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

详细信息    查看全文

• 作者：Samrat Dutta ; Bruce A. Armitage ; Yuri L. Lyubchenko
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：March 15, 2016
• 年：2016
• 卷：55
• 期：10
• 页码：1523-1528
• 全文大小：335K
• ISSN：1520-4995

文摘

Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA–PNA and PNA–DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614−5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ^MPPNA–DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ^MPPNA–DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, k_off ≈ 0.030 ± 0.01 s^–1 for γ^MPPNA–DNA hybrid duplex vs 0.375 ± 0.18 s^–1 for the DNA–DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ^MPPNA–DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ^MPPNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.