rEXPAR: An Isothermal Amplification Scheme That Is Robust to Autocatalytic Parasites

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biochemistry

Background:

• Self-replicating DNA parasites can form in isothermal amplification reactions like EXPAR.
• These parasites interfere with template-based DNA amplification and molecular programming.
• Parasite formation is a common issue in laboratory settings, hindering experimental reproducibility.

Purpose of the Study:

• To develop a strategy to prevent the formation of DNA parasites in EXPAR reactions.
• To enhance the robustness of EXPAR and related molecular programming approaches.
• To maintain compatibility with existing molecular programming toolboxes like PEN DNA.

Main Methods:

• Utilized a nicking enzyme, Nb.BssSI, with a three-letter recognition site (C, G, T) on the top strand.
• Modified the sequence design of EXPAR templates to prevent parasite formation.
• Removed dATP from the reaction solution.
• Engineered a parasite-proof bistable reaction network using the PEN DNA toolbox.

Main Results:

• Successfully prevented parasite formation by strategic nicking enzyme selection and template design.
• Demonstrated that removing dATP from the solution, in conjunction with the chosen enzyme, eliminates parasites.
• Confirmed the compatibility of the parasite-proof EXPAR system with PEN DNA programs.
• Successfully engineered a bistable reaction network resistant to parasite contamination.

Conclusions:

• Employing nicking enzymes with asymmetric recognition sites offers a robust method to eliminate DNA parasites in EXPAR.
• This approach significantly improves the reliability of isothermal amplification and molecular programming.
• The developed method is compatible with advanced molecular programming applications, paving the way for more stable synthetic biological systems.