CiteULike: mtagaya's rnai

Small interfering RNA-induced transcriptional gene silencing in human cells.

KV Morris — 2008-08-16T00:32:46-00:00

Science (New York, N.Y.), Vol. 305, No. 5688. (27 August 2004), pp. 1289-1292.

Small interfering RNA (siRNA) and microRNA silence genes at the transcriptional, posttranscriptional, and/or translational level. Using human tissue culture cells, we show that promoter-directed siRNA inhibits transcription of an integrated, proviral, elongation factor 1alpha (EF1A) promoter-green fluorescent protein reporter gene and of endogenous EF1A. Silencing was associated with DNA methylation of the targeted sequence, and it required either active transport of siRNA into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction. These results demonstrate that siRNA-directed transcriptional silencing is conserved in mammals, providing a means to inhibit mammalian gene function.

RNA interference: Hitting the on switch

Erika Check — 2007-08-24T11:04:20-00:00

Nature, Vol. 448, No. 7156. (22 August 2007), pp. 855-858.

Gene regulation for higher cells: a theory.

RJ Britten — 2005-09-21T12:21:14-00:00

Science, Vol. 165, No. 891. (25 July 1969), pp. 349-357.

Activating gene expression in mammalian cells with promoter-targeted duplex RNAs

Bethany Janowski — 2007-04-23T20:28:33-00:00

Nat Chem Biol, Vol. 3, No. 3. (March 2007), pp. 166-173.

Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.

Z Chen — 2008-08-14T00:53:58-00:00

Molecular cancer therapeutics, Vol. 7, No. 3. (March 2008), pp. 698-703.

We recently reported that synthetic dsRNAs targeting promoter regions can induce gene expression in a phenomenon referred to as dsRNA-induced gene activation/RNA activation (RNAa) [Li et al. Proc Natl Acad Sci U S A 2006;103:17337-42]. The present study investigates the in vitro antitumor activity RNAa can elicit through triggering the expression of cell cycle repressor protein p21(WAF1/CIP1) (p21) in human bladder cancer cells. Transfection of a 21-nucleotide dsRNA targeting the p21 promoter (dsP21) was used to induce p21 expression in T24 and J82 bladder cancer cell lines. Reverse transcription-PCR and Western blot analysis accessed the increase p21 mRNA and protein levels, respectively, in transfected cells. In association to p21 induction, dsP21 transfection significantly inhibited bladder cancer cell proliferation and clonogenicity. Further analysis of cell viability and cell cycle distribution revealed that dsP21 transfection also enhanced apoptotic cell death and caused an accumulation in the G(1) phase in both cell lines. In conclusion, p21 activation by RNAa has antitumor activity in vitro in bladder cancer cells. These results suggest that RNAa could be used for cancer treatment by targeted activation of tumor suppressor genes.

Small dsRNAs induce transcriptional activation in human cells.

Long-Cheng Li — 2006-11-07T13:30:13-00:00

Proc Natl Acad Sci U S A (3 November 2006)

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21(WAF1/CIP1) (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5' end of the antisense strand, or "seed" sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.

RNAi and RNAa - The Yin and Yang of RNAome.

PN Pushparaj — 2008-03-06T10:50:45-00:00

Bioinformation, Vol. 2, No. 6. (2008), pp. 235-237.

RNA interference (RNAi) is a powerful technology with huge applications for functional genomics, target identification in drug discovery and elucidation of molecular signaling pathways. Current RNAi studies have demonstrated the clinical potential of small interfering RNAs (siRNAs) in metabolic diseases, cancer, AIDS, malaria, neurodegenerative disorders, dental diseases and other illnesses. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs could effectively activate a particular gene in a sequence specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed as RNA activation (RNAa). The paradoxical concept of Yin and Yang, which describe two primal opposing but complementary principles, can potentially be applied to elucidate the complex phenomenon of RNAa/RNAi in the RNAome. This warrants a proper understanding of the RNAi/RNAa molecular pathways in living organisms before any of the small dsRNAs can potentially be exploited for therapeutics in human beings.

Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.

Aimee L Jackson — 2006-05-18T18:49:58-00:00

RNA (8 May 2006)

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect.

Paul R Clark — 2008-02-29T05:43:20-00:00

Nucleic Acids Res (20 December 2007)

Tumor necrosis factor (TNF) initiates local inflammation by triggering endothelial cells (EC) to express adhesion molecules for leukocytes such as intercellular adhesion molecule-1 (ICAM-1 or CD54). A prior study identified siRNA molecules that reduce ICAM-1 expression in cultured human umbilical vein EC (HUVEC). One of these, ISIS 121736, unexpectedly inhibits TNF-mediated up-regulation of additional molecules on EC, including E-selectin (CD62E), VCAM-1 (CD106) and HLA-A,B,C. 736 siRNA transfection was not toxic for EC nor was there any evidence of an interferon response. 736 Transfection of EC blocked multiple early TNF-related signaling events, including activation of NF-kappaB. IL-1 activation of these same pathways was not inhibited. A unifying explanation is that 736 siRNA specifically reduced expression of mRNA encoding tumor necrosis factor receptor 1 (TNFR1) as well as TNFR1 surface expression. A sequence with high identity to the 736 antisense strand (17 of 19 bases) is present within the 3'UTR of human TNFR1 mRNA. An EGFP construct incorporating the 3'UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence. Chemical modification and mismatches within the sense strand of 736 also inhibited silencing activity. In summary, an siRNA molecule selected to target ICAM-1 through its antisense strand exhibited broad anti-TNF activities. We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand. This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

Toxicogenomics of non-viral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and specificity.

S Akhtar — 2007-08-31T01:46:45-00:00

Adv Drug Deliv Rev, Vol. 59, No. 2-3. (30 March 2007), pp. 164-182.

RNA interference (RNAi) is an evolutionary conserved cellular process for the regulation of gene expression. In mammalian cells, RNAi is induced via short (21-23 nt) duplexes of RNA, termed small interfering RNA (siRNA), that can elicit highly sequence-specific gene silencing. However, synthetic siRNA duplexes are polyanionic macromolecules that do not readily enter cells and typically require the use of a delivery vector for effective gene silencing in vitro and in vivo. Choice of delivery system is usually made on its ability to enhance cellular uptake of siRNA. However, recent gene expression profiling (toxicogenomics) studies have shown that separate from their effects on cellular uptake, delivery systems can also elicit wide ranging gene changes in target cells that may impact on the 'off-target' effects of siRNA. Furthermore, if delivery systems also alter the expression of genes targeted for silencing, then siRNA activity may be compromised or enhanced depending on whether the target gene is up-regulated or down-regulated respectively. Citing recent examples from the literature, this article therefore reviews the toxicogenomics of non-viral delivery systems and highlights the importance of understanding the genomic signature of siRNA delivery reagents in terms of their impact on gene silencing activity and specificity. Such information will be essential in the selection of optimally acting siRNA-delivery system combinations for the many applications of RNA interference.

Advances in MicroRNAs: Implications for Gene Therapists.

Rebecca T Marquez — 2008-01-07T08:23:42-00:00

Hum Gene Ther (19 December 2007)

MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate the expression of as many as one-third of all human messenger RNAs (mRNAs). miRNAs are thought to be involved in diverse biological processes, including tumorigenesis. Analysis of miRNA levels may have diagnostic implications. Evidence shows that numerous viruses interact with the miRNA machinery, and that a number of viruses encode their own miRNAs. It seems likely that miRNAs will be implicated in many human diseases. Manipulation of miRNA levels by gene therapy provides an attractive new approach for therapeutic development. This review focuses on approaches to manipulate miRNA levels in cells and in vivo, and the implications for gene therapy. Furthermore, we discuss the use of endogenous miRNAs as scaffolds for the expression of RNA interference (RNAi) as well as competition between exogenous RNAi triggers and endogenous miRNAs. Because short interfering RNAs can also act as miRNAs, seed matches with the 3' untranslated regions of genes should be avoided to prevent off-target effects. Last, we discuss the use of miRNAs to avoid immune responses to viral vectors.

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.

K Ui-Tei — 2007-08-09T00:51:02-00:00

Nucleic Acids Res, Vol. 32, No. 3. (2004), pp. 936-948.

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

Theoretical design of antisense RNA structures substantially improves annealing kinetics and efficacy in human cells.

V Patzel — 2007-05-11T01:04:04-00:00

Nat Biotechnol, Vol. 16, No. 1. (January 1998), pp. 64-68.

The success of antisense therapeutics is not predictable despite their widespread use in biotechnology and molecular medicine. The relationship between RNA structure and biological effectiveness is largely not understood; however, antisense RNA-mediated effects in vivo seem to be related to annealing kinetics in vitro. This study suggests that terminal unpaired nucleotides and overall flexibility of antisense RNA directed against the human immunodeficiency virus type 1 (HIV-1) are related to fast RNA-RNA annealing in vitro as well as to strong inhibition of virus replication in human cells. Annealing rate constants of computer-selected antisense RNA species approach the values for natural antisense RNA in the order of 10(6) M-1s-1. When considering the unfavorable stability in cellular extracts of antisense RNA species that were found to anneal fast in vitro, an antisense effect against HIV-1 in human cells was observed that was 10- to 10,000-fold stronger than that measured for species predicted to anneal slowly. A computer-supported structural design of antisense RNA can serve as a platform to determine RNA-RNA association in vitro and biological effectiveness in living cells.

Sfold web server for statistical folding and rational design of nucleic acids.

Y Ding — 2005-12-14T14:31:16-00:00

Nucleic Acids Res, Vol. 32, No. Web Server issue. (1 July 2004)

The Sfold web server provides user-friendly access to Sfold, a recently developed nucleic acid folding software package, via the World Wide Web (WWW). The software is based on a new statistical sampling paradigm for the prediction of RNA secondary structure. One of the main objectives of this software is to offer computational tools for the rational design of RNA-targeting nucleic acids, which include small interfering RNAs (siRNAs), antisense oligonucleotides and trans-cleaving ribozymes for gene knock-down studies. The methodology for siRNA design is based on a combination of RNA target accessibility prediction, siRNA duplex thermodynamic properties and empirical design rules. Our approach to target accessibility evaluation is an original extension of the underlying RNA folding algorithm to account for the likely existence of a population of structures for the target mRNA. In addition to the application modules Sirna, Soligo and Sribo for siRNAs, antisense oligos and ribozymes, respectively, the module Srna offers comprehensive features for statistical representation of sampled structures. Detailed output in both graphical and text formats is available for all modules. The Sfold server is available at http://sfold.wadsworth.org and http://www.bioinfo.rpi.edu/applications/sfold.

siRNA Selection Server: an automated siRNA oligonucleotide prediction server.

B Yuan — 2007-05-10T09:19:18-00:00

Nucleic Acids Res, Vol. 32, No. Web Server issue. (1 July 2004)

The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.

Selecting effective siRNAs based on guide RNA structure

Christian Köberle1 — 2007-05-10T09:06:16-00:00

Nature Protocols, Vol. 1 (16 November 2006), pp. 1832-1839.

In RNA interference, guide RNAs direct RNA-induced silencing complexes to mRNA targets, mediating cleavage and ultimately leading to gene silencing. We have observed that unstructured guide strands, which either completely lack complementary bases or in which internal base pairing is thermodynamically unlikely, confer strongest silencing, whereas structures with base-paired ends are inactive. Thus, the structure of the guide strand represents a major determinant of small interfering RNA activity. Here we describe a detailed computational protocol for identification of unstructured guide strands for a given mRNA target sequence. Sequentially, all guide sequences with target complementarity are simulated, their corresponding structures are folded and unstructured guide strands are selected and rated according to thermodynamic parameters. Although this procedure is new and remains to be validated by the community, it allows reliable identification of highly active siRNAs that can be used for functional target validation or drug development.

