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      Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

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          Abstract

          Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.

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          Most cited references41

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          Physical Principles in the Construction of Regular Viruses

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            Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy.

            Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)N, over a wide range of salt concentrations and chain lengths (10 < or = N < or = 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be obtained between our data and the worm-like chain model provided that limited relaxations of the ssDNA conformations occur within the fluorescence lifetime of the donor. The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl.
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              A virus-based single-enzyme nanoreactor.

              Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or physically anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme experiments in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-molecule studies on their enzymatic behaviour. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.
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                Author and article information

                Journal
                J Phys Condens Matter
                J Phys Condens Matter
                cm
                JCOMEL
                Journal of Physics
                IOP Publishing
                0953-8984
                1361-648X
                10 May 2018
                11 April 2018
                : 30
                : 18
                : 184002
                Affiliations
                [1 ]Laboratory of Biomolecular Nanotechnology, MESA + Institute of Nanotechnology, University of Twente , P O Box 217, 7500 AE, Enschede, Netherlands j.j.l.m.cornelissen@ 123456utwente.nl
                [2 ]Flinders Centre for Nanoscale Science and Technology, Flinders University , GPO Box 2100, Adelaide, SA 5001, Australia
                Author information
                https://orcid.org/0000-0003-0171-3435
                https://orcid.org/0000-0002-9728-5043
                Article
                cmaab4a9 aab4a9 JPCM-110690.R1
                10.1088/1361-648X/aab4a9
                7104908
                29512513
                3486f6f1-0ab8-40a2-a394-cd8486e9afd5
                © 2018 IOP Publishing Ltd

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 15 December 2017
                : 16 February 2018
                : 7 March 2018
                Page count
                Pages: 8
                Funding
                Funded by: ERC Consolidator Grand (Protcage)
                Award ID: ERC-2013-CoG
                Categories
                Paper
                Special Issue on Viral Capsids
                Custom metadata
                1361-648X/18/184002+08$33.00
                Printed in the UK
                yes

                viruses,fluorescent cascade,viral genome,self-assembly,fluorescent probes

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