Long Lifetime of Hydrogen-Bonded DNA Basepairs by Force Spectroscopy

Abstract

Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ~2 s for two-hydrogen bonds (A-T) and ~4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements.

Chemical recognition and binding kinetics in a functionalized tunnel junction

“…This is in agreement with our measurements of the lifetime of similar complexes using atomic force microscopy, where we have observed that the zero-force lifetime of hydrogen-bonded complexes tethered in a nanogap is on the order of seconds. 12, 19 Thus, recognition-tunneling may also provide a tool for slowing DNA transport in a nanopore reader.…”

“…The off-rates are slow (corresponding to lifetimes of seconds) consistent with AFM measurements of the lifetimes of hydrogen-bonded complexes in a nanogap. 19, 12 This behavior has recently been explained as a consequence of the bond confinement in the gap. 21 The on-rates are fast, probably too fast to be measured with the techniques used here, but certainly consistent with DNA sequencing speeds of many tens of bases per second.…”

Resolving the molecular mechanism of cadherin catch bond formation

“…Similarly, AFM force spectroscopy experiments show that the lifetime of single base-pairs formed via either 2 or 3 H-bonds is B2 and 4 s, respectively 40 . Furthermore, these experiments show that H-bonds exhibit lifetimes larger than 0.1 s even in the presence of 40-60 pN load 40 .…”

Different roles of cadherins in the assembly and structural integrity of the desmosome complex

“…In order to eliminate hidden multiple unbinding events from visibly indistinguishable single unbinding events, we followed a protocol used for identifying the heterogeneity in chemical bonds or hidden multiple bonds as described previously (Fuhrmann et al, 2012). After eliminating all hidden multiple events, the forces were plotted as a histogram for each retracting velocity with bin width calculated according to a statistical method (Scott, 1979).…”