Abstract
Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing.
Highlights
Many modifications exist to alter the properties of RNA or DNA oligonucleotides to make them better suited to laboratory applications or therapeutic development [1]
We demonstrate that substitution of one or more sites in the central region of the antisense strand of an small interfering RNAs (siRNAs) duplex is compatible with the RNA interference (RNAi) machinery
Modified RNA oligonucleotides were synthesized at Alnylam Pharmaceuticals, and synthetic protocols are provided as Supplementary Information
Summary
Many modifications exist to alter the properties of RNA or DNA oligonucleotides to make them better suited to laboratory applications or therapeutic development [1]. Abasic sites occur spontaneously in cellular DNA at a frequency of approximately 1 in 300 000 bases per genome per day [2]. Because abasic sites can lead to genomic damage, synthetic abasic monomers are often used to construct model DNA strands for study of the cellular machinery for DNA damage repair. Less attention has been paid to abasic site-containing RNA (abasic RNA), even though the modification modulates physicochemical properties and biological function. Abasic RNA has been used to probe RNA structure [9] and can be compatible with RNA interference (RNAi) [10]. These studies provide a starting point for using abasic RNA as a tool for research and discovery
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