Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor

Abstract

Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

Current perspectives in intronic micro RNAs (miRNAs)

“…These two types of small RNA molecules usually interfere with expression of genes intracellularly based on their completely or partially complementary of the small RNAs to the target gene, respectively. Basically, siRNAs are double-stranded RNAs that degrade target gene transcripts based on nearly perfect complementarity between these two RNA molecules [7,8]. miRNAs, unlike the stringent complementarity of siRNAs to their RNA targets, are single-stranded that pair with target RNAs, which have partial complementarity to the miRNAs [9,10].…”

Pharmaceutical Prospects for RNA Interference

“…Because of the high target sequence specificity of RNAi, mismatches between mRNA and the siRNA duplex have been shown to strongly reduce activity (7)(8)(9)(10)(11). Mismatches in the center of the duplex, generally, have stronger effects than mismatches closer to the ends of the strand.…”

“…Holen et al showed that a shift of only three nucleotides in the position of a target region of the human tissue factor gene can have pronounced effects on siRNA silencing efficacy. The pattern of siRNA-mediated silencing of human tissue factor, as a function of the position of the target region was independent of the cell type in which it was studied (11).…”

Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells

“…[7][8][9] It was recently found that the transfection of synthetic 21-23 nt siRNAs with 2-nt 3 0 overhangs into mammalian cells effectively inhibits the expression of the endogenous genes in a sequence-specific manner. [10][11][12] These small RNA duplexes, which are chemically synthesized mimics of Dicer products, are presumably incorporated into RISC and target their cognate substrates for degradation. Lee et al 13 have reported the inhibition of human immunodeficiency virus type 1 (HIV-1) replication by the expression of small interfering RNAs targeted against HIV-1 rev in human cells.…”

“…When we examined the cellular uptake of the fluorescently labeled siRNAs, prepared with a Silencert siRNA Labeling kit (Ambion, TX, USA), with several kinds of transfection reagents (Lipofectaminet 2000, 12,18 Lipofectin, and Oligofectamine) by the FACS Calibur and CellQuest software, the FITC-labeled siRNAs encapsulated with Lipofectaminet 2000 strongly associated with the target cells (data not shown). Cotransfection of plasmids (pNL4-3) and siRNAs was carried out using Lipofectaminet 2000.…”