Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing (original) (raw)
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siRNAmod: A database of experimentally validated chemically modified siRNAs
Scientific Reports, 2016
Small interfering RNA (siRNA) technology has vast potential for functional genomics and development of therapeutics. However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. Chemical modifications in siRNAs provide means to overcome these shortcomings and improve their stability and potency. Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. Therefore, we have developed siRNAmod, a specialized databank for chemically modified siRNAs. Currently, our repository contains a total of 4894 chemically modified-siRNA sequences, comprising 128 unique chemical modifications on different positions with various permutations and combinations. It incorporates important information on siRNA sequence, chemical modification, their number and respective position, structure, simplified molecular input line entry system canonical (SMILES), efficacy of modified siRNA, target gene, cell line, experimental methods, reference etc. It is developed and hosted using Linux Apache MySQL PHP (LAMP) software bundle. Standard user-friendly browse, search facility and analysis tools are also integrated. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. siRNAmod is freely available at: http://crdd.osdd.net/servers/sirnamod. RNA interference (RNAi) phenomenon was described by A. Fire and coworkers in Caenorhabditis elegans 1. They injected double stranded RNA that potentially and specifically interfere with the endogenous gene at mRNA level leading to its silencing 1. RNA silencing pathway utilizes dicer to process long double stranded RNA (dsRNA) to 19-21 nucleotide small interfering RNAs (siRNAs) with 2-nucleotide unphosphorylated 3′ overhangs 2. RNA induced silencing complex (RISC) incorporates siRNA antisense strand, resulting in cleavage of cognate mRNA target 3 as shown in Fig. 1. siRNAs are explored extensively in gene silencing experiments and therapeutics development 4. However, their therapeutic usage is hampered due to many factors e.g. susceptibility to ribonucleases digestion, short biological half-life, lack of proper delivery vehicle, cellular uptake, immune-stimulatory effect, non-specific gene targeting and toxicity 5,6. These limitations are due to the inherent physicochemical properties of siRNAs that includes short and stiff structure, high charge density (poly-anionic), hydrophilic and liability to nuclease cleavage 5,6. To mitigate these restraints, chemical modifications in siRNAs have been substantially investigated 7. Synthetic oligonucleotides were used in the late 1970's for gene inhibition known as antisense technology 8. Modified nucleoside phosphoramidites are used in solid phase chemical synthesis of siRNA which allow site-specific incorporation of chemically modified nucleotide moiety at specific positions within the siRNA 9. Chemical modifications on siRNA are categorized based on component of nucleotide modified as ribose, base or phosphate and change in properties like biological activity, thermodynamic stability and nuclease resistance 7. Chemical modifications on sugar moiety e.g. locked nucleic acids (LNA), unlocked nucleic acids (UNA) 10,11 , 2′-deoxy, 2′-O-methyl 12 , 2′-fluoro, 2′-methoxyethyl, 2′-aminoethyl, were tested for RNAi activity 13,14. In LNA, methylene bridge is created between 2′-oxygen and 4′-carbon to increase the RNAi activity favorably 15,16 and also improves nuclease resistance 17. UNA are open ring derivatives of ribose without C-2′ and C-3′ carbon-carbon bond but structurally mimic the RNA when incorporated into duplex 18. Ribose 2′-OH and phosphorothioate modification increases stability of siRNA 19,20. Besides the 2′-OH was not found to be essential for RNAi activity 21. However, fluorine modification at 2′-OH on different nucleotide positions of siRNA maintains silencing
Nucleic Acids Research, 2009
The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNAinduced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.
Approaches for chemically synthesized siRNA and vector-mediated RNAi
Febs Letters, 2005
Successful applications of RNAi in mammalian cells depend upon effective knockdown of targeted transcripts and efficient intracellular delivery of either preformed si/shRNAs or vector expressed si/shRNAs. We have previously demonstrated that 27 base pair double stranded RNAs which are substrates for Dicer can be up to 100 times more potent than 21mer siRNAs. In this mini-review we elaborate upon the rationale and design strategies for creating Dicer substrate RNAs that provide enhanced knockdown of targeted RNAs and minimize the utilization of the sense strand as RNAi effectors. Expression of shRNAs or siRNAs in mammalian cells can be achieved via transcription from either Pol II or Pol III promoters. There are certain constrictions in designing such vectors, and these are described here. Additionally, we review strategies for inducible shRNA expression and the various viral vectors that can be used to transduce shRNA genes into a variety of cells and tissues. The overall goal of this mini-review is to provide an overview of available approaches for optimizing RNAi mediated down regulation of gene expression in mammalian cells via RNA interference. Although the primary focus is the use of RNAi mediated cleavage of targeted transcripts, it is highly probable that some of the approaches described herein will be applicable to RNAi mediated inhibition of translation and transcriptional gene silencing.
Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells
Nucleic Acids Research, 2003
Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate ef®cient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without speci®c nucleotide overhangs are highly ef®cient in gene silencing. Blocking of the 5¢-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3¢ terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2¢-O-methyl modi®cation, but not molecules with terminal modi-®cations, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2¢-O-methyl modi®cations for stability and activity. We demonstrate that 2¢-O-methyl modi®cations at speci®c positions in the molecule improve stability of siRNAs in serum and are tolerated without signi®cant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.
DEQOR: a web-based tool for the design and quality control of siRNAs
Nucleic Acids Research, 2004
RNA interference (RNAi) is a powerful tool for inhibiting the expression of a gene by mediating the degradation of the corresponding mRNA. The basis of this gene-specific inhibition is small, double-stranded RNAs (dsRNAs), also referred to as small interfering RNAs (siRNAs), that correspond in sequence to a part of the exon sequence of a silenced gene. The selection of siRNAs for a target gene is a crucial step in siRNAmediated gene silencing. According to present knowledge, siRNAs must fulfill certain properties including sequence length, GC-content and nucleotide composition. Furthermore, the cross-silencing capability of dsRNAs for other genes must be evaluated. When designing siRNAs for chemical synthesis, most of these criteria are achievable by simple sequence analysis of target mRNAs, and the specificity can be evaluated by a single BLAST search against the transcriptome of the studied organism. A different method for raising siRNAs has, however, emerged which uses enzymatic digestion to hydrolyze long pieces of dsRNA into shorter molecules. These endoribonuclease-prepared siRNAs (esiRNAs or 'diced' RNAs) are less variable in their silencing capabilities and circumvent the laborious process of sequence selection for RNAi due to a broader range of products. Though powerful, this method might be more susceptible to cross-silencing genes other than the target itself. We have developed a web-based tool that facilitates the design and quality control of siRNAs for RNAi. The program, DEQOR, uses a scoring system based on state-of-the-art parameters for siRNA design to evaluate the inhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siRNAs with high silencing potential for chemical synthesis. In addition, each siRNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of esiRNAs or chemically designed siRNAs. DEQOR is freely available at
siSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse
Nucleic Acids Research, 2013
RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others' supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2-8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificityfocused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.
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...
Multiplexing siRNAs to compress RNAi-based screen size in human cells
Nucleic Acids Research, 2007
Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (800 siRNAs, 400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.