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells.

D Yang — 2007-05-10T08:56:59-00:00

Proc Natl Acad Sci U S A, Vol. 99, No. 15. (23 July 2002), pp. 9942-9947.

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

A microRNA in a multiple-turnover RNAi enzyme complex.

G Hutvágner — 2007-05-10T08:52:29-00:00

Science, Vol. 297, No. 5589. (20 September 2002), pp. 2056-2060.

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.

Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells.

Daniel H Kim — 2006-08-30T11:54:35-00:00

Nat Struct Mol Biol (27 August 2006)

Argonaute proteins are the core components of effector complexes that facilitate RNA interference (RNAi). Small interfering RNAs (siRNAs) targeted to promoter regions mediate transcriptional gene silencing (TGS) in human cells through heterochromatin formation. RNAi effector complexes have yet to be implicated in the mechanism of mammalian TGS. Here we describe the role of the human Argonaute-1 homolog (AGO1) in directing TGS at the promoters for human immunodeficiency virus-1 coreceptor CCR5 and tumor suppressor RASSF1A. AGO1 associates with RNA polymerase II (RNAPII) and is required for histone H3 Lys9 dimethylation and TGS. AGO1, TAR RNA-binding protein-2 (7TRBP2) and Polycomb protein EZH2 colocalize to the siRNA-targeted RASSF1A promoter, implicating Polycomb silencing in the mechanism of mammalian TGS. These results establish a connection between RNAi components AGO1 and TRBP2, RNAPII transcription and Polycomb-regulated control of gene expression.

Involvement of AGO1 and AGO2 in mammalian transcriptional silencing

Bethany Janowski — 2006-09-06T16:54:13-00:00

Nature Structural & Molecular Biology, Vol. 13, No. 9. (27 August 2006), pp. 787-792.

L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells.

Nuo Yang — 2006-09-05T12:35:55-00:00

Nat Struct Mol Biol (27 August 2006)

LINE-1s, or L1s, are highly abundant retrotransposons comprising 17% of the human genome. Most L1s are retrotransposition defective; nonetheless, there are approximately 100 full-length L1s potentially capable of retrotransposition in the diploid genome. L1 retrotransposition may be detrimental to the host and thus needs to be controlled. Previous studies have identified sense and antisense promoters in the 5' UTR of full-length human L1. Here we show that the resulting bidirectional transcripts can be processed to small interfering RNAs (siRNAs) that suppress retrotransposition by an RNA interference (RNAi) mechanism. We thus provide evidence that RNAi triggered by antisense transcripts may modulate human L1 retrotransposition efficiently and economically. L1-specific siRNAs are among the first natural siRNAs reported in mammalian systems. This work may contribute to understanding the regulatory role of abundant antisense transcripts in eukaryotic genomes.

Small interfering RNAs to the rescue: blocking L1 retrotransposition

Harris Soifer — 2006-09-06T16:54:14-00:00

Nature Structural & Molecular Biology, Vol. 13, No. 9., pp. 758-759.

siRNAs can function as miRNAs.

JG Doench — 2005-03-09T21:11:43-00:00

Genes Dev, Vol. 17, No. 4. (15 February 2003), pp. 438-442.

With the discovery of RNA interference (RNAi) and related phenomena, new regulatory roles attributed to RNA continue to emerge. Here we show, in mammalian tissue culture, that a short interfering RNA (siRNA) can repress expression of a target mRNA with partially complementary binding sites in its 3' UTR, much like the demonstrated function of endogenously encoded microRNAs (miRNAs). The mechanism for this repression is cooperative, distinct from the catalytic mechanism of mRNA cleavage by siRNAs. The use of siRNAs to study translational repression holds promise for dissecting the sequence and structural determinants and general mechanism of gene repression by miRNAs.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.

