Steric Restrictions of RISC in RNA Interference Identified with Size-Expanded RNA Nucleobases

Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 ? using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.     

Designing highly active siRNAs for therapeutic applications

The discovery of RNA interference (RNAi) generated considerable interest in developing short interfering RNAs (siRNAs) for understanding basic biology and as the active agents in a new variety of therapeutics. Early studies showed that selecting an active siRNA was not as straightforward as simply picking a sequence on the target mRNA and synthesizing the siRNA complementary to that sequence. As interest in applying RNAi has increased, the methods for identifying active siRNA sequences have evolved from focusing on the simplicity of synthesis and purification, to identifying preferred target sequences and secondary structures, to predicting the thermodynamic stability of the siRNA. As more specific details of the RNAi mechanism have been defined, these have been incorporated into more complex siRNA selection algorithms, increasing the reliability of selecting active siRNAs against a single target. Ultimately, design of the best siRNA therapeutics will require design of the siRNA itself, in addition to design of the vehicle and other components necessary for it to function in vivo. In this minireview, we summarize the evolution of siRNA selection techniques with a particular focus on one issue of current importance to the field, how best to identify those siRNA sequences likely to have high activity. Approaches to designing active siRNAs through chemical and structural modifications will also be highlighted. As the understanding of how to control the activity and specificity of siRNAs improves, the potential utility of siRNAs as human therapeutics will concomitantly grow.     

RNA interference with 2′,4′-bridged nucleic acid analogues

In this study, a number of 2′,4′-BNA- and 2′,4′-BNA^NC-modified siRNAs were designed and synthesized. Their thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2′,4′-BNA- and 2′,4′-BNA^NC-modified siRNAs (named siBNA and siBNA^NC, respectively) showed very high T_m values, were remarkably stable in serum sample and showed promising RNAi properties equal to those exhibited by natural siRNAs. Thermally stable siBNAs composed of slightly modified sense and antisense strands were capable of suppressing gene expression equal to that of natural siRNA. A number of modifications on the sense strand by 2′,4′-BNA or 2′,4′-BNA^NC, either consecutively or separated by natural RNA nucleotides, is tolerable in RNAi machinery. Modifications at the Argonauate (Ago2) cleavage site of the sense strand (9–11th positions from the 5′-end of the sense strand) produced variable results depending on siRNA composition. Mostly, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on siRNA composition.     

siRNA function in RNAi: a chemical modification analysis

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.     

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.     

SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene.RNAi can be used to identify the function of genes or to knock down the targeted genes.In RNAi technology,19 bp double-stranded short interfering RNAs (siRNA) with characteristic 3' overhangs are usually used.The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA.Moreover,there are many factors influencing siRNA activity and these factors are usually nonlinear.To find the motif features and the effect on siRNA activity,we carried out a feature extraction on some published experimental data and used these features to train a backpropagation neural network (BP NN).Then,we used the trained BP NN to predict siRNA activity.     

Enzymatically Produced Pools of Canonical and Dicer-Substrate siRNA Molecules Display Comparable Gene Silencing and Antiviral Activities against Herpes Simplex Virus

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.     

Identification of sequence features that predict competition potency of siRNAs

Small interfering RNAs (siRNAs) specifically knock-down target mRNAs via RNA interference (RNAi) mechanism. During this process, introduction of excess amount of exogenous siRNAs could lead to the saturation of cellular RNAi machinery. One consequence of RNAi machinery saturation is the competition between two simultaneously introduced siRNAs, during which one siRNA loses gene silencing activity. Although competition phenomena have been well characterized, the molecular and sequence features of siRNAs that specify the competition potency remain poorly understood. Here, for the first time, we performed a large-scale siRNA competition potency analysis by measuring the competition potency of 56 different siRNAs and ranking them based on their competition potency. We have also established an algorithm to predict the competition potency of siRNAs based upon the conserved sequence features of strong and weak competitor siRNAs. The present study supports our hypothesis that the competition potency of siRNAs is specified by the 5′-half antisense sequence and provides a useful guideline to design siRNAs with minimal RNAi machinery saturation.     

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

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, 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.     

