siPools: highly complex but accurately defined siRNA pools eliminate off-target effects

Abstract

Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.

Biochemical isolation of Argonaute protein complexes by Ago-APP

“…Both inhibitors against the let-7 miRNAs released repression by two-to threefold. Depletion of individual TNRC6 proteins had even weaker effects, suggesting redundancy as observed before (31). Strikingly, our peptide fused to eYFP led to a seven-and eightfold increase in luciferase activity, suggesting highly efficient inhibition of the miRNA pathway.…”

“…4A). Finally, we compared the peptide inhibition with common miRNA antisense inhibitors directed against let-7a or let-7e and to knockdowns of the individual TNRC6 proteins using specific siRNA pools (siPools) (31) (Fig. 4B).…”

Multi-Target Inhibition of Cancer Cell Growth by SiRNA Cocktails and 5-Fluorouracil Using Effective Piperidine-Terminated Phosphorus Dendrimers

“…Regarding the significant decrease of viability of HeLa cells after treatment with 50 and 100 nM scrambled siRNA delivered by means of the AE2G3 dendrimer, we assume unintended off-target effects [42] due probably to partial complementary matching of the scrambled siRNA sequence to the mRNA sequences of other vitally important genes in HeLa cells. For instance, BLAST (Basic Local Alignment Search Tool provided by the U.S. National Center for Biotechnology Information, NCBI) search revealed partial complementarity (13 nucleotides) of Scrambled siRNA sequences to several transcripts of genes coding metabolically active proteins, such as 5-methyltetrahydrofolate-homocysteine methyltransferase (involved in the metabolism of essential amino acids), kinesin family member 3B (takes part in chromosome movement during cell division) and DNA topoisomerase I (regulates transcription).…”

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…Cells were used 2 days posttransfection. Pools of siRNA were chosen over individual sequences in order to maximize loss of target function while minimizing off‐target effects 37, 38…”