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...lexes (Bernstein et al. 2001; Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001; Knight and Bass 2001). One strand of the duplex usually remains as a mature miRNA (Khvorova et al. 2003; =-=Schwarz et al. 2003-=-). Pri-miRNA processing is a critical event in miRNA biogenesis. This initial processing event predetermines mature miRNA sequences by generating one end of mature miRNA (Lee et al. 2003; Lund et al. ...

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...a mature miRNA and is then assembled into the effector complex called miRNP Cell 125, 887–901, June 2, 2006 ª2006 Elsevier Inc. 887sor miRNA-induced silencing complex (miRISC) (Khvorova et al., 2003; =-=Schwarz et al., 2003-=-). RNA interference (RNAi), the gene silencing mechanism that is mediated by small RNAs, is now a powerful genetic tool in mammalian systems. Effective and stable gene knockdown can be achieved by the...

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...ric siRNA targeting the human sod1 mRNA (Figure 1A). This siRNA loads one strand (red) to the near exclusion of the other (blue) because it contains an unpaired nucleotide at the 5# end of the guide (=-=Schwarz et al., 2003-=-). Upon incubation in Drosophila embryo lysate, the radiolabeled passenger strand was converted to a 9 nt, 5#-radiolabeledCell 608 Figure 1. The siRNA Passenger Strand, but Not the Guide, Is Cleaved ...

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... 32, No. 3 DOI: 10.1093/nar/gkh247 Nucleic Acids Research, Vol. 32 No. 3 ã Oxford University Press 2004; all rights reserved Published online February 9, 2004 at E kigaku-K yoshitsu (U N IV O F T O K Y O ) on O ctober 17, 2010 nar.oxfordjournals.org D ow nloaded from sequence features except for the fourth condition brought about the least levels of RNAi in mammalian cells. Essentially the same rules for siRNA sequence preference appeared to apply for siRNA-based RNAi in chick embryos and DNAbased RNAi in mammalian cells. During the course of the preparation of this manuscript, Schwarz et al. (29) showed sequence conditions resembling the above to some extent to be necessary for RISC formation and subsequent target RNA cleavage in Drosophila embryonic extracts. Khvorova et al. (30) indicated that both the enhanced ¯exibility at the siRNA end including the 5¢ AS end and low internal energy across the duplex are strongly correlated with siRNA and microRNA (miRNA) functions. The similarity in siRNA sequence requirements for in vivo RNAi in mammalian cells [this work, (30)] and in vitro Drosophila RNAi (29) could re¯ect underlying similarities between the RNAi mechanisms in insects and ani...

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...n methodology is based on target accessibility evaluation (5,13), typical design rules and published empirical rules, and stability rules for siRNA duplex ends and for the region of the cleavage site =-=(14,15)-=-. Recently, it has been reported that functional siRNA duplexes tend to have lower stability on the 5 0 -antisense end ( 4 bp) than on the 5 0 - sense end (14,15). It is proposed that both the absolut...

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...certain degree of asymmetry in the abundance of these miRNAs. In the case of miR-302b and miR-302c, the ratios between the cloning frequencies of the two sides were 22.5:1 and 10:1, respectively. According to the standard nomenclature (Ambros et al., 2003), the less abundant miRNA of each pair was designated with an asterisk mark. Recent studies using siRNA duplexes demonstrated that this asymmetry comes from asymmetric degradation of the opposing strand following Dicer processing. According to this, the strand with the less stable 5V end has a better chance to survive (Khvorova et al., 2003; Schwarz et al., 2003). Sixty-nine percent of our novel miRNA precursors are clearly in agreement with this, indicating that this rule is generally valid although the mechanism of strand selection for miRNA may be more complex than that for siRNA. It is intriguing that ES cells are such rich sources of novel miRNAs. Previous cloning efforts intensively searched for miRNA genes using cultured mammalian cells and adult tissues. It would be an interesting quest to look for additional miRNAs in other rare cell types. For instance, smallRNA expression profiling of various stem cells or mouse embryos from different devel...

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... confirmed by a modified small RNA cloning approach (Table S1). Although siRNAs are generated in the double-stranded form, often only one of the strands is incorporated into RISC and can be detected (=-=Schwarz et al., 2003-=-; Khvorova et al., 2003). Twenty-four nucleotide SRO5-P5CDH natsiRNA was detected with an antisense (relative to SRO5) probe but not with a sense probe (Figure 1C), suggesting that the sense strand as...

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...mbers (Liu et al., 2004; Meister et al., 2004). The thermodynamic properties of the double-stranded intermediate influence which strand will complex with the Argonaute protein (Khvorova et al., 2003; =-=Schwarz et al., 2003-=-). In extreme cases, as with most miRNAs, only one of the two strands is incorporated. In contrast, usually both strands of an siRNA duplex can be found in Argonaute complexes, Cell 129, 69–82, April ...

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...RNA or miRNA duplex and activation of RISC requires the removal of the passenger strand (Khvorova et al., 2003; Leuschner et al., 2006; Matranga et al., 2005; Miyoshi et al., 2005; Rand et al., 2005; =-=Schwarz et al., 2003-=-). Activated RISC is a ribonucleoprotein complex and consists in its core of an Argonaute protein and a single-stranded siRNA or miRNA, which acts as a guide to target complementary sequences within m...