Construction of Ligand-Responsive MicroRNAs that Operate Through Inhibition of Drosha Processing
MicroRNAs (miRNAs) offer powerful tools for targeted gene silencing in almost all eukaryotes. These tools have received considerable attention for their utility in both fundamental genetic studies and as therapeutic agents. Rendering individual microRNAs responsive to endogenous or exogenously applied molecules (or ligands) can improve the stringency of silencing and can mediate autonomous control. This chapter describes the construction of ligand-responsive miRNAs that undergo reduced processing and subsequent gene silencing when bound by the recognized ligand. Following a simple set of rules, the engineered microRNAs can be readily modified to target different sequences and to bind different ligands. Individual miRNAs also can be incorporated into the same transcript for tunable, multi-gene silencing.
Key wordsAptamers Drosha Gene regulation Mammalian cells MicroRNA Riboswitch RNA interference
This work was supported by North Carolina State University (start-up funds to CLB), the National Science Foundation (fellowship to RJB), the National Institutes of Health (RC1GM091298), and the Defense Advanced Research Projects Agency (HR0011-11-2-0002).