Improvements in siRNA properties mediated by 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)

RNA interference (RNAi) has emerged recently as an efficient mechanism for specific gene silencing. Short double-stranded small interfering RNAs (siRNAs) are now widely used for cellular or drug target validation; however, their use for silencing clinically relevant genes in a therapeutic setting remains problematic because of their unfavourable metabolic stability and pharmacokinetic properties. To address some of these concerns, we have investigated the properties of siRNA modified with 2'-deoxy-2'-fluoro-beta-d-arabinonucleotide units (araF-N or FANA units). Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA. We also show that the incorporation of FANA units into siRNA duplexes increases activity and substantially enhances serum stability of the siRNA. A fully modified sense 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) strand when hybridized to an antisense RNA (i.e. FANA/RNA hybrid) was shown to be 4-fold more potent and had longer half-life in serum (approximately 6 h) compared with an unmodified siRNA (<15 min). While incorporation of FANA units is well tolerated throughout the sense strand of the duplex, modifications can also be included at the 5' or 3' ends of the antisense strand, in striking contrast to other commonly used chemical modifications. Taken together, these results offer preliminary evidence of the therapeutic potential of FANA modified siRNAs.     

Small interfering RNAs containing full 2'-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary single-strand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2'-O-methyl (2'-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2'-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2'-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.     

Effect of target secondary structure on RNAi efficiency

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. However, the levels of knockdown vary greatly. Here, we examine the effect of target disruption energy, a novel measure of target accessibility, along with other parameters that may affect RNAi efficiency. Based on target secondary structures predicted by the Sfold program, the target disruption energy represents the free energy cost for local alteration of the target structure to allow target binding by the siRNA guide strand. In analyses of 100 siRNAs and 101 shRNAs targeted to 103 endogenous human genes, we find that the disruption energy is an important determinant of RNAi activity and the asymmetry of siRNA duplex asymmetry is important for facilitating the assembly of the RNA-induced silencing complex (RISC). We estimate that target accessibility and duplex asymmetry can improve the target knockdown level significantly by nearly 40% and 26%, respectively. In the RNAi pathway, RISC assembly precedes target binding by the siRNA guide strand. Thus, our findings suggest that duplex asymmetry has significant upstream effect on RISC assembly and target accessibility has strong downstream effect on target recognition. The results of the analyses suggest criteria for improving the design of siRNAs and shRNAs.     

Synthesis and properties of double-stranded RNA-bindable oligodiaminogalactose derivatives conjugated with vitamin E

RNA interference (RNAi) is a gene-regulating system that is controlled by external short interfering RNAs (siRNAs). Sequence selective gene silencing by siRNA shows promise in clinical research. However, there have been few efficient methods for delivering siRNAs to target cells. In this study, we propose a novel type of RNA duplex-bindable molecule with an oligodiaminosaccharide structure. These 2,6-diamino-2,6-dideoxy-(1-4)-β-d-galactopyranose oligomers (oligodiaminogalactoses; ODAGals) conjugated with α-tocopherol (vitamin E; VE) or a VE analog were designed as novel siRNA-bindable molecules that can be utilized to deliver RNAi drugs to the liver. Among these compounds, the VE analog-bound ODAGal was suggested to bind to RNA duplexes without inhibiting RNAi activity.     

Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries

Use of highly potent small interfering RNAs (siRNAs) can substantially reduce dose-dependent cytotoxic and off-target effects. We developed a genetic forward approach by fusing the cytosine deaminase gene with targets for the robust identification of highly potent siRNAs from RNA interference (RNAi) libraries that were directly delivered into cells via bacterial invasion. We demonstrated that two simple drug selection cycles performed conveniently in a single container predominately enriched two siRNAs targeting the MVP gene (siMVP) and one siRNA targeting the egfp gene (siEGFP) in surviving cells and these proved to be the most effective siRNAs reported. Furthermore, the potent siRNAs isolated from the surviving cells possessed noncellular toxic characteristics. Interestingly, the length of highly potent siMVPs identified could be as short as 16-mer, and increasing the length of their native sequences dramatically reduced RNAi potency. These results suggest that the current approach can robustly discover the most potent and nontoxic siRNAs in the surviving cells, and thus has great potential in facilitating RNAi applications by minimizing the dose-dependent and sequence nonspecific side effects of siRNAs.     

Inhibition of respiratory viruses by nasally administered siRNA

Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are two respiratory pathogens of paramount medical significance that exert high mortality. At present, there is no reliable vaccine or antiviral drug against either virus. Using an RNA interference (RNAi) approach, we show that individual as well as joint infection by RSV and PIV can be specifically prevented and inhibited by short interfering RNAs (siRNAs), instilled intranasally in the mouse, with or without transfection reagents. The degree of protection matched the antiviral activity of the siRNA in cell culture, allowing an avenue for quick screening of an efficacious siRNA. When targeting both viruses in a joint infection, excess of one siRNA moderated the inhibitory effect of the other, suggesting competition for the RNAi machinery. Our results suggest that, if properly designed, low dosages of inhaled siRNA might offer a fast, potent and easily administrable antiviral regimen against respiratory viral diseases in humans.