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

ABSTRACT: 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 cooper

SUBMITTER: Kong L 

PROVIDER: S-EPMC12923505 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature