A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers

Abstract

MicroRNAs (miRNAs) are crucial regulators of gene expression at the post-transcriptional level, offering valuable insights into disease mechanisms and prospects for targeted therapeutic interventions. Herein, we present a class of miRNA-induced light-up RNA sensors (miLS) that are founded on the toehold mediated principle and employ the fluorogenic RNA aptamers Pepper and Squash as imaging modules. By incorporating a sensor switch to disrupt the stabilizing stem of these aptamers, our design offers enhanced flexibility and convertibility for different target miRNAs and aptamers. These sensors detect multiple miRNA targets (miR-21 and miR-122) with detection limits of 0.48 and 0.2 nM, respectively, while achieving a robust signal-to-noise ratio of up to 44 times. Capitalizing on the distinct fluorescence imaging channels afforded by Pepper-HBC620 (red) and Squash-DFHBI-1T (green), we establish an orthogonal miRNA activation imaging platform, enabling the simultaneous visualization of different intracellular miRNAs in living cells. Our dual-color orthogonal miLS imaging platform provides a powerful tool for sequence-specific miRNA imaging in different cells, opening up new avenues for studying the intricate functions of RNA in living cells.