Modulation of thermal stability can enhance the potency of siRNA

Abstract

During RNA-induced silencing complex (RISC) assembly the guide (or antisense) strand has to separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process was found to be facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without prior cleavage. Here we show that the potency of siRNA can be improved by modulating the internal thermodynamic stability profile with chemical modifications. Using a model siRNA targeting the firefly luciferase gene with subnanomolar IC50, we found that placement of thermally destabilizing modifications, such as non-canonical bases like 2,4-difluorotoluene or single base pair mismatches in the central region of the sense strand (9–12 nt), significantly improve the potency. For this particular siRNA, the strongest correlation between the decrease in thermal stability and the increase in potency was found at position 10. Controls with stabilized sugar-phosphate backbone indicate that enzymatic cleavage of the sense strand prior to strand dissociation is not required for silencing activity. Similar potency-enhancing effects were observed as this approach was applied to other functional siRNAs targeting a different site on the firefly luciferase transcript or endogenously expressed PTEN.

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo

“…A variety of chemical modifications are compatible with siRNA activity, including 29-fluoro, 29-OMe, 2,4-difluorotoluene, phosphorothioate, and others . Such modifications have the potential to reduce immunostimulation, enhance serum stability, increase specificity, and improve activity Addepalli et al 2010). Manipulation of the naturally occurring 21-mer design has revealed that not only longer duplexes, such as dsiRNA, but also shorter duplexes and duplexes with altered overhangs participate in RNAi (Chu and Rana 2008;Chang et al 2009).…”

Strategies for Designing and Validating Immunostimulatory siRNAs

“…This was recently applied to increase the immuno stimulatory properties of an siRNA targeting H5N1 in fl uenza in chicken cells ( 9 ) , but could also be applicable to mammalian studies. Interestingly, when such a mismatch is introduced between bases 9 and 12 from the 5 ¢ -end of the passenger strand, it can also result in increased RNAi potency ( 23 ) . Preservation of the targeting strand is however essential, as single mismatches with the target can ablate silencing ef fi cacy of the duplex ( 24 ) .…”

New antitumor 6-chloropurine nucleosides inducing apoptosis and G2/M cell cycle arrest

“…The configuration of the anomeric center as well as the purine substitution pattern could be confirmed by the observed chemical shifts in the 1 H and 13 C NMR spectra and the 3 other β-hexopyranosyl nucleosides [22][23][24]. The distinction between N 7 or N 9 substitution at the purine scaffold can be achieved by the chemical shift of purine carbon 5.…”

“…Nucleoside synthesis can be performed by a two steps procedure, starting with the acetylation [12] or halogenation [13,14] of suitable precursors, followed by reaction with the heterocyclic base. A direct access to nucleosides can be gained by Lewis acid activation with tin chloride [15,16] or TMSOTf [17] of methyl glycosides in a reaction employing a persilylated purine.…”