1-GFP labelled D.d. cell attached to the cantilever subjected to a pulling force of 0.2 nN. NanoWorld Arrow-TL2 tipless cantilevers were used.
Fig. 2 A from “Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study” by N. Kamprad et al.: Cell parametrization: β, angle between the normal on the cell membrane and the cell axis; R1, contact radius between the cell and substrate; R0, equatorial cell radius; R2, contact radius between the cell and cantilever, ϕ1 contact angle towards the substrate; ϕ2, contact angle between the cell and cantilever, in the background is a section of the confocal image in B. B: morphology of the carA-1-GFP labelled D.d. cell attached to the cantilever subjected to a pulling force of 0.2 nN.
*Nadine Kamprad, Hannes Witt, Marcel Schröder, Christian Titus Kreis, Oliver Bäumchen, Andreas Janshoff, Marco Tarantola Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study Nanoscale, 2018, 10, 22504-22519 DOI: 10.1039/C8NR07107A To read the full article follow this external link: https://pubs.rsc.org/en/content/articlehtml/2018/nr/c8nr07107a Open Access The article “Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study” by Nadine Kamprad, Hannes Witt, Marcel Schröder, Christian Titus Kreis, Oliver Bäumchen, Andreas Janshoff and Marco Tarantola 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 http://creativecommons.org/licenses/by/3.0/.