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Despite its biological significance, the real\u2010time 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\u2010activated Error\u2010Robust (SUPER) platform, an in situ, split\u2010DNAzyme\u2010based system enabling precise imaging of mRNA splicing events in live cells. SUPER employs an identical parental DNAzyme reassembled via isoform\u2010specific intron\u2010exon junctions, providing high\u2010fidelity discrimination of closely related splicing variants. Its dual\u2010site\u2010activated fluorescence design ensures error\u2010robust, background\u2010minimized 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\u2010target transcripts. This approach further allows for real\u2010time 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\u2010associated diseases by enabling isoform\u2010selective gene regulation while mitigating oligonucleotide toxicity.<\/jats:p>","DOI":"10.1002\/advs.202517895","type":"journal-article","created":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T10:51:56Z","timestamp":1768992716000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Modular DNAzyme for Precise Visualization and Intervention of Alternative Splicing Isoforms in Live Cells"],"prefix":"10.1002","volume":"13","author":[{"given":"Mengru","family":"Lin","sequence":"first","affiliation":[{"name":"College of Health Science and Engineering Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine Hubei University  Wuhan Hubei P. R. 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