On the art of identifying effective and specific siRNAs

Abstract

Small interfering RNAs (siRNAs) have been widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents. Because not all siRNAs that are cognate to a given target mRNA are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective siRNAs. Furthermore, because target-complementary siRNAs can also target other mRNAs containing sequence segments that are partially complementary to the siRNA, most computational tools include ways to reduce potential off-target effects in the siRNA selection process. Though these methods facilitate selection of functional siRNAs, they do not yet alleviate the need for experimental validation. This perspective provides a practical guide based on current wisdom for selecting siRNAs.

Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

“…RNA interference (RNAi) or cells derived from knock-out animals should be very useful here. However, major efforts are required due to a large number of potential remodeling enzymes, off-target effects, and compensatory phenomena (71). Second, once the enzymes involved in remodeling have been identified, studies with model membranes with systematically varied compositions are needed to define the factors responsible for the high specificity of the remodeling process.…”

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

“…siRNA selection starts with a bioinformatic design based on a combination of general rules [113], Tom Tuschl's rules and rational design [114]. These algorithms predict the siRNA potency, even if, at that time, this technique is perfectible and always necessitates an experimental validation [115]. Even though siRNA can mediate mRNA silencing in a highly specific way, attention must be paid to a potential interference with mRNA sharing partial homology with the target, called off-target genes or to the development of an immunostimulatory effect correlated to interferon response induction.…”

Function-based gene identification using enzymatically generated normalized shRNA library and massive parallel sequencing

“…RISC containing the guide (antisense) strand of siRNA causes endonucleolytic cleavage of target mRNA (1). Synthetic siRNA duplexes and shRNA templates are usually developed through rational design based on sequencebased rules that undergo continuous modification and optimization (2). The efficacy of the published rules for siRNA design remains controversial, and their applicability to shRNA is still uncertain (3).…”