Cooperative Mismatch Discrimination by PNA and DNAzyme Enables High-Fidelity Cleavage of Plasmid DNA

Peptide nucleic acid (PNA) and DNAzymes have recently been used to develop an artificial DNA nuclease system named PNA-assisted double-stranded DNA nicking by DNAzymes (PANDA) for genetic engineering. Interestingly, the PANDA system demonstrated a higher sequence fidelity than CRISPR/Cas9, with the ability to discriminate single-nucleotide mismatches. To evaluate the source of PANDA's sequence fidelity, we conducted kinetic experiments that separately examined the kinetics of PNA invasion and DNAzyme cleavage, each under rate-limiting conditions. Our results show that PNA serves as an initial mismatch "inspector," while DNAzyme adds complementary specificity during the cleavage process. Notably, PNA and DNAzyme recognize mismatches at opposite ends of their binding regions, enabling cooperative discrimination of mismatches across the entire target site, including regions that are typically difficult to distinguish by other methods. This dual recognition mechanism enhances PANDA's sequence fidelity, particularly in single-nucleotide mismatch discrimination. These findings establish PANDA as a promising molecular tool for precise, targeted DNA manipulation, offering a robust platform for applications that require stringent sequence specificity.