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. 2009 Jan;37(1):e8.
doi: 10.1093/nar/gkn953. Epub 2008 Nov 29.

Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries

Zhen Li  1 , Yves Fortin, Shi-Hsiang Shen
Affiliations

Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries

Zhen Li et al. Nucleic Acids Res. 2009 Jan.

Abstract

Use of highly potent small interfering RNAs (siRNAs) can substantially reduce dose-dependent cytotoxic and off-target effects. We developed a genetic forward approach by fusing the cytosine deaminase gene with targets for the robust identification of highly potent siRNAs from RNA interference (RNAi) libraries that were directly delivered into cells via bacterial invasion. We demonstrated that two simple drug selection cycles performed conveniently in a single container predominately enriched two siRNAs targets the MVP gene (siMVP) and one siRNA targets the egfp gene (siEGFP) in surviving cells and these proved to be the most effective siRNAs reported. Furthermore, the potent siRNAs isolated from the surviving cells possessed noncellular toxic characteristics. Interestingly, the length of highly potent siMVPs identified could be as short as 16-mer, and increasing the length of their native sequences dramatically reduced RNAi potency. These results suggest that the current approach can robustly discover the most potent and nontoxic siRNAs in the surviving cells, and thus has great potential in facilitating RNAi applications by minimizing the dose-dependent and sequence nonspecific side effects of siRNAs.

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Figures

Figure 1.
Figure 1.
Schematic of protocol for forward and robust selection of effective siRNAs in a single container. Mammalian cells are transfected with pCMV-CD-taget gene constructs. Target genes are digested. siRNA libraries are constructed from DNase I-digested target genes and transformed into E. coli BT203, and delivered into cells via bacterial invasion. Upon drug (5FC) selection, potent and nontoxic siRNAs are immediately identified in surviving cells.
Figure 2.
Figure 2.
Dose-responses of cells to 5FC. HEK 293 cells stably expressing the CD-MVP hybrid transcript were transfected with pEGFP-C1 for EGFP expression. After 24 h, the pEGFP-C1 transfected cells were incubated with E. coli BT-203 that had been transformed with vector pHippy-SspI (without siMVP library) at a bacteria-to-cell ratio (m.o.i.) of 20 at 37°C for 2 h. After washing, cells were re-fed with medium containing 25 µg/ml of gentamicin for overnight, and then incubated with 5FC at various concentrations. Photomicrographs of surviving cells showing EGFP-expressing were taken 48 h after addition of 5FC. Upper panel, GFP Images; lower panel, Optical Images.
Figure 3.
Figure 3.
The positions and numbers of siMVPs identified in each drug selection cycle. HeLa cells expressing the CD-MVP hybrid transcript were incubated with E. coli BT-203 containing the siMVP library for delivery of siMVPs into cells at an m.o.i. of 20 at 37°C for 2 h. After washing as before, 5FC was added to cells at 150 µM concentration. At 48 h after adding 5FC plasmid DNAs containing siMVPs were extracted from surviving cells and transformed into E. coli DH5α. About 30–40 colonies were randomly picked up from the transformants, and plasmid DNAs were reisolated from the cultures of individual colonies for PCR amplification of siMVPs using two primers located the H1 and U6 promoter regions, respectively. The sequences of siMVPs amplified from individual colonies were determined by DNA sequencing. At the meantime, plasmid DNA mixtures were reextracted from the transformed E. coli DH5α, and directly transformed into E. coli BT-203 for the second cycle of bacterial invasion and forward drug selection, and so repeatedly for the third selection cycle.
Figure 4.
Figure 4.
Evaluation of RNAi potency. (A) HeLa cells were transfected with chemically synthesized siMVPs at indicated concentrations with similar transfection efficiency measured as coexpressed luciferase activities. Cell lysates were subjected to western blot analysis with anti-MVP after transfection for 48 h. (B) HeLa cells were transfected with 60 ng pEGFP-MVP, 1.2 ng pRLSV40 (Rluc) and chemically synthesized siMVPs at indicated concentrations. (C) As with (B), HeLa cells were transfected with indicated concentrations of siMVP-684 derivatives synthesized in vitro. (D) As with (B) and (C), HeLa cells were transfected with indicated concentrations of siEGFPs synthesized in vitro. Results in (B), (C) and (D) were obtained from three independent experiments each performed in triplicate. RNAi potency of siMVPs was normalized by Rluc activity for transfection efficiency against unrelated siRNA control (siHB1). Data are mean ± SD.
Figure 5.
Figure 5.
Evaluation of siMVP cytotoxicity. (A) siMVP-containing plasmids were isolated either directly from the library (selection cycle 0), or from cells that survived in each selection cycle with 150 μM 5FC, or from dead cells resulted in the first cycle of bacterial invasion and drug selection and transformed into E. coli. For toxicity evaluation, 30 colonies were randomly selected from each transformation for reisolation of siMVP-containing plasmids. HeLa cells in 96-well plate transfected in triplicate with siMVP-containing plasmids (0.2 μg) prepared from individual colonies. (B) HeLa cells transfected with 10 nM of siRNAs synthesized in vitro. Cell survival values represent the mean of three independent transfections each performed in triplicate. Five highly toxic siMVPs, siMVP-28, siMVP-34, siMVP-52, siMVP-59 and siMVP-61, were identified from dead cells (A) after screening the transformed100 colonies. Their toxicity was compared with most toxic SRD5a2-3 reported. Cellular viability assays in both (A) and (B) were performed at 72-h after transfection. The dotted line represents the 75% cell viability threshold. Data in (A) and (B) are mean ± SD.
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