Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification (original) (raw)

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA

Nature Biotechnology, 2005

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications 1. Many nucleic acids, including double-(dsRNA) 2 and single-stranded RNA (ssRNA) 3-5 , can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system 6,7. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.

siRNA and Innate Immunity

Oligonucleotides, 2009

Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of infl ammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modifi cation approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

Dissecting RNA-Interference Pathway with Small Molecules

Chemistry & Biology, 2005

guides the siRISC* complex to the target mRNA [6, 7]. The guide antisense-strand base pairs with the target Department of Biochemistry and Molecular Pharmacology mRNA, forming an A-form helical geometry recognized by RISC* [3, 8]. In the final step, RISC* cleaves the target University of Massachusetts Medical School Worcester, Massachusetts 01605 mRNA [9]. RISC* can then be recycled to cleave another mRNA [10]. Recently, structural and biochemical studies defined Ago2 as the catalytic engine of RISC* that mediates the Summary cleavage of the target RNA in human cells [11-13]. Other components of RISC required for RNAi have re-RNA interference (RNAi) is a process whereby shortmained elusive, including the putative RNA helicase ininterfering RNAs (siRNA) silence gene expression in volved in unwinding siRNA. To identify new components a sequence-specific manner. We have screened a chemiof the RNAi pathway, we developed a screen for small cal library of substituted dihydropteridinones and idenmolecules that block ATP-dependent steps of RNAi. tified a nontoxic, cell permeable, and reversible inhibi-Two small molecules identified in this screen were furtor of the RNAi pathway in human cells. Biochemical ther characterized and were shown to specifically inand fluorescence resonance-energy transfer experihibit an early step in the RNAi pathway. ments demonstrated that one of the compounds, named ATPA-18, inhibited siRNA unwinding that oc-Results and Discussion curred within 6 hr of siRNA transfection. Extracts prepared from ATPA-18-treated cells also exhibited a We took a small-molecule-based approach to dissect decrease in target RNA cleavage by activated RNAthe ATP-dependent steps of the RNAi pathway. We syninduced silencing complex (RISC*). Interestingly, when thesized a small chemical library of substituted dihyactivated RISC*, which harbors unwound antisense dropteridinones as ATP analogs that contain both rigid siRNA, was treated with ATPA-18 in vitro, target RNA and flexible scaffolds (C.U.D. and T.M.R., unpublished cleavage was not affected, indicating that this comdata) and screened their activities in a dual-fluorespound inhibited siRNA unwinding or steps upstream cence assay for RNAi induction [3]. In this assay, plasof unwinding in the RNAi pathway. Our results also mids harboring enhanced green-and red-fluorescent establish the timing of siRNA unwinding and show proteins (EGFP and RFP, respectively) were cotransthat siRNA helicase activity is required for RNAi. fected into HeLa cells with EGFP siRNA that targets ATPA-18 analogs will therefore provide a new class of EGFP mRNA for degradation. The ratio of EGFP/RFP small molecules for studying RNAi mechanisms in a fluorescence in the presence of siRNA was calculated variety of model organisms and deciphering in vivo and normalized to the EGFP/RFP ratio of mock treated genetic functions through reverse genetics. cells. RNAi activity was then compared to RNAi in cells transfected with EGFP siRNA and treated with an ATP Introduction analog from the dihydropteridinones library. The screen was designed to identify small molecule probes that RNA interference (RNAi), the process by which mRNA would specifically inhibit ATP-dependent events ocis targeted for degradation by 21-23 nucleotide shortcurring during RNAi and, in doing so, inhibit RNAi. We interfering RNA (siRNA), has become key to establishidentified two nontoxic compounds that showed concening connections between gene structure and function tration-dependent inhibition of RNAi in the dual-fluoin human cells by reverse genetics (reviewed in [1, 2]). rescence assay. The structure of these compounds, Because of its broad applications in biology and medidesignated ATPA-18 and ATPA-21, and their associated cine, understanding the fundamental mechanism of inhibitory activities are shown in Figure 1B. RNAi phenomenon is of great importance. The current To investigate whether ATPA-18 and ATPA-21 can inmodel for how RNAi occurs in vivo is shown in Figure hibit RNAi against endogeneously expressed mRNA, 1A. In the first step of RNAi induction, the 5# ends of we tested their effect on RNAi targeted to human CDK9 the siRNA duplex are phosphorylated, resulting in the mRNA. CDK9 is the cyclin-dependent kinase comformation of an RNA-induced silencing complex (RISC)ponent of the P-TEFb, CDK9-cyclinT1 complex, which siRNA complex [3, 4]. Additional ATP-dependent events is involved in regulating transcription elongation [14]. take place next involving siRNA unwinding from the 5# CDK9 double-stranded siRNA wholly complementary end of the antisense strand and activation of RISC to CDK9 mRNA knocked down CDK9 protein levels substantially, unlike mock or mismatched CDK9 siRNA

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo

RNA, 2012

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed “Dicer-substrate siRNA” (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as d...

A SHORT COMMUNICATION ON THERAPEUTIC IMPORTANCE OF siRNA INDUCED GENE SILENCING

Small interfering ribonucleic acid (siRNAs) are small interfering molecules which are messenger ribonucleic acid (mRNA) specific have many potential therapeutic role and by approaching techniques like microarray make possible of genome wide study. Small Interfering RNA have short 20-24 bp double stranded deoxyribonucleic acid (dsDNA) with phosphorylated 5’ ends and hydroxylated 3’ ends with two overhanging nucleotides. siRNA can also be introduced by transfection. Introduction of too much small interfering ribonucleic acid (siRNA) can results in nonspecific events due to activation of innate immune response.

Functional comparison of single- and double-stranded siRNAs in mammalian cells

Biochemical and Biophysical Research Communications, 2004

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25 bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double-and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.

Gene Silencing Activity of siRNA Molecules Containing Phosphorodithioate Substitutions

ACS Chemical Biology, 2012

Chemically synthesized small interfering RNAs (siRNAs) have been widely used to identify gene function and hold great potential in providing a new class of therapeutics. Chemical modifications are desired for therapeutic applications to improve siRNA efficacy. Appropriately protected ribonucleoside-3′-yl S-[β-(benzoylmercapto)ethyl] pyrrolidinothiophosphoramidite monomers were prepared for the synthesis of siRNA containing phosphorodithioate (PS2) substitutions in which the two non-bridging oxygen atoms are replaced by sulfur atoms. A series of siRNAs containing PS2 substitutions have been strategically designed, synthesized and evaluated for their gene silencing activities. These PS2-siRNA duplexes exhibit an A-form helical structure similar to unmodified siRNA. The effect of PS2 substitutions on gene silencing activity is positiondependent, with certain PS2-siRNAs showing significantly higher activity than unmodified siRNA. The relative gene silencing activities of siRNAs containing either PS2 or phosphoromonothioate (PS) linkages at identical positions are variable and depend on the sites of modification. 5′-Phosphorylation of PS2-siRNAs has little or no effect on gene silencing activity. Incorporation of PS2 substitutions into siRNA duplexes increases their serum stability. These results offer preliminary evidence of the potential value of PS2 modified siRNAs.