A Fire — 2005-12-16T15:55:55-00:00

Nature, Vol. 391, No. 6669. (19 February 1998), pp. 806-811.

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.

Identification and characterization of small RNAs involved in RNA silencing

Alexei Aravin — 2006-09-17T18:29:22-00:00

FEBS Letters, Vol. 579, No. 26. (31 October 2005), pp. 5830-5840.

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.

Prevalence of off-target effects in Drosophila RNA interference screens

Yong Ma — 2006-09-10T17:37:58-00:00

Nature (10 September 2006)

Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes.

Xiang Liu — 2006-07-06T14:20:04-00:00

RNA (14 June 2006)

In Drosophila melanogaster, the Dicer-2/R2D2 complex initiates RNA interference (RNAi) by processing long double-stranded RNA (dsRNA) into small interfering RNA (siRNA). Recent biochemical studies suggest that the Dcr-2/R2D2 complex also facilitates incorporation of siRNA into the RNA-induced silencing complex (siRISC). Here we present genetic evidence that R2D2 and Dcr-2 are both required for loading siRNA onto the siRISC complex. Consistent with this, only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, can efficiently interact with duplex siRNA. Furthermore, both dsRNA-binding domains of R2D2 are critical for binding to siRNA and promoting assembly of the siRISC complexes.

Cell type–specific delivery of siRNAs with aptamer-siRNA chimeras

James Mcnamara — 2006-08-10T13:04:26-00:00

Nature Biotechnology, Vol. 24, No. 8. (25 June 2006), pp. 1005-1015.

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells.

S Saxena — 2006-07-11T09:23:04-00:00

J Biol Chem, Vol. 278, No. 45. (7 November 2003), pp. 44312-44319.

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3'-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5'-phosphate but not a 3'-hydroxyl group. G.U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3-4 base mismatches and additional G.U mismatches.

Mismatched siRNAs downregulate mRNAs as a function of target site location.

Scott E Martin — 2006-06-13T17:30:38-00:00

FEBS Lett (5 June 2006)

In mammalian cells, RNA interference can be mediated by synthetic duplex RNAs, termed small interfering RNAs (siRNAs), which assist in cleaving completely complementary mRNA transcripts. MicroRNAs (miRNAs) are endogenous small RNAs that assist in translationally repressing mRNAs with regions of partial complementarity, but may also reduce transcript levels. Since miRNAs predominantly interact with the 3' UTRs of transcripts, we sought to ask if mismatched siRNAs mimicking miRNAs affect cognate mRNA levels as a function of target site location. We find that mismatched siRNAs targeting the 3' UTRs of two endogenous transcripts yield a greater reduction in mRNA levels than those targeting the coding region. Our findings demonstrate the importance of target site location within endogenous mRNAs for small RNAs associated with RNAi.

Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

KH Chung — 2006-07-06T09:29:48-00:00

Nucleic Acids Res, Vol. 34, No. 7. (2006)

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction.

CI An — 2006-06-09T01:27:44-00:00

RNA, Vol. 12, No. 5. (May 2006), pp. 710-716.

Recent studies have uncovered extensive presence and functions of small noncoding RNAs in gene regulation in eukaryotes. In particular, RNA interference (RNAi) has been the subject of significant investigations for its unique role in post-transcriptional gene regulation and utility as a tool for artificial gene knockdown. Here, we describe a novel strategy for post-transcriptional gene regulation in mammalian cells in which RNAi is specifically modulated through RNA aptamer-small molecule interaction. Incorporation of an RNA aptamer for theophylline in the loop region of a short hairpin RNA (shRNA) designed to silence fluorescent reporter genes led to dose-dependent inhibition of RNAi by theophylline. shRNA cleavage experiments using recombinant Dicer demonstrated that theophylline inhibited cleavage of an aptamer-fused shRNA by Dicer in vitro. Inhibition of siRNA production by theophylline was also observed in vivo. The results presented here provide the first evidence of specific RNA-small molecule interaction affecting RNAi, and a novel strategy to regulate mammalian gene expression by small molecules without engineered proteins.

