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

## TL;DR

A new DNAzyme system called SUPER allows real-time tracking and targeted intervention of alternative splicing in live cells, offering potential for studying and treating splicing-related diseases.

## Contribution

The SUPER platform introduces a split-DNAzyme system for high-fidelity, real-time imaging and regulation of spliced mRNA isoforms in live cells.

## Key findings

- SUPER enables precise imaging of alternative splicing events like Bcl-xL/Bcl-xS in live cells with minimal background.
- The system allows isoform-selective gene regulation by activating therapeutic aptamers without off-target effects.
- SUPER tracks variant integrity and decay through real-time monitoring of probe colocalization changes.

## 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.

A Stringent dUPlex‐activated Error Robust (SUPER) DNAzyme system enables real‐time imaging of alternative mRNA splicing (e.g., Bcl‐xL/Bcl‐xS) in living cells via target‐triggered split‐DNAzyme reassembly and dual‐color fluorescence. It also achieves mRNA‐selective knockdown through DNAzyme‐based gene regulation, serving as a versatile tool for splicing dynamics research and RNA‐guided precision medicine.