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.

The stability of RNA in aqueous systems is critical for multiple environmental applications including evaluating the environmental fate of RNA interference pesticides and interpreting viral genetic marker abundance for wastewater-based epidemiology. In addition to biological processes, abiotic reactions may also contribute to RNA loss. In particular, some metals are known to dramatically accelerate rates of RNA hydrolysis under certain conditions (i.e., 37 °C or higher temperatures, 0.15–100 mM metal concentrations). In this study, we investigated the extent to which metals catalyze RNA hydrolysis under environmentally relevant conditions. At ambient temperature, neutral pH, and ∼10 μM metal concentrations, we determined that metals that are stronger Lewis acids (i.e., lead, copper) catalyzed single-stranded (ss)­RNA, whereas metals that are weaker Lewis acids (i.e., zinc, nickel) did not. In contrast, double-stranded (ds)­RNA resisted hydrolysis by all metals. While lead and copper catalyzed ssRNA hydrolysis at ambient temperature and neutral pH values, other factors such as lowering the solution pH and including inorganic and organic ligands reduced the rates of these reactions. Considering these factors along with sub-micromolar metal concentrations typical of environmental systems, we determined that both ssRNA and dsRNA are unlikely to undergo significant metal-catalyzed hydrolysis in most environmental aqueous systems.

No abstract

As an essential biomolecule for life, RNA is ubiquitous across environmental systems where it plays a central role in biogeochemical processes and emerging technologies. The persistence of RNA in soils and sediments is thought to be limited by enzymatic or microbial degradation, which occurs on timescales that are orders of magnitude faster than known abiotic pathways. Herein, we unveil a previously unreported abiotic pathway by which RNA rapidly hydrolyzes on the timescale of hours upon adsorption to iron (oxyhydr)oxide minerals such as goethite (α-FeOOH). The hydrolysis products were consistent with iron present in the minerals acting as a Lewis acid to accelerate sequence-independent hydrolysis of phosphodiester bonds comprising the RNA backbone. In contrast to acid-or base-catalyzed RNA hydrolysis in solution, mineral-catalyzed hydrolysis was fastest at circumneutral pH, which allowed for both sufficient RNA adsorption and hydroxide concentration. In addition to goethite, we observed that RNA hydrolysis was also catalyzed by hematite (α-Fe 2 O 3 ) but not by aluminum-containing minerals (e.g., montmorillonite). Given the extensive adsorption of nucleic acids to environmental surfaces, we anticipate previously overlooked mineral-catalyzed hydrolysis of RNA may be prevalent particularly in iron-rich soils and sediments, which must be considered across biogeochemical applications of nucleic acid analysis in environmental systems.

The adsorption of DNA at mineral-water interfaces is well-established to increase its persistence in soils and sediments; however, adsorbed RNA in similar environments degrades rapidly, in some cases outpacing solution-phase degradation occurring over hours to days. Herein, we elucidate a novel abiotic mechanism by which RNA, but not DNA, degrades upon adsorption to surfaces of iron (oxyhydr)oxides such as goethite (α-FeOOH) that are abundant in soils and sediments. Upon adsorption to goethite, both single-stranded and double-stranded RNA hydrolyzed on the timescale of hours under environmentally relevant physicochemical conditions. The reaction products were consistent with iron present in goethite acting as a Lewis acid to accelerate non-selective hydrolysis of phosphodiester bonds comprising the RNA backbone. In contrast to well-established acid- or base-catalyzed RNA hydrolysis in solution, mineral-catalyzed hydrolysis was fastest at circumneutral pH, which allowed for both sufficient RNA adsorption and hydroxide concentration. We further confirmed that contact of the RNA with the mineral surface is necessary for hydrolysis to occur by demonstrating RNA degradation was inhibited by compact RNA conformation at elevated ionic strength and competitive adsorption with orthophosphate and organic matter. In addition to goethite, we observed RNA hydrolysis was also catalyzed by hematite (α-Fe2O3), but not by aluminum-containing minerals (e.g., montmorillonite). Given the extensive adsorption of nucleic acids to environmental surfaces, we anticipate previously overlooked mineral-catalyzed hydrolysis of RNA may be prevalent particularly in iron-rich soils and sediments, which must be considered across biogeochemical applications of nucleic acid analysis in environmental systems.