Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single-Stranded RNA

Abstract

Phosphodiester bonds in the backbones of double-stranded (ds)­RNA and single-stranded (ss)­RNA are known to undergo alkaline hydrolysis. Consequently, dsRNA agents used in emerging RNA interference (RNAi) products have been assumed to exhibit low chemical persistence in solutions. However, the impact of the duplex structure of dsRNA on alkaline hydrolysis has not yet been evaluated. In this study, we demonstrated that dsRNA undergoes orders-of-magnitude slower alkaline hydrolysis than ssRNA. Furthermore, we observed that dsRNA remains intact for multiple months at neutral pH, challenging the assumption that dsRNA is chemically unstable. In systems enabling both enzymatic degradation and alkaline hydrolysis of dsRNA, we found that increasing pH effectively attenuated enzymatic degradation without inducing alkaline hydrolysis that was observed for ssRNA. Overall, our findings demonstrated, for the first time, that key degradation pathways of dsRNA significantly differ from those of ssRNA. Consideration of the unique properties of dsRNA will enable greater control of dsRNA stability during the application of emerging RNAi technology and more accurate assessment of its fate in environmental and biological systems, as well as provide insights into broader application areas including dsRNA isolation, detection and inactivation of dsRNA viruses, and prebiotic molecular evolution.

Metal-Catalyzed Hydrolysis of RNA in Aqueous Environments

“…At the lower lead concentrations tested, we were able to determine that the loss of measurable ssRNA followed first-order kinetics in the presence of lead (Figure S6), similar to ssRNA loss according to the first-order kinetics that we previously observed for base-catalyzed hydrolysis . The ssRNA loss rate increased dramatically with lead concentration.…”

“…Materials and supplies are detailed in Section S1. We prepared 1006 nucleotides (nt) ssRNA and 1000 base pairs (bp) dsRNA (sequences available in Section S2) according to a previously described protocol , (details in Section S3.1). To conduct experiments isolating RNA degradation by abiotic processes, we used previously established protocols to avoid the presence of ribonucleases and microbes (details in Section S3.2).…”

Intra-strand symmetries and asymmetries in bacterial DNA: Evolutive features or relics of primordial genomes?

“…In particular, the widely accepted hypothesis of the RNA world that would precede 5 the emergence of DNA genomes [58,59,73] leads to suppose that any interpretation for the S-H model as the process underlying the formation of primitive genomes should possibly be based on evidences of as RNA or even other pre-RNA candidates polymerize [12,50,75]. In particular, it would be necessary more research on how double-stranded polymers polymerizes [6,75,78].…”

First Detection of Rotavirus C in Asymptomatic Pigs from India with Unexpected Dominance and Characterization of the Structural and Nonstructural Genes

“…The main limitation of the current study is long term storage of the clinical specimens. Reduction in RVC detection rate possibly will be negligible as double stranded RNA are known to be more stable and reported to have wide applications for accurate assessment in environmental and biological systems [34]. Failure to characterize complete genome of the remaining study strains was mostly due to low viral load as the infection were subclinical as well as shortage in amount of few fecal specimens.…”