Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors

Abstract

MicroRNAs (miRNAs) are ubiquitous regulators of gene expression that contribute to almost any cellular process. Methods for managing of miRNA activity are attracting increasing attention in relation to diverse experimental and therapeutic applications. DNA-encoded miRNA inhibitors expressed from plasmid or virus-based vectors provide persistent miRNA suppression and options of tissue-directed micromanaging. In this report, we explore the potential of exploiting short, hairpin-shaped RNAs for simultaneous suppression of two or more miRNAs. Based on the "Tough Decoy" (TuD) design, we create dual-targeting hairpins carrying two miRNA recognition sites and demonstrate potent co-suppression of different pairs of unrelated miRNAs by a single DNA-encoded inhibitor RNA. In addition, enhanced miRNA suppression is achieved by expression of RNA polymerase II-transcribed inhibitors carrying clustered TuD hairpins with up to a total of eight miRNA recognition sites. Notably, by expressing clustered TuD inhibitors harboring a single recognition site for each of a total of six miRNAs, we document robust parallel suppression of multiple miRNAs by inhibitor RNA molecules encoded by a single expression cassette. These findings unveil a new potential of TuD-based miRNA inhibitors and pave the way for standardizing synchronized suppression of families or clusters of miRNAs.

Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys

“…The possibility of encoding a single perfect site obviates uncertainties about the ideal position of the artificial mismatches and thus facilitates TuD design. Notably, two recent studies support this notion by showing that miRNAs also can be inhibited potently by a perfectly matched TuD (35) and that one or two miRNA binding sites work equally well (58). In turn, this finding emphasizes that our AAV vectors for custom TuD expression are also useful for specific inhibition of cellular miRNAs, as we already have exemplified in the present study with miR-122.…”

“…Again, the modular design of our AAV vectors will facilitate and accelerate this process. Moreover, it may be interesting to adapt a strategy from the Mikkelsen laboratory, which expressed clustered TuDs from a RNA polymerase II promoter (55,58), to gain spatiotemporal control over RNAi specificity.…”

“…Ideally, in the future, this approach will be combined with further strategies to increase on-targeting or to prevent off-targeting, such as bioinformatical identification of "safe" sequences, rational design of shRNAs with inherent strand biases, AAV capsid engineering, or TuD concatamerization for better inhibition (25,26,43,58). Moreover, our technology readily complements high-throughput strategies that also aim at identifying potent shRNAs with reduced off-targeting potential, such as a massively parallel sensor assay or screening of ultracomplex, high-coverage shRNA libraries (20,(62)(63)(64).…”

Antiangiogenic Eye Gene Therapy

“…This allows VEGF to bind to the otherwise less preferable mFlt1, suggesting expression of ectopic miRNA-10 target sequence molecules as a novel tool to reduce CNV. For efficient elimination of a specific miRNA, or for simultaneous targeting of several different miRNAs, the target sequences could be expressed either as clustered tough decoys[89][90][91] or circular miRNA sponges 86. These examples of using shRNA and miRNA target sequences expressed from a viral vector may also contribute to the development of effective anti-VEGF therapy.…”

miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico

“…In should be noted that other alternative approaches are currently used to efectively inhibit miRNAs: I) TuDs (“tough decoys”), similarly to miRNA sponges, contain also MBSs, but they are placed in a single-stranded region of short stem-loop1746; II) antagomiRs - contain sequence with full complementarity to the target miRNA(s)47; III) “mask” RNA - that binds to the target mRNA and protects it from recognition by the miRNA48. Bak and colleagues performed a side-by-side comparison of seven different DNA-encoded miRNA inhibitors including antagomiRs, TuDs, miRNA sponges, and “mask” RNA and concluded that TuDs and bulged miRNA sponges are the most potent miRNA inhibitors49.…”