A computational study of off-target effects of RNA interference.

S Qiu — 2006-05-11T06:44:29-00:00

Nucleic Acids Res, Vol. 33, No. 6. (2005), pp. 1834-1847.

RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of approximately 21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, 'off-target effects' compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100-400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5' termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of approximately 10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity.

Aimee L Jackson — 2006-05-11T06:29:15-00:00

RNA (8 May 2006)

Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended ("off-target") consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3' UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with dependence on siRNA concentration similar to that of silencing of the target transcript. Thus, short stretches of sequence complementarity to the siRNA or shRNA seed region are key to the silencing of unintended transcripts.

Off-target effects by siRNA can induce toxic phenotype.

Yuriy Fedorov — 2006-05-11T06:28:06-00:00

RNA (8 May 2006)

Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA.

Many commonly used siRNAs risk off-target activity.

O Snøve — 2006-05-11T06:22:48-00:00

Biochem Biophys Res Commun, Vol. 319, No. 1. (18 June 2004), pp. 256-263.

Using small interfering RNA (siRNA) to induce sequence specific gene silencing is fast becoming a standard tool in functional genomics. As siRNAs in some cases tolerate mismatches with the mRNA target, knockdown of genes other than the intended target could make results difficult to interpret. In an investigation of 359 published siRNA sequences, we have found that about 75% of them have a risk of eliciting non-specific effects. A possible cause for this is the popular BLAST search engine, which is inappropriate for such short oligos as siRNAs. Furthermore, we used new special purpose hardware to do a transcriptome-wide screening of all possible siRNAs, and show that many unique siRNAs exist per target even if several mismatches are allowed. Hence, we argue that the risk of off-target effects is unnecessary and should be avoided in future siRNA design.

Accelerated off-target search algorithm for siRNA.

T Yamada — 2006-05-11T06:22:23-00:00

Bioinformatics, Vol. 21, No. 8. (15 April 2005), pp. 1316-1324.

MOTIVATION: Designing highly effective short interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi) is one of the hottest topics in molecular biology. The relationship between siRNA sequences and RNAi activity has been studied extensively to establish rules for selecting highly effective sequences. However, there is a pressing need to compute siRNA sequences that minimize off-target silencing effects efficiently and to match any non-targeted sequences with mismatches. RESULTS: The enumeration of potential cross-hybridization candidates is non-trivial, because siRNA sequences are short, ca. 19 nt in length, and at least three mismatches with non-targets are required. With at least three mismatches, there are typically four or five contiguous matches, so that a BLAST search frequently overlooks off-target candidates. By contrast, existing accurate approaches are expensive to execute; thus we need to develop an accurate, efficient algorithm that uses seed hashing, the pigeonhole principle, and combinatorics to identify mismatch patterns. Tests show that our method can list potential cross-hybridization candidates for any siRNA sequence of selected human gene rapidly, outperforming traditional methods by orders of magnitude in terms of computational performance. AVAILABILITY: http://design.RNAi.jp CONTACT: yamada@cb.k.u-tokyo.ac.jp.

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.

X Lin — 2006-04-19T08:25:38-00:00

Nucleic Acids Res, Vol. 33, No. 14. (2005), pp. 4527-4535.

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three 'top hits' from our screen have been demonstrated to result from off-target gene silencing. Two of the three 'siRNA hits' were found to directly trigger down-regulation of hif-1alpha mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1alpha mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1alpha was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3'-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving 'hits-follow-up' strategies for future large-scale knockdown experiments.

3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets

Amanda Birmingham — 2006-02-18T12:38:11-00:00

Nature Methods, Vol. 3, No. 3. (17 February 2006), pp. 199-204.