Kinetic measurements of the fluorescent protein synthesis facilitate determination of the miRNA activity.

Oncogenesis is inevitably associated with microRNA expression deregulation. Thus, development of both miRNA targeting substances or small RNA for ant-cancer therapy have been reported. Specifically, repression of the miR-16-1-3p and miR-16-2-3p activities play pivotal roles in osteosarcoma and many other cancers. The majority of miRNA sensors use protein degradation to measure miRNA activities. Here we report miRNA sensors that use fluorescent protein synthesis rather than degradation to measure miRNA activity. Specifically, miR-16-1-3p and miR-16-2-3p sensors consist of the bidirectional tet-On system driving the expression of the Katusha2S protein that is regulated by the RNA interference and GFP as a reference. These sensors specifically detect mir16-1-3p and mir16-2-3p small RNA mimics in the osteosarcoma cell line after doxycycline induction. Kinetic measurements of the reporter responses to the miRNA mimics revealed that pre-induced sensors reach significant differences from the control faster, within 2h than the sensors that were induced after mimic transfection. Thus, kinetic measurements of the fluorescent protein synthesis during doxycycline induction of the tet-On system are feasible for determination of the small RNA activity.