Selection of Allosteric DNAzymes for Theophylline Sensing by Expression-SELEX

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Aptamers, DNA or RNA molecules, bind ligands but require expression platforms for signal transduction.
• DNA aptamers offer stability and cost-effectiveness but face similar transduction challenges.
• Expression-SELEX integrates DNAzyme expression platforms into selection for direct signal generation.

Purpose of the Study:

• To explore the broad applicability of Expression-SELEX for discovering diverse ligand-dependent DNAzymes.
• To identify theophylline-responsive allosteric DNAzymes as replacements for RNA aptamers.
• To develop functional biosensors for clinical detection using these DNAzymes.

Main Methods:

• Applied Expression-SELEX to isolate theophylline-responsive DNAzymes.
• Screened 12,880 unique candidates for ligand-dependent cleavage activity.
• Incorporated fluorophore-quencher pairs for enhanced liquid-phase biosensor assays.

Main Results:

• Identified top 10 candidates with significant theophylline-dependent cleavage and specificity against caffeine.
• Demonstrated effective discrimination between theophylline and caffeine.
• Developed optimized biosensors functional in 1%-5% urine, detectable via PAGE gel and liquid-phase assays.

Conclusions:

• Expression-SELEX is a versatile method for rapidly generating ligand-responsive DNAzymes.
• Developed DNAzyme biosensors show potential as cost-effective diagnostic tools.
• These biosensors are suitable for clinical detection and biomedical applications.