Summary A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)exp) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation producing “c9RAN proteins”. Since neutralizing r(GGGGCC)exp could inhibit these potentially toxic events, we sought to identify small molecule binders of r(GGGGCC)exp. Chemical and enzymatic probing of r(GGGGCC)8 indicate it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)exp were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)66 and neurons trans-differentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)exp-binding small molecules as a possible c9FTD/ALS therapeutic, and suggest c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC)exp.

In the original version of this paper, panels C and D in the legend of Figure 4 were referred to in reverse order. The phrase ''either with (C) or without (D) mS'' should have been ''either without (C) or with (D) mS.'' This has now been corrected in the article online.

Short-interfering RNAs (siRNAs) are common tools in molecular biology, however the development of RNAi-based therapeutics is limited by immunostimulatory and non-specific effects mediated by off-target RNA-binding proteins. PKR and ADAR1 are two proteins implicated in RNAi off-target effects, and share a common means of interaction with siRNAs through double-stranded RNA binding motifs (dsRBMs). Here we report the site-specific introduction of N2- propargyl 2-aminopurine into siRNAs and subsequent conversion to two bulky products via copper-catalyzed azide alkyne cycloaddition (CuAAC) with either Nazidoacetyl-mannosamine azide or N-ethylpiperidine azide. We observed position-specific effects on RNAi activity for modifications made to both the passenger and guide strands. These findings are rationalized in light of recent structural studies of components of the RNA-induced silencing complex (RISC) and RISC-loading complex (RLC). The most active siRNAs were assayed for binding affinity to the RNA-dependent protein kinase (PKR) and adenosine deaminase that acts on RNA 1 (ADAR1). PKR binding was significantly reduced by multiple modifications, regardless of size, and ADAR1 binding was reduced in a position and size-sensitive manner. Our findings present a strategy for designing siRNAs that reduce off-target dsRBM-protein binding while retaining native RNAi activity.

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).

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.