Probing of miniPEGγ-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 PNA-DNA hybrid duplex. 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. (2011) Journal of Organic Chemistry 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) 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 (γMPPNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that γMPPNA-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, koff ∼ 0.030 ± 0.01 sec-1 for γMPPNA-DNA hybrid duplex vs. 0.375 ± 0.18 sec-1 for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γMPPNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γMPPNA 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.

Calcium increases titin N2A binding to F-actin and regulated thin filaments

“…N2A constructs were attached to AFM tips (Fig. 1 ) using standard procedures for thiol-maleimide crosslinking 26 , 27 . Silicon nitride AFM tips (MSNL, Bruker Inc., k = 30–40 pN/nm) were amino (APTES) functionalized following a previously published protocol 28 .…”

“…Measurements in the absence of Ca 2+ were performed by adding 700 µM EGTA (Sigma Aldrich) to the probing buffer to eliminate free Ca 2+ . During experiments, the AFM tip was brought in contact with the surface until a triggering force of 100 pN was applied and held for 0.5 s, allowing the N2A construct to interact with F-actin on the surface 26 . The tip was then retracted by 300 nm at constant, physiologically relevant velocities between 300–2000 nm/sec 17 .…”

“…For each pulling velocity, ~100 force curves showing N2A-actin interactions, identified by polymer-like extension of the N2A constructs, were collected as described previously 26 , 27 . Each force curve was fit with the extensible worm-like chain (WLC) model to estimate contour length (L c ), persistence length (L p ), and rupture force (F), with L c and L p as free parameters.…”

Molecular Force Sensors: From Fundamental Concepts toward Applications in Cell Biology

“…The rupture force of dsDNA‐based force sensors, for example, plateaus at a force of ≈65 pN where the DNA molecule undergoes an unfolding transition (B–S transition) . Increasing the rupture force above this value is not possible when using the four natural bases A, T, C, and G, but may be achieved when using a peptide nucleic acid backbone or when substituting individual pyrimidine bases with their propynyl derivatives . All currently used intracellular MFSs employ fluorescent proteins for the FRET readout.…”

In Situ Atomic Force Microscopy Studies on Nucleation and Self-Assembly of Biogenic and Bio-Inspired Materials

“…For example, by (nano) indenting or stretching materials, their mechanical properties, such as adhesion and elasticity, can be precisely measured and mapped on the surface [100][101][102][103][104]. AFMbased nanoindentation and force spectroscopy has been applied to study many biological and bioinspired systems, including bacterial adhesive material [105], viruses [106][107][108][109][110][111][112][113][114][115][116][117][118], proteins [119][120][121][122][123][124][125], nucleic acids [126][127][128][129][130], cells [131][132][133], and plant cell walls [134]. The mechanical properties of the materials can be connected to the processes of material nucleation and self-assembly [100][101][102][103].…”