Development of Proximity-Activated Programmable Multicomponent Nucleic Acid Enzymes for Simultaneous Visualization of Multiple mRNA Splicing Variants in Living Cells

RNA splicing is a key regulatory process of gene expression that can increase the transcriptome complexity. Simultaneous monitoring of multiple splicing variants in living cells is critical for gaining new insight into cell development. Herein, we demonstrate the development of proximity-activated, programmable multicomponent nucleic acid enzymes (MNAzymes) for the simultaneous visualization of multiple RNA splicing variants (i.e., BRCA1 WT and BRCA1 Δ11q) in living cells. The presence of BRCA1 WT and BRCA1 Δ11q can specifically bring their corresponding partzymes into the proximity of each other to form two active MNAzyme motifs. Subsequently, the active sites of reporter probes 1 and 2 are cyclically cleaved by two activated MNAzyme motifs, respectively, to release abundant Cy3 and Cy5 fluorescent molecules, generating enhanced fluorescence signals for the simultaneous detection of BRCA1 WT and BRCA1 Δ11q <i>in vitro</i> and <i>in vivo</i>. Notably, this assay can be simply and isothermally conducted in a one-step format without the necessity for unstable protein enzymes, precise temperature control, and complex operation procedures. This method can sensitively detect 2.46 fM BRCA1 WT and 2.77 fM BRCA1 Δ11q and accurately distinguish breast cancer patients from healthy individuals by measuring target BRCA1 splicing variants from the tissue samples. Moreover, this method can real-time image BRCA1 splicing variants in living cells and can be extended to detect other cellular target RNAs (e.g., miRNAs, piRNAs, lncRNAs, and circRNAs) by simply changing the sequences of substrate arms, holding promising applications in clinical diagnosis and precise therapy.