Precision discrimination of single nucleotide polymorphisms via chemically engineered PNA-mediated padlock probe...

Highly sensitive and specific detection of single nucleotide polymorphisms (SNPs) in genomic DNA is of great significance in disease diagnosis and personalized medicine. Here, we developed a fluorescent method for highly specific detection of SNPs in mixed-sequence KRAS double-stranded DNA (dsDNA) under physiological conditions by combining peptide nucleic acid (PNA)-directed padlock probe assembly with rolling circle amplification (RCA). The core of this approach involves the selective opening of dsDNA using an acridine-linked cyclopentane PNA (Acr-PNA_cyp4), followed by hybridization of a padlock probe and subsequent RCA. The resulting RCA products are then detected through hybridization with PNA molecular beacon (PNA MB) probes. Making use of the high specificity of PNA-mediated strand invasion (PMSI) and ligation reactions in conjunction with the robust amplification capability of RCA technology, the method achieves precise detection of target dsDNA at concentrations as low as 0.66 fM, enabling effective discrimination among different mutation types. Furthermore, the approach has been successfully applied to the detection of KRAS mutations in colorectal cancer cells, confirming its practical utility in biological samples. This method does not require sophisticated thermal cycling equipment, highlighting its potential for use in clinical settings and resource-limited environments for the detection of low-abundance disease-related mutations.