HS-AFM video of Covid’s Docking Method

Johannes Kepler University in Linz Austria has published a High-Speed Atomic Force Microscopy video of human lectin CLEC4G binding to glycans on a SARS-CoV-2 spike. This video was recorded by Daniel Canena and Peter Hinterdorfer and is, according to the two researchers, the first short film of the active structure the virus uses to attach to cell

Congratulations!

NanoWorld Ultra-Short AFM Cantilevers of the USC-F1.2-k0.15 type were used for the HS-AFM video.

Please follow this external link to the Johannes Keppler University webpage to watch the video: https://www.jku.at/en/news-events/news/detail/news/film-of-covids-docking-method/ or have a look on Youtube

NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)
NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)

High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action

The current pandemic is not the only health threat worldwide. Another worry is the increasing antibiotic resistance which increases the fear to run out of effective antibiotics.

This is one of the reasons why antimicrobial peptides (AMPs) are gaining more and more interest.

The lipopeptide Daptomycin ( DAP ) has been therapeutically used as a last resort antibiotic against multidrug-resistant enterococci and staphylococci in the past. Unfortunately, some strains have become resistant to Dap in recent years. There still is a knowledge-gap on the details of Dap activity. It is therefore important to understand the structure-activity relationships of AMPs on membranes in order to develop more antibiotics of this type as a countermeasure to the spread of resistance.*

High Speed Atomic Force Microscopy ( HS-AFM ) makes it possible to observe dynamic biological processes on a molecular level.

In the article “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” Francesca Zuttion, Adai Colom, Stefan Matile, Denes Farago, Frédérique Pompeo, Janos Kokavecz, Anne Galinier, James Sturgis and Ignacio Casuso describe how, by using the possibilities offered by high speed atomic force microscopy, they were able to confirm some up until now hypothetical models and additionally detected some previously unknown molecular mechanisms. *

The HS-AFM imaging made it possible for the authors to observe the development of the dynamics of interaction at the molecular-level over several hours. *

They investigated the lipopeptide Daptomycin under infection-like conditions and could confirm Dap oligomerization and the existence of half pores. *

They also mimicked bacterial resistance conditions by increasing the CL-content in the membrane. *

By correlating the results of other research techniques such as FRET, SANS, NMR, CD or electrophysiology techniques with the results they achieved with high speed atomic force microscopy F. Zuttion et al. were able to confirm several, previously, hypothetical models, and detect several unknown molecular mechanisms. *

It is to be hoped that the possibilities offered by HS-AFM imaging will stimulate new models and insight on the structure-activity relationship of membrane-interacting molecules and also open up the possiblity to increase the throughput of screening of molecular candidates considerably. *

NanoWorld USC ( Ultra-Short AFM Cantilevers) of the USC-F1.2-k0.15 type, which are specially designed for the use in high speed atomic force microscopy, were used for the HS-AFM imaging described in the article cited below. These AFM probes have a typical resonance frequency of 1200 kHz and have a wear resistant AFM tip made from high density carbon.

Figure 4 Sub-MIC Dap on POPG at 37 °C. Tens of minutes from “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” by Francesca Zuttion et al. NanoWorld Ultra-Short AFM Cantilevers USC-F1.2-k0.15 AFM probes for HS-AFM imaging were used.
Figure 4 Sub-MIC Dap on POPG at 37 °C. Tens of minutes from “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” by Francesca Zuttion et al.
Intermediate stages a A new structure appeared: dimples, zones of thinner membrane thickness, whose diameter was in the range 7 ± 2 nm. Most dimples diffuse, but some remained static (colour scale: 3 nm). Movie details: frame rate 97 ms; zoom of a full image of 150 nm × 90 nm and 256 × 160 pixels. b The dimple diffusion consisted of swinging trajectories, implying membrane-mediated dimple-dimple attraction (colour scale: 3 nm). b, right, Energy profile of the interaction of the dimples obtained derived from 120 centre-to-centre distance measurements that contains as the oligomers two energy minima. Movie details: frame rate 83 ms; full image of 150 nm × 150 nm and 256 × 256 pixels. c In some membrane zones, clusters of dimples, reminiscent of cubic phases, developed (colour scale: 4 nm). Movie details: frame rate 74 ms; full image of 90 nm × 60 nm and 256 × 160 pixels. d The clusters of dimples were moderately dynamical in time, with moderate internal rearrangements (colour scale: 4 nm). Movie details: frame rate 74 ms; full image of 25 nm × 16 nm and 256 × 160 pixels. e The other deformation found was elongated-humps on top of the POPG membrane. e, left, An elongated-hump in the proximity of a cluster of dimples (colour scale: 4 nm). e, right, A close-up and a profile of an elongated-hump. Additional images of elongated-humps on Supplementary Fig. 1. Movie details: frame rate 479 ms; zoom of full image of 250 nm × 200 nm and 300 × 256 pixels. f It was observed that the dimples and the elongated-humps fused and gave yield to pores of toroidal structure where a protruding ring surrounds the pore (colour scale: 4 nm). Movie details: frame rate 74 ms; full image of 40 nm × 40 nm and 256 × 160 pixels.

*Francesca Zuttion, Adai Colom, Stefan Matile, Denes Farago, Frédérique Pompeo, Janos Kokavecz, Anne Galinier, James Sturgis and Ignacio Casuso
High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action
Nature Communications volume 11, Article number: 6312 (2020)
DOI: https://doi.org/10.1038/s41467-020-19710-z

Please follow this external link to read the full article: https://rdcu.be/ciaW2

Open Access : The article “High-speed atomic force microscopy highlights new molecular mechanism of daptomycin action” by Francesca Zuttion, Adai Colom, Stefan Matile, Denes Farago, Frédérique Pompeo, Janos Kokavecz, Anne Galinier, James Sturgis and Ignacio Casuso 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 https://creativecommons.org/licenses/by/4.0/.

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/.