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Abstract
Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (Spodoptera litura, Locusta migratoria, Periplaneta americana, and Zophobas atratus) from different orders were characterized using a modified assay method. The results revealed that all insect dsRNA degrading nucleases tested showed high activity in alkaline environments at optimal Mg2+ concentrations and elevated temperatures. We also found that enzymes from different insects varied in terms of their optimal reaction conditions and kinetic parameters. Whole body enzyme activity differed dramatically between insect species, although enzymes with higher substrate affinities (lower Km) were usually balanced by a smaller Vmax to maintain a proper level of degradative capacity. Furthermore, enzyme activities varied significantly between the four tested tissues (whole body, gut, hemolymph, and carcass) of the insect species. All the insects tested showed several hundred-fold higher dsRNA degrading activity in their gut than in other tissues. Reaction environment analysis demonstrated that physiological conditions in the prepared gut fluid and serum of different insects were not necessarily optimal for dsRNA degrading nuclease activity. Our data describe the biochemical characteristics and tissue distributions of dsRNA degrading activities in various insects, not only explaining why oral delivery of dsRNA often produces lower RNAi effects than injection of dsRNA, but also suggesting that dsRNA-degrading activities are regulated by physiological conditions. These results allow for a better understanding of the properties of dsRNA degrading nucleases, and will aid in the development of successful RNAi strategies in insects.
Highlights
Since the first demonstration of long, double stranded RNAs mediating RNA interference (RNAi) in Caenorhabditis elegans (Fire et al, 1998), RNAi technology has been widely used in scientific research, clinical applications, and agricultural pest control in the past decades (Baum et al, 2007; Price and Gatehouse, 2008; Bellés, 2010; Burand and Hunter, 2013; Biochemical Comparison of Double stranded RNAs (dsRNA) Degrading NucleasesZhang et al, 2017)
RNA interference is achieved by administration of dsRNA to the target site, where it is processed into small interfering RNA by Dicer enzyme and coupled with Argonaute protein to form an RNA-induced silencing complex (RISC), which combines with complementary mRNA to induce target RNA degradation (Meister and Tuschl, 2004)
When tobacco caterpillar gut fluid was used as the nuclease source and incubated with the 24 bp dsEGFP substrates labeled with these fluorescent and quencher combinations at a final concentration of 0.5 μM dsRNA, the fluorescence intensity was measured at excitation and emission wavelengths of 494 and 519 nm for fluorophore FAM, 552 and 570 nm for fluorophore Cy3, and 650 and 670 nm for fluorophore Cy5, respectively
Summary
Since the first demonstration of long, double stranded RNAs (dsRNA) mediating RNA interference (RNAi) in Caenorhabditis elegans (Fire et al, 1998), RNAi technology has been widely used in scientific research, clinical applications, and agricultural pest control in the past decades (Baum et al, 2007; Price and Gatehouse, 2008; Bellés, 2010; Burand and Hunter, 2013; Biochemical Comparison of dsRNA Degrading NucleasesZhang et al, 2017). Since the first demonstration of long, double stranded RNAs (dsRNA) mediating RNA interference (RNAi) in Caenorhabditis elegans (Fire et al, 1998), RNAi technology has been widely used in scientific research, clinical applications, and agricultural pest control in the past decades Systemic RNAi efficiency variation among different insects has been a barrier to the application of RNAi as an effective tool for functional genomic research, and to the biological control of pests by dsRNA-expressing transgenic plants and gene-specific dsRNA insecticides (Terenius et al, 2011; Zhang et al, 2013; Kola et al, 2015; Joga et al, 2016). It is necessary to better understand the factors underlying the diversity of dsRNA degrading activities in the hemolymph and gut lumen of different insect species
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