CRISPR/Cas13 system-based entropy-driven DNAzyme switch powered DNA walking system for sensitive and direct rotavirus...

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• DNA walker strategies face challenges in design complexity, sequence dependence, and amplification efficiency.
• Existing methods often struggle to balance specificity and sensitivity in molecular detection.

Purpose of the Study:

• To develop an innovative DNA probe (LW probe) integrating locked DNAzyme and entropy-driven amplification (EDA) with a DNAzyme-powered DNA walker.
• To enhance the sensitivity and specificity of rotavirus detection by synergizing EDA and DNA walker mechanisms.

Main Methods:

• Designed a double-stranded "LW probe" that remains inactive until target rotavirus sequences are present.
• Utilized Cas13a/crRNA trans-cleavage activity to activate the LW probe, initiating EDA and releasing a DNAzyme segment.
• Employed the released DNAzyme to cleave substrate probes on gold nanoparticles, generating fluorescence signals for detection.

Main Results:

• The integrated system demonstrated high specificity, capable of discriminating single-base mismatched sequences.
• Achieved remarkable sensitivity with a low detection limit of 2.7 fM for rotavirus.
• The proposed method outperformed or matched previous DNA walker-based systems in sensitivity and stability.

Conclusions:

• The synergistic combination of EDA and DNAzyme-driven DNA walkers offers a powerful platform for sensitive and specific molecular detection.
• This approach provides a promising tool for the detection of low-abundance targets like rotavirus, with applications in disease diagnosis and biomedical research.