Non-nucleotide Modification of Anti-miRNA Oligonucleotides

MicroRNAs (miRNAs) are important modulators of gene expression. Synthetic anti-microRNA oligonucleotides (AMOs, or anti-miRs) are a form of steric-blocking antisense oligonucleotides (ASOs) that inhibit miRNA function through high-affinity binding and subsequent inactivation and/or degradation of the targeted miRNA. AMOs are a primary tool used to empirically determine the biological targets of a miRNA and can also be used therapeutically when overexpression of a miRNA contributes to a disease state. Chemical modification of synthetic AMOs enhance potency by protecting the oligonucleotide from nuclease degradation and by increasing binding affinity to the target miRNA. A new steric-blocking ASO modification strategy with favorable properties for use in AMOs was recently developed that combines use of high-affinity 2'-O-methyl RNA with terminally positioned non-nucleotide "ZEN" modifiers. This protocol describes use of ZEN AMOs in a dual-luciferase reporter assay as a simplified means to validate AMO performance or to quickly test putative miRNA binding sites in target sequences. This protocol also describes a method using Western blot analysis for quantifying the level of upregulation of proteins made from an mRNA that is thought to be under miRNA regulation, following inhibition of that miRNA by ZEN AMO treatment.