Functional polarity is introduced by Dicer processing of short substrate RNAs

Abstract

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes (1). Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3′-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3′-overhang with 3′-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3′-overhang resides on the antisense (AS) strand and dicing proceeds to the ‘right’ (‘R’) and one where the 3′-overhang resides on the sense (S) strand and dicing proceeds to the ‘left’ (‘L’). Interestingly, the ‘R’ version of the asymmetric 27mer is generally more potent in reducing target gene levels than the ‘L’ version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the ‘R’ form favoring S strand and the ‘L’ form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

Novel Cell type-specific aptamer-siRNA delivery system for HIV-1 therapy

“…In contrast, Ch L-2 and Ch 2 have poorer efficacy (<70%) and strand selectivity (R = 1.9 and 1.6, respectively). These data suggest that the aptamer-27 mer siRNA chimeras indeed enhance the RNAi efficacy and potent, consistent with previous studies in our laboratory 29,30 .…”

“…However, in the absence of lipofection, gene silencing from Ch L-1 and Ch 1 was specific to CHO-gp160 expressing cells and no inhibition of luciferase was observed in CHO-EE cells. Interestingly, Ch L-1 and Ch 1 which are linked to the 27 mer duplex RNA showed somewhat greater efficacy than chimeras Ch L-2 and Ch 2, consistent with our previous observations of Dicer substrates enhancing RNAi 29,30 .…”

“…Based upon our own previous studies 29, 30 , we tested the aptamer as a chimeric transcript with a Dicer substrate RNA duplex (25-30 nt). We utilized a 27 mer siRNA in which one strand is covalently attached to the aptamer, and the second strand is base paired to the upper strand and compared a 27 base pair dsRNA with a 21 base pair dsRNA fused to the aptamer in an analogous fashion.…”

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation

“…The DsiRNAs were designed (64,65), synthesized, and validated (45,51) by Integrated DNA Technologies. All accompanying control sequences (Scr) were also generated by Integrated DNA Technologies.…”

Noncanonical Synthetic RNAi Inducers

“…This review focuses on current strategies of development of siRNA structural modifications for promoting better gene silencing with low risk of side effects, with particular focus on longer siRNA duplexes 25-30 nt in length that mimic Dicer substrates (Dicer substrate siRNA (dsiRNA)) [15][16][17][18][19][20]. Special attention has been paid to the long double-stranded RNA duplexes, which induced effective gene silencing and did not require Dicer-mediated processing of the substrate into smaller units: trimer RNA (tsiRNA) with 63 nt in length and tripartite-interfering RNA (tiRNA) with 38 nt in length [21,22].…”