Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals

In this study a natural miRNA precursor was modified by replacing the natural terminal loop with a regulatory sensor domain, the TetR-binding aptamer to enable the ligand-dependent control of the processing of miRNA precursor to mature miRNAs. The TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. The design proved to be widely applicable, allowing to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage could be inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence could be shown. Thus, this work describes the development of a RNA-based tool that is capable of robustly and reversibly controlling miRNA abundance