Topology-Gated λ Exonuclease Enables Amplification-Free Signal Boosting

Area of Science:

• Molecular Biology
• Biochemistry
• CRISPR Technology

Background:

• Amplification-free detection is crucial for CRISPR-based RNA diagnostics.
• Lambda exonuclease's enzymatic properties were not fully understood in cyclic reactions.

Purpose of the Study:

• To identify and characterize a novel topological property of lambda exonuclease for signal amplification.
• To develop an amplification-free CRISPR diagnostic system using this property.

Main Methods:

• Biochemical and structural analyses of lambda exonuclease.
• Design of topology-gated dumbbell probes for CRISPR/Cas13.
• Development and application of the Topo-CRISPR system for RNA detection.

Main Results:

• Identified a self-sustained cyclic cleavage-reforming process in lambda exonuclease dependent on substrate topology.
• Developed Topo-CRISPR, achieving attomolar sensitivity in 25 minutes without preamplification.
• Demonstrated robust detection of enterovirus RNA, miR-21, and ciR1445 in clinical samples, comparable to RT-qPCR.

Conclusions:

• Lambda exonuclease acts as a topology-driven signal amplifier through a cyclic reaction.
• Topo-CRISPR offers a novel, amplification-free framework for ultrasensitive molecular sensing.
• The system is adaptable for detecting both nucleic acid and non-nucleic acid targets.