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Orders Norzivirales and Timlovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Wei Y et al. (2008) generated a method for packaging long, more than 2,000 nucleotides, RNA sequences, which was referred to as armored L-RNA technology. It made it possible to pack the armored L-RNA of 2248 bases containing six gene fragments: hepatitis C virus, SARS-CoV1, SARS-CoV2, and SARS-CoV3, avian influenza virus matrix gene, and H5N1 avian influenza virus. In parallel, Wei B et al. (2008) improved the MS2 VLP-based armored RNA by increasing the number and affinity of the pac site in exogenous RNA and of the sequence encoding coat protein. Such one-plasmid expression system allowed the extension of the length of the armored RNA to 1891 bases and included SARS-CoV1, SARS-CoV2, SARS-CoV3, HCV, and influenza H5N1 fragments. The armored L-RNA technology with the improved pac site was used by the elaboration of the branched DNA assay by the HIV-1 detection (Zhan et al. 2009). This laboratory also presented protocols for the MS2 VLP-based armored RNA detection for enterovirus 71 and coxsackievirus A16 (Song et al. 2011) and avian influenza H7N9 virus (Sun Yu et al. 2013).
Non-Vaccine VLPs
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
To enhance the capacity capabilities of the approach, Wei Y et al. (2008) generated a method for packaging long, more than 2000 nucleotides, RNA sequences, which was referred to as armored L-RNA technology. To do this, they applied the two-plasmid coexpression system, in which the maturation and coat proteins were expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop, or pac site, was transcribed from another plasmid. It made it possible to pack the 3V armored L-RNA of 2248 bases containing six gene fragments: hepatitis C virus, severe acute respiratory syndrome coronavirus SARS-CoV1, SARS-CoV2, and SARS-CoV3, avian influenza virus matrix gene, and H5N1 avian influenza virus. The 3V armored L-RNA functioned successfully as a calibrator for multiple virus assays (Wei Y et al. 2008). In parallel, Wei B et al. (2008) improved the MS2 VLP-based armored RNA by increasing the number and affinity of the pac site in exogenous RNA and of the sequence encoding coat protein. In this obstacle, the one-plasmid expression system allowed the extension of the length of the armored RNA to 1891 bases, in the case when two C-variant pac sites were used. The C-variant pac site differed from that of the wild type by the substitution of U−5 with C, which yielded a significantly higher affinity for coat protein than wild-type pac site in vitro, as described in Chapter 16. Therefore, the armored RNA with the 1891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1200 bases target RNA was packaged. The armored RNA contained SARS-CoV1, SARS-CoV2, SARS-CoV3, HCV, and influenza H5N1 fragments (Wei B et al. 2008). The armored L-RNA technology with the improved pac site was used by the elaboration of the branched DNA assay by the HIV-1 detection (Zhan et al. 2009). This laboratory also presented protocols for the MS2 VLP-based armored RNA detection for enterovirus 71 and coxsackievirus A16 (Song et al. 2011) and avian influenza H7N9 virus (Sun et al. 2013).
Strategies for improving the specificity of siRNAs for enhanced therapeutic potential
Published in Expert Opinion on Drug Discovery, 2018
Aditya Kiran Gatta, Raghu Chandrashekhar Hariharapura, Nayanabhirama Udupa, Meka Sreenivasa Reddy, Venkata Rao Josyula
The most sensitive and high throughput approaches are the Real-Time PCR and the microarray analysis [126,127]. Another method to assess the efficiency of knockdown is the luminescence-based ‘branched DNA assay.’ A branched DNA assay requires magnetic beads, probes (capture probe, target probe), preamplifier, and an amplifier. It measures the gene expression levels in the luminescence-based manner [128]. The potential candidate siRNAs were chosen for further studies, after evaluating their gene silencing potential by cell culture methods.