Mechanism of DNA multimerization caused by strand-displacement DNA polymerases

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Bst DNA polymerase is crucial for isothermal amplification reactions.
• A side reaction, multimerization (MM), can occur under specific conditions.
• MM compromises the accuracy and reliability of DNA/RNA diagnostic tests.

Purpose of the Study:

• To elucidate the mechanism of multimerization (MM) in strand-displacement DNA polymerases.
• To identify factors influencing the efficiency of MM.
• To provide insights for improving the reliability of molecular diagnostics.

Main Methods:

• Detailed mechanistic study of multimerization.
• Utilized molecular modeling with specific steric and thermodynamic characteristics.
• Investigated the role of DNA breathing, enzyme envelopment, and 3'-end convergence.

Main Results:

• MM involves enzyme envelopment by synthesized DNA, 3'-end convergence, and DNA slippage.
• Initiation of MM is rare, but amplified trigger structures lead to nonspecific amplicons.
• Highest MM efficiency observed with DNA templates and conditions promoting DNA breathing, enzyme envelopment, and 3'-end convergence.

Conclusions:

• The proposed mechanism explains multimerization in Bst DNA polymerase reactions.
• Understanding MM is critical for enhancing the specificity and reliability of isothermal amplification-based diagnostics.
• Optimizing reaction conditions to minimize DNA breathing and enzyme-DNA interactions can reduce MM.