Metal-Catalyzed Hydrolysis of RNA in Aqueous Environments

Abstract

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.

RNA hydrolysis at mineral-water interfaces

“…To compare intermediate products, we plotted the intensity of a gel image as a function of migration distance beyond the original ssRNA (29). Shorter ssRNA molecules generated by abiotic alkaline hydrolysis fell along a smooth distribution of lengths with no discernable specific peaks (Figure 1D), consistent also with products from abiotic metal ion-catalyzed hydrolysis (26). In contrast, enzymes from representative sources (i.e., human saliva, soil) each generate multiple specific peaks due to preferential scission of specific sequences (Figure 1E).…”

“…The apparent first-order rate constant of dsRNA hydrolysis (i.e., 0.052(±0.001) h -1 ) was ~3-fold slower (Figure S10) than ssRNA of a comparable length and sequence (Section S2). The duplex structure of dsRNA is known to impede abiotic hydrolysis catalyzed by bases or metal ions (25,26). Notably, rates for the hydrolysis of dsRNA, if detected at all, by these solution-phase reactions were at least one magnitude slower than for ssRNA, prohibiting these abiotic reactions from contributing significantly to dsRNA degradation in the environment.…”

“…Relative to DNA, the presence of the 2′-hydroxyl group on the RNA structure reduces its chemical stability by facilitating hydrolysis of the phosphodiester backbone. However, despite this structural instability, solution-phase abiotic RNA hydrolysis catalyzed by hydroxide or metal ions has been found to be too slow to contribute to RNA degradation on relevant timescales (i.e., days) in environmental systems (25,26), suggesting that loss of dissolved RNA predominantly results from microbial nucleases (27). Like DNA, RNA has been observed to strongly adsorb to minerals (e.g., iron (oxyhydr)oxides) (28) and to undergo rapid and extensive adsorption to particles in soils (29,30).…”

“…We hypothesized that the apparent instability of adsorbed RNA may be attributable to the hydrolysis of phosphodiester bonds occurring at mineral-water interfaces. Notably, RNA hydrolysis is established to be catalyzed by dissolved metal ions acting as Lewis acids (26,31), albeit at concentrations far higher than encountered in environmental systems (26). However, metals in certain minerals (i.e., iron (oxyhydr)oxides such as goethite) that are ubiquitous in soils and sediments may facilitate an analogous reaction for RNA adsorbed on mineral surfaces.…”

Influence of diverse storage conditions of double‐stranded RNAin vitro on the RNA interference efficiency in vivo insect Tribolium castaneum

“…40,41 RNA molecules are therefore generally recommended to be stored at ultra-low temperatures and multiple freeze-thaw cycles should be avoided for dsRNA. Although duplex structure of dsRNA is generally considered more stable than ssRNA molecules, 42,43 chemically synthesized dsRNA molecules are usually kept at −80 or −20 °C according to most RNAi research publications and manufacturers' protocols. However, in actual wet laboratory experiments, researchers are usually uncertain whether dsRNA molecules are still active after a period of storage and unavoidable freeze-thaw cycles due to little available information.…”