Chemical and Structural Diversity of siRNA Molecules

Nawrot, Barbara; Sipa, Katarzyna

doi:10.2174/156802606777303658

Cited by 50 publications

(36 citation statements)

References 91 publications

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“…This modification pattern was most uniformly active when a single strand was modified, and ASm was used for most of the studies reported herein. Given the extent to which functional performance of chemically modified siRNAs and DsiRNAs varies with sequence context, we would not be surprised if nuclease resistance without compromising potency of the RNA duplex as a trigger of RNAi (Nawrot and Sipa, 2006;Zhang et al, 2006). Modification of a few residues is usually well tolerated; however, widespread modification of the siRNA can lead to reduced activity compared to unmodified RNA.…”

Section: Discussionmentioning

confidence: 99%

Chemical Modification Patterns Compatible with High Potency Dicer-Substrate Small Interfering RNAs

et al. 2008

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Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

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Section: Discussionmentioning

confidence: 99%

Chemical Modification Patterns Compatible with High Potency Dicer-Substrate Small Interfering RNAs

et al. 2008

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“…While chemical modifications of siRNAs and Dicersubstrate RNAs have been extensively studied (Nawrot and Sipa 2006;Zhang et al 2006;Collingwood et al 2008;Robbins et al 2009), synthetic sshRNAs have not been similarly studied. Because of their different structures and probable differences in how they are processed by the RNAi machinery, modifications that have proven effective for siRNAs or Dicer-substrate RNAs may not necessarily be effective with sshRNAs.…”

Section: Introductionmentioning

confidence: 99%

Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs

Ge¹,

Dallas²,

Ilves³

et al. 2009

RNA

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Small hairpin RNAs (shRNAs) with 19-base-pair, or shorter, stems (short shRNAs [sshRNAs]) have been found to constitute a class whose mechanism of action appears to be distinct from that of small interfering RNAs (siRNAs) or longer shRNAs. These sshRNAs can be as active as canonical siRNAs or longer shRNAs. Their activity is affected by whether the antisense strand is positioned 59 or 39 to the loop (L or R sshRNAs, respectively). Dicer seems not to be involved in the processing of sshRNAs, although the mechanism of target gene suppression by these hairpins is through Ago2-mediated mRNA cleavage. In this study, the effects of chemical modifications on the potency, serum stability, and innate immune response of sshRNAs were investigated. Deoxynucleotide substitution and 29-O-methyl (29-OMe) modification in the sense strand and loop did not affect silencing activity, but, unlike with siRNAs, when placed in the antisense strand these modifications were detrimental. Conjugation with bulky groups at the 59-end of L sshRNAs or 39-end of R sshRNAs had a negative impact on the potency. Unmodified sshRNAs in dimer form or with blunt ends were immunostimulatory. Some modifications such as 39-end conjugation and phosphorothioate linkages on the backbone of the sshRNAs could also induce inflammatory cytokine production. However, 29-OMe substitution of sshRNAs abrogated the innate immune response and improved the serum stability of the hairpins.

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“…One approach to reach this goal is the introduction of chemical modifications into siRNA duplexes (Manoharan 2004;Fougerolles et al 2005;Uprichard 2005;Nawrot and Sipa 2006). Research efforts in this field are focused on improving three of the most important siRNAs features: (1) cell membrane permeability, (2) in vivo stability, and (3) specificity of their silencing action.…”

Section: Introductionmentioning

confidence: 99%

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Sipa

Sochacka

Kaźmierczak-Barańska

et al. 2007

RNA

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128

126

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A series of nucleobase-modified siRNA duplexes containing ''rare'' nucleosides, 2-thiouridine (s 2 U), pseudouridine (C), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s 2 U or C units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DTm 3.9 and 6.6°C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s 2 U and C units at their 39-ends and with a D unit at their 59-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s 2 U and C units at their 59-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 39-adjacent to the mutation site. Thermally stable siRNA molecules containing several s 2 U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.

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Chemical and Structural Diversity of siRNA Molecules

Cited by 50 publications

References 91 publications

Chemical Modification Patterns Compatible with High Potency Dicer-Substrate Small Interfering RNAs

Chemical Modification Patterns Compatible with High Potency Dicer-Substrate Small Interfering RNAs

Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

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