GC Content and Thermal Stability of Double-Stranded RNA: Fragments of Microsporidia Vairimorpha ceranae and Nosema bombycis AT-Rich Genes Are Sensitive to Standard Heat Treatment

TL;DR

This study shows that AT-rich dsRNA from microsporidia is sensitive to heat, leading to degradation and loss of RNAi activity, which can be mitigated with cations.

Contribution

The study reveals the thermal sensitivity of AT-rich dsRNA from microsporidia and identifies cations as stabilizers to prevent degradation during heat treatment.

Findings

AT-rich dsRNA from microsporidia degrades at 95°C, unlike control fragments with normal GC content.

Mono- and divalent cations stabilize dsRNA during heating, preserving RNAi activity.

Thermal destruction of dsRNA reduces pest-suppressing activity in Colorado potato beetle larvae.

Abstract

Heating at 95 °C or boiling E. coli HT115 (DE3) cells is often used to extract heterologous dsRNA or kill bacteria, although these temperatures cause dsRNA denaturation and destruction. In this study, we examined the risk of degradation of dsRNA fragments of AT-rich genes at high temperature. The expression of dsRNA fragments of AT-rich genes encoding DNA replication enzymes from the microsporidia Vairimorpha ceranae and Nosema bombycis in E. coli HT115 (DE3) was accompanied by heating the bacteria at 95 °C for 30 min. In contrast to four control fragments with normal GC content, the AT-rich dsRNAs of microsporidia were destroyed by this treatment. The in vitro synthesis and heating of the studied dsRNAs showed the degradation of both microsporidia and control fragments. The thermal degradation of in vitro-synthesized control dsRNA with a normal GC content of 47.6% was prevented by the addition of 2 × YT media, NaCl, or low concentrations of MgSO4. This demonstrates the important role of mono- and divalent cations in stabilizing heated fragments and helps explain the preservation of their integrity and RNAi-initiating activity despite the treatment of bacteria at temperatures that denature dsRNA. Feeding Colorado potato beetle larvae with the same in vitro-synthesized dsRNA containing fragments of three Leptinotarsa decemlineata genes showed that their thermal destruction was accompanied by a decrease in pest-suppressing activity. No dsRNA degradation was observed at 80 °C or after E. coli sonication, and these treatments, as well as increasing cation content, may help to avoid the degradation of heat-sensitive dsRNA.

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