Abstract
Alternative splicing is a fundamental mechanism that enhances proteomic diversity and modulates gene function, with its dysregulation being a hallmark of numerous diseases. Despite its biological significance, the real-time monitoring of spliced mRNA isoforms in living cells remains challenging due to limited specificity and sensitivity in existing methods. Herein, we present a Stringent dUPlex-activated Error-Robust (SUPER) platform, an in situ, split-DNAzyme-based system enabling precise imaging of mRNA splicing events in live cells. SUPER employs an identical parental DNAzyme reassembled via isoform-specific intron-exon junctions, providing high-fidelity discrimination of closely related splicing variants. Its dual-site-activated fluorescence design ensures error-robust, background-minimized imaging with spatial colocalization as an intrinsic validation mechanism. Beyond dynamic isoform profiling, the programmable nature of SUPER enables its conversion into a spatially confined catalytic antenna, locally activating therapeutic aptamers without affecting off-target transcripts. This approach further allows for real-time tracking of variant integrity and decay by monitoring subtle changes in probe colocalization. Our platform offers a powerful tool for dissecting splicing mechanisms and holds promise for therapeutic intervention in splicing-associated diseases by enabling isoform-selective gene regulation while mitigating oligonucleotide toxicity.