Mismatch-sensitive DNA hybridization controlled by inchworm-type peptide nucleic acid–PEG conjugates

Abstract

The duplex-forming behavior of an inchworm-type PNA–PEG conjugate (i-PPc), engineered for the selective recognition of point mutations in DNA, was assessed through thermodynamic analysis employing UV melting curves and circular dichroism spectroscopy. The i-PPc demonstrated the ability to form stable duplexes exclusively with fully complementary DNA sequences, while no hybridization with single-base mismatched sequences. This binary on/off hybridization behavior was maintained even under physiologically relevant conditions (37 °C), thereby illustrating the exceptional point mutation discrimination capability of i-PPc. The behavior observed can be ascribed to the distinctive structure of i-PPc, wherein two PNA segments, possessing intrinsically different duplex-forming stabilities—high and low—are covalently linked via a flexible PEG linker. The high-stability PNA segment functions as the primary recognition domain for point mutations, thereby defining the sequence specificity of duplex formation. Conversely, the low-stability segment contributes cooperatively to the overall duplex stabilization only when the high-stability segment successfully hybridizes with the target DNA. This cooperative mechanism underlies the sequence-selective duplex formation of i-PPc, highlighting its potential as a highly specific probe for DNA mutation diagnostics. These findings indicate that i-PPc represents a promising platform for point mutation detection and nucleic acid-based molecular diagnostics grounded in DNA hybridization under physiological conditions.