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

Abstract

MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with and sequester specific miRNAs to prevent them from interacting with their target messenger (m)RNAs. Due to the high specificity of miRNA sponges and strong inhibition of target miRNAs, these molecules have become increasingly applied in miRNA loss-of-function studies. However, improperly designed sponge constructs may sequester off-target miRNAs; thus, it has become increasingly important to develop a tool for miRNA sponge construct design and testing. In this study, we introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences. Furthermore, we also provide an example, supplemented with experimental data, of how to use this tool. Using miRNAsong, we designed and tested a sponge for miR-145 inhibition, and cloned the sequence into an inducible lentiviral vector. We found that established cell lines expressing miR-145 sponge strongly inhibited miR-145, thus demonstrating the usability of miRNAsong tool for sponge generation. URL: http://www.med.muni.cz/histology/miRNAsong/.

Figure 1. Schematic representation of miRNA sponge mechanism and design.

Sponge transcript (black colour) contains high-affinity MBSs that sequester target miRNA (red colour).

Figure 2. Expression of miR-145 sponge upon cumate induction and efficiency of miR-145 inhibition.

(a) Cell morphology and GFP expression upon cumate-induced expression of miR-145 sponge, as observed by light and fluorescent microscopy. Scale bars represent 100 μm. (b) Quantification of sponge transcript levels in Ago2-immunoprecipitated (Ago2 IP) and control IgG (IgG IP) fractions, as demonstrated by RT-qPCR. Data are depicted as mean + SD of triplicates. The length of the transcript was checked by standard agarose electrophoresis (graph inset). Full-length gel is presented in Supplementary Fig. 1. (c) Western blot analysis of Ago2 levels in Ago2 IP and control IgG IP fractions. Full-length blot is presented in Supplementary Fig. 2. (d) Expression of c-Myc and Sox2 upon miR-145 sponge induction, as determined by western blot analysis. Experiments were performed on three different HEK293T clones expressing miR-145 sponge or empty vector. One representative western blot is shown. Full-length blot is presented in Supplementary Fig. 3. Quantification of western blots is provided under each western blot. Data represents fold change relative to an empty vector control without cumate ± SD. β-Actin was used as a loading control. (e) Levels of MYC and SOX2 transcripts associated with Ago2 upon miR-145 sponge expression. Data are depicted as mean ± SD of triplicates.

Figure 3. Strategy to ligate miR-145 sponge oligo duplexes into the vector.

MBS, miRNA-binding sites.