<i>E. coli</i> RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

Grünberg, Sebastian; Coxam, Baptiste; Chen, Tien-Hao; Dai, Nan; Saleh, Lana; Corrêa, Ivan R.; Nichols, Nicole M.; Yigit, Erbay

doi:10.1093/nar/gkab284

Cited by 13 publications

(9 citation statements)

References 38 publications

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“…If the heating is carried out after the addition of the lysis reagent, the sample is heated to 60–70°C for 10–30 min ( Sarathi et al, 2008 ; Ahn et al, 2019 ). A single heating step after the addition of the lysis reagent is to be preferred, due to possible degradation of the dsRNA in a high-temperature environment rich in polyvalent ions ( Ai et al, 2018 ) and the possibly increased dsRNase activity of bacterial single-stranded RNases under non-physiological conditions ( Grünberg et al, 2021 ). The sonication treatment causes shearing of the bacterial cells through acoustic cavitation.…”

Section: Discussionmentioning

confidence: 99%

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

Verdonckt

Broeck

2022

Front. Physiol.

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RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates.

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Section: Discussionmentioning

confidence: 99%

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

Verdonckt

Broeck

2022

Front. Physiol.

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“…Many nucleases require Ca 2+ as a cofactor to stabilize their quaternary structures. − UTSA-280, constructed from sodium squarate and Ca 2+ , was selected as the first enzyme carrier. However, in this case, another divalent metal ion (Mg 2+ , Mn 2+ , or Co 2+ ) is required to promote DNase I activity .…”

Section: Methodsmentioning

confidence: 99%

Self-Activated Nucleases Formulation Based on Metal–Organic Frameworks with Enhanced Activity and Stability

An,

Xie,

Ren

et al. 2023

ACS Materials Lett.

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Nucleases are widely used tools for cleaving phosphodiester bonds in DNA in molecular biology research, medical diagnosis, and disease treatment. However, their inherently fragile nature limits their practical applications. Metal–organic frameworks (MOFs) have emerged as solid supports for enzyme immobilization. But, the immobilized enzymes usually exhibit low catalytic activity and low enzyme availability. In this study, for the first time in the literature, we developed a self-activated nuclease formulation based on squaric acid-based MOFs for efficient enzyme activation and protection from harsh environments. Deoxyribonuclease I (DNase I) and its cofactors (Ca2+ and Mg2+, Mn2+, or Co2+) were encapsulated in a suitable MOF via a water-based de novo approach. These MOF carriers improved the stability of DNase I against perturbation environments (heating, treatment with an organic solvent, vortexing, and freeze–thawing) and released the enzymes and their cofactors during catalysis, enabling the conversion of the enzyme from its inactive to active form. Moreover, the squarate ligands disintegrated from the MOFs can serve as metal chelators, protecting DNase I from the inhibitory effects of Na+ and K+. This multifunctional nuclease formulation combines enzyme protection, self-activation, and customized catalysis. This formulation was also applied to other nucleases, alkaline phosphatase (ALP) and deoxyribonuclease II (DNase II), which exhibited enhanced activity and stabilities. This study opens a new avenue for the facile preparation, storage, and transportation of nucleases, thus promoting their practical applications.

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“…The solutions were vortexed and then briefly centrifuged to spin down any liquid droplets and incubated without shaking for 2.0−2.6 h at ambient laboratory temperature, which were measured using a Durac Plus thermometer (ThermoFisher) and reported in the corresponding figure captions. After RNA-DOM incubation, we applied RNase I at 5 U (for ssRNA) or 10 U (for dsRNA, which RNase I is less active toward 72 ) to generate sufficient amounts of 3′-NMPs over the duration of the degradation experiments (up to 9 h).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Binding of Dissolved Organic Matter to RNA and Protection from Nuclease-Mediated Degradation

Chatterjee,

Zhang,

Parker

2023

Environ. Sci. Technol.

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The persistence of RNA in environmental systems is an important parameter for emerging applications, including ecological surveys, wastewater-based epidemiology, and RNA interference biopesticides. RNA persistence is controlled by its rate of biodegradation, particularly by extracellular enzymes, although the specific factors determining this rate have not been characterized. Due to prior work suggesting that nucleic acids−specifically DNA−interact with dissolved organic matter (DOM), we hypothesized that DOM may bind RNA and impede its biodegradation in natural systems. We first adapted a technique previously used to assess RNA-protein binding to differentiate RNA that is bound at all sites by DOM from RNA that is unbound or partially bound by DOM. Results from this technique suggested that humic acids bound RNA more extensively than fulvic acids. At concentrations of 8−10 mg C /L, humic acids were also found to be more effective than fulvic acids at suppressing enzymatic degradation of RNA. In surface water and soil extract containing DOM, RNA degradation was suppressed by 39−46% relative to pH-adjusted controls. Due to the ability of DOM to both bind and suppress the enzymatic degradation of RNA, RNA biodegradation may be slowed in environmental systems with high DOM concentrations, which may increase its persistence.

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E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

Cited by 13 publications

References 38 publications

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

Self-Activated Nucleases Formulation Based on Metal–Organic Frameworks with Enhanced Activity and Stability

Binding of Dissolved Organic Matter to RNA and Protection from Nuclease-Mediated Degradation

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