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

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Alternative splicing generates proteomic diversity but its dysregulation is linked to diseases.
• Existing methods for monitoring spliced mRNA isoforms in real-time lack specificity and sensitivity.
• Precise live-cell imaging of mRNA splicing events is crucial for understanding gene function and disease mechanisms.

Purpose of the Study:

• To develop a novel platform for precise, real-time imaging of mRNA splicing events in live cells.
• To overcome the limitations of existing methods in specificity and sensitivity for monitoring splicing variants.
• To explore the potential of this platform for therapeutic interventions in splicing-associated diseases.

Main Methods:

• Developed the Stringent dUPlex-activated Error-Robust (SUPER) platform, a split-DNAzyme-based system.
• Utilized isoform-specific intron-exon junctions for high-fidelity discrimination of splicing variants.
• Employed a dual-site-activated fluorescence design with spatial colocalization for error-robust imaging.

Main Results:

• Achieved precise imaging of mRNA splicing events in live cells with high specificity and sensitivity.
• Demonstrated the platform's ability to dynamically profile mRNA isoforms and track variant integrity.
• Showcased the programmable nature of SUPER for localized therapeutic aptamer activation and monitoring.

Conclusions:

• The SUPER platform provides a powerful tool for dissecting splicing mechanisms in real-time.
• This technology enables isoform-selective gene regulation for potential therapeutic applications in splicing-associated diseases.
• SUPER offers a promising approach to mitigate oligonucleotide toxicity while enabling targeted gene regulation.