A Novel Synthetic Toehold Switch for MicroRNA Detection in Mammalian Cells.

Abstract

MicroRNAs (miRNA or miR) are short noncoding RNA of about 21-23 nucleotides that play critical roles in multiple aspects of biological processes by mediating translational repression through targeting messenger RNA (mRNA). Conventional methods for miRNA detection, including RT-PCR and Northern blot, are limited due to the requirement of cell disruption. Here, we developed a novel synthetic toehold switch, inspired by the toehold switches developed for bacterial systems, to detect endogenous and exogenously expressed miRNAs in mammalian cells, including HEK 293, HeLa, and MDA-MB-231 cells. Transforming growth factor β-induced miR-155 expression in MDA-MB-231 cells could be detected by the synthetic toehold switch. The experimental results showed the dynamic range of current design of toehold switch is about two. Furthermore, we tested multiplex detection of miR-155 and miR-21 in HEK 293 cells by using miR-155 and miR-21 toehold switches. These toehold switches provide a modest level of orthogonality and could be optimized to achieve a better dynamic range. Our experimental results demonstrate the capability of miRNA toehold switch for detecting and visualizing miRNA expression in mammalian cells, which may potentially lead to new therapeutic or diagnostic applications.