Quantitative Analysis of the Nanopore Translocation Dynamics of Simple Structured Polynucleotides

Abstract

Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using α-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through α-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding.

Diffusive dynamics of DNA unzipping in a nanopore

“…This suggests that the first maximum, related to the unzipping, has impact on the total unzipping/translocation time. This is consistent with Schink et al, [21] where the unzipping times were strongly correlated with the hairpin structure. We conclude that the fine-smoothed PMF better represents the underlying molecular mechanism of unzipping.…”

“…Two energy maxima were found, one of them corresponding to the unzipping and the other to the translocation barriers caused by friction and nonbonded interactions with the pore walls. Thus, our CG model is more detailed than the one by Schink et al, [21] because the interactions with the pore walls are modeled explicitly and there is no fitting parameter. Dependence of the diffusion coefficient and effective charge on the DNA position along the pore axis has also been determined.…”

“…Computer simulations help to understand the detailed mechanism of translocation or unzipping, for example, interactions with the pore walls, analyte conformation, and unzipping sequence. [19,20] The unzipping kinetics of different hairpins were studied by Schink et al [21] and Viasnoff et al, [22] who described the unzipping as motion on an effective potential surface. However, the electrolyte and the pore walls were not treated explicitly.…”

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

“…Moving the ssDNA within the αHL pore is associated with a free energy of translocation, F(m) ( Figure 4C). The free energy is assumed to contain only one term 63 caused by the interaction with the applied potential. As a consequence of the small diameter of αHL's inner 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 constriction, the potential is assumed to drop linearly and completely over this region ( Figure 4B).…”

Topology sorting and characterization of folded polymers using nano-pores

“…We also asked if translocation time and chain topology are correlated. This technology has been subject to intense research for simple-structured polynucleotides [38]; however, the current study is the first to use nano-pores to systematically measure contact arrangements of folded molecules [13] (Fig. 1).…”