Probing of PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Abstract

Peptide nucleic acids (PNA) are synthetic polymers the neutral peptide backbone of which provides elevated stability to PNA-PNA and hybrid PNA-DNA duplex. It was demonstrated recently (J.Org.Chem.2011, 76, 5614-5627) that additional modification of the backbone such as incorporation of diethylene glycol (miniPEG) further increases the duplex thermal stability, so the hybrid duplex with 10 miniPEG units (γ-PNA) has the melting temperature 23°C higher compared to the regular PNA-DNA duplex. Here we applied AFM force spectroscopy to probe the strength of the γ-PNA-DNA duplex (5′GAGTAG GTAG-3’) containing ten miniPEG-modified units. Single-stranded PNA and DNA oligonucleotides containing terminal thiol groups were immobilized on amino-functionalized AFM tip and mica substrate, respectively via bifunctional PEG tethers and the interaction between the DNA and γ-PNA polymers was analyzed by multiple approach retraction cycles over various locations on the mica substrate. Such single molecule probing experiments produced force curves with well-defined rupture events corresponding to the dissociation of the duplex formed during the approach step. The experiments were performed at various pulling rates (300-3000 nm/sec) enabled us to characterize the hybrid duplex stability using dynamic force spectroscopy (DFS) approach. The DFS measurements yielded rupture forces varying in the range 60-70 pN. The data analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 10−9 sec−1 and rupture distance x-1∼ 1.7 nm. Similar published DFS measurements of DNA duplex are characterized by koff with ∼ 108 times less values, which is in line with elevated stabilities of the γ-PNA-DNA duplexes compared with the DNA duplexes.The work was supported by NIH grants 5P01 GM091743-03 and 5R01 GM096039-04 to YLL. γ-PNA was synthesized at PNA innovation.