Nwokeoji, A.O., Kumar, S., Kilby, P.M. et al. (3 more authors) (2019) Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC. Analyst, 144 (16). pp. 4985-4994. ISSN 0003-2654
Abstract
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. We 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. Moreover, further analysis under non-denaturing conditions revealed the presence of heterogeneous dsRNA species. IP-RP-HPLC fractionation and AFM analysis revealed that these alternative RNA species do not arise from different lengths of individual dsRNA molecules in the product, but represent misannealed RNA species that present as larger assemblies or multimeric forms of the RNA. These results for the first time provide direct structural insight into dsRNA produced both in vivo in bacterial systems and in vitro, highlighting the structural heterogeneity of RNA produced. These results are the first example of detailed characterisation of the different forms of dsRNA from two production systems and establish atomic force microscopy as an important tool for the characterisation of long dsRNA.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 The Royal Society of Chemistry. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence (http://creativecommons.org/licenses/by/3.0/). |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) The University of Sheffield > Faculty of Science (Sheffield) > Department of Physics and Astronomy (Sheffield) |
Funding Information: | Funder Grant number Syngenta Ltd PO: 8700549715 Biotechnology and Biological Sciences Research Council (BBSRC) BB/M012166/1 Engineering and Physical Sciences Research Council (EPSRC) EP/M027430/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 30 Jul 2019 11:52 |
Last Modified: | 08 Feb 2021 15:03 |
Status: | Published |
Publisher: | Royal Society of Chemistry (RSC) |
Refereed: | Yes |
Identification Number: | 10.1039/c9an00954j |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:149124 |