8-Oxoguanosine Switches Modulate the Activity of Alkylated siRNAs by Controlling Steric Effects in the Major versus Minor Grooves

Abstract

Small interfering double-stranded RNAs have been synthesized bearing one or more base modifications at nucleotide positions 4, 11 and/or 16 in the guide strand. The chemically modified base is an N2-alkyl-8-oxo-7,8-dihydroguanine (alkyl = propyl, benzyl) that can alternatively pair in a Watson-Crick sense opposite cytosine (C) or as a Hoogsteen pair opposite adenine (A). Cellular delivery with C opposite led to effective targeting of A-containing but not C-containing mRNA sequences in a dual luciferase assay with RNA interference levels that were generally as good as or better than unmodified sequences. The higher activity is ascribed to an inhibitory effect of the alkyl group projecting into the minor groove of double-stranded RNA preventing off-target binding to proteins such as PKR (RNA-activated protein kinase).

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

“…4 In addition, we reported the effect of N 2 -alkylated 8-oxo-7,8-dihydro-2′-deoxyguanosine analogs at specific positions in the guide strand opposite A in the target, a pairing believed to place the N 2 group in the major groove. 23 …”

“…4 In addition, we reported the effect of N 2 -alkylated 8-oxo-7,8-dihydro-2′-deoxyguanosine analogs at specific positions in the guide strand opposite A in the target, a pairing believed to place the N 2 group in the major groove. 23 However, a systematic study focusing on the effect of solitary major groove modifications at different guide strand positions has not been reported. Furthermore, for this study we chose to modify the purine 7-position because, like the C5 of pyrimidines, this site is located in the major groove of duplex structures and not involved in Watson-Crick base pairing.…”

Chemical Modification of siRNA Bases To Probe and Enhance RNA Interference

“…In recent work, we showed that N 2 -alkyl-8-oxo-2′-deoxyguanosine analogues (alkyl = propyl, benzyl) ( 23 ), adopting either the syn or anti conformation depending upon their base-pairing partner, can be used as the switchable base to introduce a steric blockade to protein binding in one form (C opposite) vs. the other (A opposite) (see Figure 2). 33,34 …”

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

“…28,32,33 Kool and coworkers, and Manoharan, Egli and coworkers reported substitution of pyrimidines (uridine) in the guide strands of siRNAs with more hydrophobic aromatic ring systems (e.g. 2,4-difluorobenzene 29 and 2,4-difluorotoluene 30,36 ), although here silencing efficacy varied depending on the position of the substitution.…”