Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs

Abstract

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5′-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3′-termini of both strands, the addition of a UNA at the 5′-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

“…In addition, it could be very interesting combining our approach of siRNA passenger strand modification to generate thermodynamically stabilized siRNA duplexes with guide strand modifications. Very interesting combinations could be siRNA guide strand modifications in the seed region, which have been described to cause reduced siRNA off-target activity, for example, 29-O-methyl nucleotides (Jackson et al 2006a) or UNA nucleotides (Bramsen et al 2010;Laursen et al 2010;Vaish et al 2010). Since siRNAs are being used as drugs our siRNA selection parameters may also have an impact on siRNA-based therapy.…”

Asymmetric siRNA: New Strategy to Improve Specificity and Reduce Off-Target Gene Expression

“…One of the major concerns is off-target gene-expression effects. A variety of modifications of siRNA chemistry or structure have been introduced to minimize the off-target effect (Martinez et al, 2002;Chiu and Rana, 2003;Kim et al, 2004;Siolas et al, 2004;Judge et al, 2005;Sun et al, 2008;Bramsen et al, 2009;Vaish et al, 2011). It has been recently reported that modifying siRNAs with unlocked nucleobase analog could markedly reduce the off-target effects (Vaish et al, 2011).…”

“…Two potential mechanisms resulting in off-target silencing have been elucidated (Vaish et al, 2011). One is accidental incorporation of passenger strand that could guide RISC to cleave the sequence complementary to its normal target.…”

“…One of the main obstacles for the clinical use of siRNA is unintended off-target effects (Castanotto and Rossi, 2009), i.e., the down-regulation or up-regulation of genes other than the target. To address this issue, a variety of modifications have been introduced to siRNA (Soutschek et al, 2004;Bramsen et al, 2009;Vaish et al, 2011), most of which took advantage of chemical substitutions or modifications. However, it has also been reported that siRNAs with a shortened passenger strand could similarly function well and reduce off-target gene-expression effects (Sano et al, 2008;Sun et al, 2008;Chang et al, 2009).…”

RNAi Therapeutic Potentials and Prospects in CNS Disease

“…In vivo nuclease resistance of this structure is enhanced [42]. In contrast to LNA, derivatives of RNA without C2′-C3′ sugar bonds (unlocked nucleic acid-UNA) destabilize a sequence structure [43]. Substitution of pentose with hexose monosaccharides, such as cyclohexenyl, anitrol, and arabinose, was applied to develop CeNA, ANA, and 2′-F-ANA [44], subsequently resulting in enhanced stability of siRNA in vivo [45].…”