Study of PNA–DNA hybridization by AFM-based single-molecule force spectroscopy

Abstract

Abstract The PNA–DNA interaction was probed with single-molecule AFM force measurements. The PNA sequence with only six thymine bases, p(T)6, could form stable hybrids with the complementary DNA sequence of d(A)6. Rupture of the p(T)6–d(A)6 hybrids required forces of around 148 pN, which was larger than the forces to unbind short DNA duplex that were usually below 100 pN. Fitting into the rupture force–loading rate correlation produced kinetic parameters that describe the PNA–DNA interaction, which were the thermal force scale (fβ) to be 17.5 pN, the distance to the energy barrier for dissociation (Δx) to be 0.23 nm, and the dissociation rate constant at zero force (koff) to be 2.5 s−1, respectively. The results highlighted the stronger binding affinity between PNA and DNA than between DNA and DNA. And by changing the DNA sequence to d(AAGAAA) with a single-base mismatch, no specific rupture events were observed, confirming the high sensitivity of PNA–DNA hybridization to base mismatch. This single-molecule force study probes into the energetic and kinetic aspects of PNA–DNA interactions that are not accessible by conventional bulk methods, leading to deeper understanding of PNA–DNA interactions.