Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Xu, Yan; Zhang, Hong Yan; Thormeyer, Dorit; Larsson, Ola; Du, Quan; Elmén, Joacim; Wahlestedt, Claes; Liang, Zicai

doi:10.1016/s0006-291x(03)01024-6

Cited by 58 publications

(43 citation statements)

References 22 publications

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“…Due to the different targeting sites used for siRNA and AS-DON, it is hard to compare their inhibitory effects on CVB3 replication. Overall, under optimal conditions it seems that CVB3-specific siRNAs are more effective than AS-ODNs in terms of potency, efficacy, and duration, which is consistent with other studies (2,19,48). Sequence specificity of siRNA is very stringent, as single base pair mismatches between the siRNA and its target sequence dramatically reduce the silencing capability (3,12,14).…”

Section: Discussionsupporting

confidence: 86%

“…Several studies focusing on the relationship between secondary structure and siRNA effects showed that the secondary structure at least in part influences the efficiency of siRNAs (22,44,51). Conversely, it has been reported that the secondary structure of the target mRNA does not appear to have a strong effect on gene silencing (22,48). Whether secondary structure plays a role in siRNA machinery binding is still debated.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

Cheung

Zhang

Chau

et al. 2005

J Virol

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

Cheung

Zhang

Chau

et al. 2005

J Virol

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“…[11][12][13][14][15] Here, we present the first report to investigate the efficacies of RNAi in cultured nucleus pulposus cells from rats and humans in vitro, using siRNA duplexes targeting Firefly luciferase and green fluorescent protein (GFP).…”

Section: Introductionmentioning

confidence: 99%

Prolonged down regulation of specific gene expression in nucleus pulposus cell mediated by RNA interference in vitro

Kakutani

Nishida

Uno³

et al. 2006

Journal Orthopaedic Research

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To investigate the efficacies and the longevity of RNA interference in nucleus pulposus cells from rat and human, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids were cotransfected with siRNA targeting Firefly luciferase to the nucleus pulposus cells extracted from Sprague Dawley rats and scoliosis patients. The inhibitory effects were evaluated by dual luciferase assay for 3 weeks. Proliferation activity of fibroblast-like cells extracted from the subcutaneous tissue of Sprague Dawley rats and the nucleus pulposus cells were measured by proliferation assay (WST-8 assay) every 2 days after plating. The expression of Firefly luciferase was drastically inhibited both in rats (94.7%) and in humans (93.7%). The inhibitory effects were maintained for 2 weeks and had disappeared completely by 3 weeks. The proliferation activity of nucleus pulposus cells was significantly lower than fibroblast-like cells. We have shown, for the first time, siRNA-mediated gene silencing in rat and human disc cells for a relatively sustained period, probably due to the stability of the nucleus pulposus cells in terms of cell proliferation. The demonstration of this study may allow further exploration of the use of siRNA for scientific research and the treatment of disc degenerative diseases. ß

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“…The siRNA used in our screen is siFL867-885, which can effectively suppress the firefly luciferase reporter activity at 10 nM siRNA duplexes [8]. This system has been reported to be a robust siRNA screening system [8]. As a starting point, we diluted siFL867-885 to a concentration of 8.4×10…”

mentioning

confidence: 99%

Enhancement of RNAi by a small molecule antibiotic enoxacin

Zhang

2008

Cell Res

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RNAi has become a mainstream molecular tool for assessing the functions of genes in mammalian cells [1]. Large-scale RNA interference-based analyses are often complicated by false positive and negative hits due to off-target effects [2] and interferon response [3], which can be attributed at least in part to the use of high concentrations of siRNA. Lowering the amounts of siRNAs and shRNAs can effectively and expediently mitigate the off-target effect and interferon response [4]. However, in RNAi experiments, lowering the concentration of siRNA is often accompanied by a lower knockdown efficiency. One of the key factors affecting RNAi efficiency is the stability of double-stranded siRNA. We reasoned that measures that could stabilize double-stranded RNA may lead to increased RNAi efficiency. Given that RNAi requires a number of cellular proteins, it should be possible, at least in theory, to regulate the efficiency of RNAi using small organic molecules. To stabilize ds-RNA used in RNAi, we therefore envisioned the following methods: (1) to design more stable siRNA (screening for certain sequences, modification of RNA); (2) to increase the activity of ds-RNA protective proteins (such as ds-RNAbinding proteins, or simply binding domains); (3) to inhibit the activity of ds-RNA-dissolving proteins (such as RNA helicases); (4) to stabilize ds-RNA and/or its protein complex with small organic compounds. Our longstanding interest in drug-nucleic acids interaction [5] led us to search for potential small molecular regulators of RNAi. We hypothesized that inhibitors of RNA helicases may increase the stability of double-stranded siRNA, so as to enhance RNAi efficiency. Since a large family of fluoroquinolone antibiotics target bacterial DNA gyrase complexed with the targeted DNA possibly in A-form (similar to RNA) [6] and since they also exhibit antiviral activity through interference with Tat-TAR interaction [7], we decided to screen a library of commercially available fluoroquinolone antibiotics, with the hope that some of the analogs may cross-inhibit relevant human RNA helicases. Herein, we report that enoxacin, one of the fluoroquinolone antibiotics known to inhibit bacterial gyrase and topoisomerase IV with minimal effects on their mammalian counterparts, can increase RNAi efficiency. We have found that enoxacin can reduce the concentrations of siRNA by 2~5-fold for the same RNAi knockdown efficiency.A dual-luciferase reporter assay system was used to screen small organic compounds that were capable of enhancing RNAi efficiency. The siRNA used in our screen is siFL867-885, which can effectively suppress the firefly luciferase reporter activity at 10 nM siRNA duplexes [8]. This system has been reported to be a robust siRNA screening system [8]. As a starting point, we diluted siFL867-885 to a concentration of 8.4×10-10 M, and at this concentration the RNAi knockdown was partial. A small library of fifteen widely used fluoroquinolone antibiotics ( Figure 1B) was screened using the dual-luciferase reporter system. By s...

show abstract

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Cited by 58 publications

References 22 publications

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

Prolonged down regulation of specific gene expression in nucleus pulposus cell mediated by RNA interference in vitro

Enhancement of RNAi by a small molecule antibiotic enoxacin

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