A Modular DNAzyme for Precise Visualization and Intervention of Alternative Splicing Isoforms in Live Cells

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.