Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells.

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO2), cycloheximide (CHX) and Lipofectamine 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi. Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO2. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.

Antisense tools for functional studies of human Argonaute proteins.

The Argonaute proteins play essential roles in development and cellular metabolism in many organisms, including plants, flies, worms, and mammals. Whereas in organisms such as Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute mutant strains allowed the study of their biological functions, in mammals the application of this approach is limited by its difficulty and in the specific case of Ago2 gene, by the lethality of such mutation. Hence, in human cells, functional studies of Ago proteins relied on phenotypic suppression using small interfering RNA (siRNA) which involves Ago proteins and the RNA interference mechanism. This bears the danger of undesired or unknown interference effects which may lead to misleading results. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with processes in which Agos are involved. Different functional observations in the use of siRNA versus asONs indicate the relevance of this assumption. We developed asONs specific for the four human Agos (hAgos) and compared their activities with those obtained by siRNA. We confirm that hAgo2 is involved in microRNA (miRNA)- and in siRNA-mediated silencing pathways, while the other hAgos play a role only in miRNA-based gene regulation. Using combinations of asONs we found that the simultaneous down-regulation of hAgo1, hAgo2, and hAgo4 led to the strongest decrease in miRNA activity, indicating a main role of these proteins.

Phosphorothioate-stimulated uptake of siRNA by mammalian cells: a novel route for delivery.

The efficient delivery of biologically functional short interfering RNA (siRNA) in vivo remains a widely unresolved technical problem in therapeutic drug development. The repertoire of concepts for the cellular uptake of oligonucleotide-based tools and drugs has been extended by the mechanistically novel finding that phosphorothioate (PS)-modified single-stranded oligodeoxyribonucleotides (ON) promote the intracellular accumulation of naked extra-cellular siRNA in a variety of cell types. This mode of delivery gives rise to substantial intracellular amounts of siRNA, up to 10(4) siRNA molecules per cell. Conversely, the moderate biological effectiveness strongly indicates that intracellular release of siRNA from sub-cellular compartments where it seems to be trapped is a necessary step towards efficient target suppression. Here, we summarize key characteristics of the PS-stimulated cellular uptake of siRNA and describe concepts for the increase of intracellular delivery of biologically functional siRNA.

Increased RNAi is related to intracellular release of siRNA via a covalently attached signal peptide.

In the last decade short interfering RNA (siRNA) became an important means for functional genomics and the development of gene-specific drugs. However, major technical hurdles in the application of siRNA include its cellular delivery followed by its intracellular trafficking and its release in order to enter the RNA interference (RNAi) machinery. The novel phosphorothioate-stimulated cellular uptake of siRNA contrasts other known delivery systems because it involves a caveosomal pathway in which large amounts of siRNA are delivered to the perinuclear environment, leading to measurable though moderate target suppression. Limited efficacy seems to be related to intracellular trapping of siRNA. To study the role of intracellular trafficking of siRNA for biological effectiveness we studied whether a signal peptide for trans-membrane transport of bacterial protein toxins, which is covalently attached to siRNA, can promote its release from the perinuclear space into the cytoplasm and thereby enhance its biological effectiveness. We show that attachment of the peptide TQIENLKEKG to lamin A/C-directed siRNA improves target inhibition after its PS-stimulated delivery. This is related to increased efflux of the siRNA-peptide conjugate from the ER-specific perinuclear sites. In summary, this study strongly suggests that intracellular release of siRNA leads to increased biological effectiveness. Thus covalent peptide-siRNA conjugates are proposed as new tools to study the relationship between intracellular transport and efficacy of siRNA.

Cellular uptake and intracellular release are major obstacles to the therapeutic application of siRNA: novel options by phosphorothioate-stimulated delivery.

The cellular uptake of oligomeric nucleic acid-based tools and drugs including small-interfering RNA (siRNA) represents a major technical hurdle for the biologic effectiveness and therapeutic success in vivo. Subsequent to cellular delivery it is crucial to direct siRNA to the cellular location where it enters the RNA interference pathway. Here the authors summarise evidence that functionally active siRNA represents a minor fraction in the order of 1% of total siRNA inside a given target cell. Exploiting possibilities of steering intracellular release or trafficking of siRNA bears the potential of substantially increasing the biological activity of siRNA. The recently described phosphorothioate stimulated cellular delivery of siRNA makes use of the caveolar system ending in the Golgi apparatus, which contrasts all other known delivery systems. Therefore, it represents an attractive alternative to study whether promoted intracellular release is related to increased target suppression and, thus, increased phenotypic biologic effectiveness.

A hexanucleotide selected for increased cellular uptake in cis contains a highly active CpG-motif in human B cells and primary peripheral blood mononuclear cells.

The relationship between immunostimulation of human B cells by cytosine-phosphate-guanosine (CpG) -containing oligonucleotides and their physical cellular uptake is of mechanistic interest and a prerequisite for rational improvements of the therapeutic potential of CpG-harbouring oligonucleotides. Here, a combinatorial approach was used to identify nucleotide sequence motifs that facilitate increased cellular uptake in mammalian cells. Oligonucleotides harbouring the selected hexanucleotide TCGTGT in cis show increased cellular uptake. This motif contains a CpG dinucleotide within a sequence context that shows a very strong CpG-specific stimulatory activity on human B cells. Here we describe the influence of concentration, length and sequence position of the unmethylated CpG dinucleotide on immunostimulation. A comparison between phosphorothioate-derivatives and unmodified TCGTGT-containing oligonucleotides strongly indicates a great CpG-specificity for the unmodified CpG-harbouring oligonucleotides but not for the phosphorothioate versions. This work describes a link between the physical cellular uptake of naked oligonucleotides harbouring the selected cellular uptake motif TCGTGT, its strong CpG-specific stimulation of human B cells and its relationship with the sequence context of CpG and its cellular uptake.