Promiscuous 8-Alkoxyadenosines in the Guide Strand of an SiRNA: Modulation of Silencing Efficacy and Off-Pathway Protein Binding

Abstract

8-Alkoxyadenosines have the potential to exist in anti or syn conformations around the glycosidic bond when paired opposite to U or G in the complementary strands, thereby placing the sterically demanding 8-alkoxy groups in the major or minor groove, respectively, of duplex RNA. These modified bases were used as ‘base switches’ in the guide strands of an siRNA to prevent off-pathway protein binding during delivery via placement of the alkoxy group in the minor groove, while maintaining significant RNAi efficacy by orienting the alkoxy group in the major groove. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions--two in the seed region, one at the cleavage junction and another nearer to the 3′-end of the guide strand. Thermal stabilities of the corresponding siRNA duplexes showed that U is preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified positive control siRNAs, singly modified siRNAs knocked down the target mRNA efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. SiRNAs singly modified at positions 6 and 10 of the guide strand were found to be effective in blocking binding to the RNA-dependent protein kinase PKR, a cytoplasmic dsRNA-binding protein implicated in sequence independent off target effects.

Biophysical properties and thermal stability of oligonucleotides of RNA containing 7,8‐dihydro‐8‐hydroxyadenosine

“…We are in the process of expanding this study to other more structurally complex structures to explore the generality of the results presented in this study. We are also interested in incorporating this lesion into functional RNAs e.g., miRNAs and siRNAs, to explore the outcomes inflicted by this lesion on biologically relevant processes.…”

Propargylated Purine Deoxynucleosides: New Tools for Fluorescence Imaging Strategies

“…Synthesis of 4 required prior protection of both hydroxyl moieties as tert-butyldimethylsilyl (TBDMS) ethers using TBDMSCl and imidazole in dry dimethylformamide (DMF) at rt for 24 h, affording 14 in 81% yield. Nucleophilic substitution of the 8-bromine with propargyl alcohol was accomplished using nBuLi in tetrahydrofuran (THF) at −40 °C to rt for 24 h affording 15 in 91% yield [14]. Deprotection of both silyl groups using tetrabutylammonium fluoride (TBAF) in THF at rt for 3 h furnished 4 in 85% yield.…”

Small Molecule-Based Pattern Recognition To Classify RNA Structure

“…The silyl acetal was selectively removed using hydrogen fluoride (4), which was then 5′-O-4,4′-dimethoxytrityl (DMT) protected (5). 60 The 3′-O-phosphoramidite was installed via a microwave-assisted synthesis protocol developed by Krishnamurthy and co-workers to afford BFU-phosphoramidite 6. 61,62 The overall yield from commercial starting materials was 35–45%, as compared to the ∼28% overall yield reported for an alternate method reported after the start of this work.…”