Lead-start isothermal polymerase amplification controlled by DNAzymatic switches

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Standard polymerase chain reaction (PCR) uses heat activation for specificity, but this is incompatible with isothermal amplification.
• Isothermal amplification, suitable for point-of-care (PoC) tests, traditionally lacks precise control, limiting its quantitative capabilities.
• Existing methods struggle with on-demand activation and deactivation of DNA polymerases at ambient temperatures.

Purpose of the Study:

• To develop a novel isothermal amplification method with on-demand activation and deactivation at room temperature.
• To enable quantitative detection using isothermal amplification for point-of-care applications.
• To achieve high specificity and sensitivity in DNA detection using a lead ion-activated system.

Main Methods:

• Developed a lead-start isothermal amplification technique utilizing lead ion (Pb²⁺) for DNA polymerase activation.
• Correlated DNA polymerase inhibition by TQ30 aptamer with Pb²⁺-responsive GR5 DNAzyme strand cleavage.
• Investigated Pb²⁺ as an initiator or terminator of isothermal DNA amplification based on interconnector type.

Main Results:

• Achieved highly Pb²⁺-specific activation of DNA polymerase, increasing enzymatic activity over 25-fold.
• Demonstrated quantitative analysis and comparison of multiple reactions due to simultaneous start and end times.
• Successfully quantified human papillomavirus type 16 and SARS-CoV-2 genes in clinical samples (serum, saliva) with a limit of detection as low as 0.1 nM.

Conclusions:

• Lead-start isothermal amplification offers precise, quantitative, and specific DNA detection at room temperature.
• The developed method is highly applicable for field-based point-of-care tests without requiring purification.
• This innovation advances isothermal amplification for sensitive and on-demand molecular diagnostics.