Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA (original) (raw)
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European Journal of Pharmacology, 2005
To date there are only few reports of the use of small interfering RNA (siRNA) in whole animals and most of these are restricted to systemic application of siRNAs targeting the liver. In our present studies we have investigated whether siRNAs can be used against a central target after intracerebroventricular (i.c.v.) application and compared their effects with those of antisense oligonucleotides. For this purpose we designed different siRNA and antisense oligonucleotide molecules against the rat hypothalamic melanocortin MC 4 receptor and selected the siRNA and antisense oligonucleotide with the highest efficacy in vitro. We observed that siRNA, encompassing the same gene sequence as antisense oligonucleotides, induced a stronger inhibition of melanocortin MC 4 receptor expression than antisense oligonucleotides. When fluorescencelabeled siRNA were applied i.c.v. in rats no label was detected in brain tissue in spite of the use of cell detergents to improve the delivery. In contrast to these findings the i.c.v. administered fluorescence-labeled antisense oligonucleotides reached the brain structures expressing melanocortin MC 4 receptor and were taken up by the cells in these areas. In summary it seems as if 'naked' antisense oligonucleotides have an advantage over 'naked' siRNA for experiments in vivo. The development of optimized vector systems seems to be a prerequisite before siRNA can be regarded as a suitable experimental tool for in vivo studies.
Rna & Oligonucleotide Therapeutics
2010
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Single-Stranded siRNAs Activate RNAi in Animals
Cell, 2012
The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5 0 phosphate to achieve activity in vivo, and we developed a metabolically stable 5 0 -(E)-vinylphosphonate (5 0 -VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.
PLOS ONE, 2015
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.
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.
Oligonucleotides, 2008
VIEL ET AL. 202 with different chemically modi ed [ 18 F]siRNAs, and (3) the in uence of siRNA chemical modi cations on their interference activity in vitro and in vivo. Materials and Methods Chemicals including oligonucleotides Chemicals were purchased from standard commercial sources (Aldrich-, Fluka-, or Sigma, France) and were used without further puri cation, unless stated otherwise. Oligonucleotides were obtained from Eurogentec (Belgium) or from Dharmacon (USA). The sequence and the chemistry of the oligonucleotides used are shown in Table 1. Annealing of the siRNAs was achieved by incubating equimolar quantities of sense (S) and antisense (AS) strands in annealing buffer (100 mM NaCl) at 90°C for 1 minute, followed by 10 minutes at room temperature. The sequences of luciferase-targeted siRNA (S: 5′-CGU ACG CGG AAU ACU UCG AUU-3'; AS: 5′-UCG AAG UAU UCC GCG UAC GUU-3′) were designed as previously described (Elbashir et al., 2001). For oligonucleotides 2′F(AS/S) and 2′F(AS), all pyrimidines were 2′-deoxy-2′-uoro modied. For oligonucleotides 2′OMe(AS/S), 2′OMe(AS)/2′OH(S), and 2′OH(AS)/2′OMe(S), a 2′-deoxy-2′-O-methyl-modi ed base was introduced after every two bases in the two strands of the siRNA, the AS strand only, or the S strand only, respectively. Two negative control sequences were tested. One of them was furnished by Dharmacon (negative control 1).
Nucleic Acids Research, 2002
Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisenseinduced phenotypes is sometimes dif®cult, and chemical modi®cations that improve the potency and speci®city of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding af®nity, up to 10°C per substitution, making LNAs an exciting option for the optimization of antisense ef®cacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNAcontaining oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA±DNA chimeras complementary to the 5¢untranslated region, the region surrounding the start codon and the coding region of mRNA, and identi®ed effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for ef®ciently inhibiting gene expression inside cells.