MicroRNAs are short, endogenous RNAs that direct posttranscriptional regulation of gene expression vital for many developmental and cellular functions. Implicated in the pathogenesis of several human diseases, this group of RNAs provides interesting targets for therapeutic intervention. Anti-microRNA oligonucleotides constitute a class of synthetic antisense oligonucleotides used to interfere with microRNAs. In this study, we investigate the effects of chemical modifications and truncations on activity and specificity of anti-microRNA oligonucleotides targeting microRNA-21. We observed an increased activity but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination. 1. Introduction Originally identified in Caenorhabditis elegans and subsequently established in a number of additional organisms including mammalian cells [1–3], RNA interference (RNAi) is a posttranscriptional gene-silencing process targeting single-stranded RNA sequences. Whereas several classes of RNAi effectors have been identified, siRNAs and microRNAs (miRNAs) are the best characterized. miRNAs interact with transcript sequences possessing partial or full complementarity, promoting gene repression of the targeted transcripts. Dysregulation of miRNAs has been implicated in human developmental disorders and diseases, including several forms of cancer [4–6]. Manipulation of miRNAs, utilizing synthetic, chemically modified oligonucleotides (ONs) targeting select miRNAs, presents an approach to both elucidate the role of miRNA dysregulation in human disease and discover novel therapies for many pathological conditions. miRNAs have previously been shown to possess the ability to regulate multiple functionally related mRNAs, such as sets of metabolic genes [7, 8], a powerful feature that may enable miRNA-based therapeutics to circumvent redundant mechanisms that might otherwise bypass single inhibited targets. miRNA-21 (miR21) is potentially a very interesting target for future therapeutic applications. It has widespread regulatory functions and has been implicated in a variety of
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