DNA-Based Microrna Sensors

Studies over the past decade have demonstrated that differential expression of microRNA (miRNA) plays an important role in the pathogenesis of various cancers. Studies have also shown that miRNA affects cancer cells' response to treatment. Here we designed and built DNA-based sensors to study endogenous miRNA in mammalian cells. We constructed both direct and indirect sensors which report miRNA concentrations by producing a fluorescent protein. High miRNA concentration results in low fluorescence signal for the direct sensors and high fluorescence signal for the indirect sensors. We optimized our sensors in transient transfection experiments in HEK293 and TET-ON cells using fluorescent microscopy and flow cytometry measurements. By titrating synthetic miRNAs and using sensors for endogenous miRNA (hsa-miR-16, hsa-miR-17, hsa-miR-10b, and hsa-miR-146a), we show that the sensors report with high precision the miRNA concentrations. We also report time-lapse fluorescent microscopy experiments using inducible synthetic miRNAs. These results show that our sensors can reliably track miRNA concentrations. Finally, we use time-lapse data to build a quantitative model of our sensors and their response to miRNA. Using this model we created a map that relates binding affinity vs. miRNA abundance vs. sensor output. In conclusion, we present DNA-based miRNA sensors which reliably report and track miRNA concentrations.