DSIR: Assessing the Design of Highly Potent siRNA by Testing a Set of Cancer-Relevant Target Genes

Abstract

Chemically synthesized small interfering RNA (siRNA) is a widespread molecular tool used to knock down genes in mammalian cells. However, designing potent siRNA remains challenging. Among tools predicting siRNA efficacy, very few have been validated on endogenous targets in realistic experimental conditions. We previously described a tool to assist efficient siRNA design (DSIR, Designer of siRNA), which focuses on intrinsic features of the siRNA sequence. Here, we evaluated DSIR’s performance by systematically investigating the potency of the siRNA it designs to target ten cancer-related genes. mRNA knockdown was measured by quantitative RT-PCR in cell-based assays, revealing that over 60% of siRNA sequences designed by DSIR silenced their target genes by at least 70%. Silencing efficacy was sustained even when low siRNA concentrations were used. This systematic analysis revealed in particular that, for a subset of genes, the efficiency of siRNA constructs significantly increases when the sequence is located closer to the 5′-end of the target gene coding sequence, suggesting the distance to the 5′-end as a new feature for siRNA potency prediction. A new version of DSIR incorporating these new findings, as well as the list of validated siRNA against the tested cancer genes, has been made available on the web (http://biodev.extra.cea.fr/DSIR).

Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

“…7). Only a small region (429 bp) of the BIRC5 gene screened, which was too short to be designed and found siRNAs with high knockdown efficiency using existing computer algorithms [37,[42][43]. Screening SURER libraries overcomes the shortcomings of computer design algorithms.…”

“…To confirm the activity of BIRC clones, three siRNAs were chemically synthesized based on the sequences of clones 2, 38, and 42 (c-2, c-38, c-42) and assayed for knockdown of the endogenous BIRC5 gene in HepG2 cells, and the three designed [37] and synthetic siRNAs (s-1, s-2, s-3) used for comparison. Levels of BIRC5 mRNA and protein were determined in siRNA treated cells 48 h after transfection using RT-qPCR and western blots.…”

iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

“…Thus, these tools take into consideration the context sequences of siRNA and its accessibility to target mRNA. Tools of this type include siDirect [18], DEQOR [19], AsiDesigner [20], DSIR [21], and DEsi [22]. Although the abovementioned RNA design tools have been useful, they are limited to designing single RNA molecules which in turn limits their applications.…”

In vivo siRNA distribution and pharmacokinetics assessed by nuclear imaging are modulated according to radiolabelling site

“…Moreover, CK2 inhibition has been shown to have anti-tumor effect in human-glioblastoma xenografts [19,20]. In this study, we used an siRNA targeting the beta subunit of CK2 (CK2β), previously reported to inhibit at least 70% of CK2β activity [21]. Control and therapeutic siRNAs have been modified to allow 125 I radiolabelling on different sites: 3' or 5' end of the sense or antisense strand.…”