A TtAgo-Driven Autocatalytic Circuit with Thermal-Enhanced Kinetics for One-Pot Nucleic Acid Detection

Area of Science:

• Biochemistry
• Molecular Biology
• Diagnostics

Background:

• Catalytic DNA circuits show promise for nucleic acid diagnostics but face challenges with slow kinetics and non-universal one-pot methods.
• Existing methods often struggle with efficiency and broad applicability across diverse sample types.

Purpose of the Study:

• To develop a universal, one-pot catalytic DNA circuit for sensitive and rapid detection of DNA and RNA biomarkers.
• To enhance reaction kinetics and amplification efficiency for improved diagnostic capabilities.

Main Methods:

• Developed TACTIC (Thermus thermophilus Argonaute protein-driven autocatalytic circuit) utilizing TtAgo protein for heat-activated cleavage and amplification.
• Constructed an autocatalytic positive-feedback circuit with explosive regeneration of target mimics.
• Integrated TACTIC with machine learning for biomarker profiling and cancer diagnostics.

Main Results:

• Achieved a 381% increase in amplification efficiency with attomolar (aM) detection sensitivity.
• Enabled rapid, one-pot detection of bacterial DNA, mutant mRNA, and four extracellular vesicle-derived miRNAs (EV miRNAs) in under 30 minutes.
• Established machine learning-driven diagnostic and staging models for breast cancer using EV miRNA expression patterns.

Conclusions:

• TACTIC provides a universal and efficient platform for sensitive nucleic acid detection in clinical samples.
• The developed circuit accelerates reaction kinetics and improves amplification efficiency, overcoming limitations of previous methods.
• This advancement offers new possibilities for higher-order catalytic circuits and expands the toolkit for accurate nucleic acid biomarker detection and disease diagnosis.