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. 2016 Feb 16;6:20979. doi: 10.1038/srep20979

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Copyright © 2016, Macmillan Publishers Limited

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(A) The artificial intron is shown flanked by a splice donor (DS) and an acceptor site (AS) and contains a branch-point domain (BrP), a poly-pyrimidine tract (PPT) and pre-miRNA. The miRNA sponge or artificial miRNA sequence is inserted inside the intron, located between the 5-splice site and the BrP. The intronic miRNA is co-transcribed within a precursor messenger RNA (pre-mRNA) of NS1 driven by the pNS1 promoter and cleaved out of the pre-mRNA by RNA splicing. Although the exons are ligated to form a mature messenger RNA (mRNA) for NS1 protein synthesis, the spliced intron with the pre-miRNA is further processed into mature miRNA by Dicer. (B) The strategy to generate the anti-let-7 and anti-miR-210 constructs. Alignment of anti-let-7 and anti-miR-210 sequences with aal-let-7 and aal-miR-210, respectively. Complete matching of the seed regions with the anti-miRNA sequence and tail regions are shown. Both let-7 and miR-210 sponges contain three repeat antisense constructs (red letters) that can bind to aal-let-7 and aal-miR-210, respectively. (C) Sequences and predicted precursor structures for the miRNA-based artificial miRNAs used in this study. The mature artificial miRNAs are shown in red, and their related target mRNA sequences are in blue. The target sequences locations are shown below.