Enhancement of RNAi by a small molecule antibiotic enoxacin
Cell Research |
Enhancement of RNAi by a small molecule antibiotic enoxacin
Qiangzhe Zhang 0
Caihong Zhang 0
Zhen Xi 0
0 State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University , Tianjin 300071, China Cell Research (2008) 18:1077-1079
RNAi has become a mainstream molecular tool for assessing the functions of genes in mammalian cells . Large-scale RNA interference-based analyses are often complicated by false positive and negative hits due to off-target effects  and interferon response , 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 . 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  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) [
] and since they also exhibit antiviral
activity through interference with Tat-TAR interaction
], 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
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
]. This system has been reported to be a robust siRNA
screening system [
]. 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 statistical analysis of
experimental data (Figure 1A), seven compounds were found
to increase RNAi efficiency (Figure 1B, the compounds
shown with asterisk (*)). Among these 7
fluoroquinolones exhibiting statistically significant RNAi-enhancing
activities, enoxacin and norfloxacin were more active
than others at the same concentration. We then chose
enoxacin and norfloxacin for further characterization. It
npg Enhancement of RNAi by a small molecule antibiotic enoxacin
was found that the RNAi-enhancing activity of enoxacin
was dose-dependent (see Supplementary information,
Figure S1). The EC50 of enoxacin as an RNAi enhancer
is about 30 mM. This concentration is far below the IC50
of enoxacin for inhibition of topoisomerase II in human
macrophages (IC50 = 1 485 mM) (see Supplementary
information, Figure S2) [
]. At 120 mM, enoxacin started
to cause non-specific reduction of the firefly luciferase
activity (see Supplementary information, Figure S3). The
RNAi efficiency of different concentrations of siRNA
(siFL867-885) in the presence or absence of enoxacin
was also examined. At 12 h post-transfection, HEK-293
cells were treated with or without 50 mM enoxacin. An
optimum RNAi-enhancing effect by enoxacin was
observed when the siRNA concentration was at 8.4?10?10
M. Generally, enoxacin can reduce the amount of siRNA
by 2~5-fold to achieve the same RNAi knockdown
efficiency (Figure 1C).
In summary, we have demonstrated that certain
fluoroquinolone antibiotics such as enoxacin, in addition to
their powerful clinic use for the treatment of infections in
humans and animals [
], can be used to increase RNAi
efficiency. Enoxacin can significantly reduce the amount
of siRNA (by 2~5-fold) to achieve the same RNAi
efficacy. The precise mechanism of this RNAi enhancement
remains unclear at present. While our manuscript was in
preparation, a similar finding with more detailed analysis
was reported by Jin and colleagues [
], who proposed
that enoxacin acts by potentially increasing RISC
loading efficiency through a mechanism depending on the
protein factor TRBP. Nevertheless, one cannot rule out
the possibility that the effect of enoxacin on RNAi is
due to the cross-interaction with human RNA helicases
and the stabilization of RNAi molecules, especially in
view of the finding that human RNA helicase A (RHA)
is an active RISC component and functions in RISC as
an siRNA loading factor [
]. Further mechanistic study
(such as QSAR, photolabelling and affinity purification)
is being actively pursued in our lab to elucidate the
molecular basis of the observation reported here. Other
dsRNA stabilizing agents are still waiting to be explored
for the regulation of RNAi. We believe that the finding
of small molecular RNAi enhancers could potentially be
used as a tool in genome-scale loss-of-function screening
with RNAi, especially in cases that are highly dependent
on the use of relatively low concentrations of siRNA and
shRNA. It may also be a useful molecular probe to shed
new light on the RNAi process. (Experimental materials
and methods are depicted in the Supplementary
information, Data S1)
We thank Jun O Liu from Johns Hopkins School
of Medicine for critical discussions and proof-reading
of the manuscript. This work was supported by the
National Key Project for Basic Research of China
(Supplementary information is linked to the online version of
the paper on the Cell Research website.)
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