DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering

Abstract

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanoscale pores is under intense investigation for single-molecule DNA sequencing and analysis. Here we show that the precise patterning of the entry and the middle section of the ClyA nanopore with positive charges are crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. The dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double stranded DNA is diffusion-limited while the capture of single stranded DNA is reaction-limited. KeywordsClyA nanopore; DNA rotaxane; protein engineering; DNA sequencing; bacteriophage DNA sliding The translocation of DNA across specialized proteins is an important biological process. Bacteria use secretin channels in their pili to uptake or transfer DNA, while viruses such as phages use their portal proteins to either pack genomic DNA into the viral capsid or to eject it into target cells. In the three domains of life, β-clamp proteins form a toroid structure that encircles and slides along DNA to aid the function of DNA processing enzymes. Portal proteins in all known bacteriophages (λ, P22, T4, T3, T7, SPP1, P2 and ϕ29)1 and many components of secretin channels2 form dodecameric rings. The available crystal structures of portal proteins (ϕ29,3 SPP1,4 P225 and T46) have revealed the presence of a central channel through which dsDNA must both enter during packaging and exit during injection. While the interior channel lining is mostly negatively charged, a few positively charged amino acids, arranged as rings along the length of the pore, can also be found. A similar

Multi-compartment encapsulation of communicating droplets and droplet networks in hydrogel as a model for artificial cells

“…Owing to its evolving importance as a nanopore sensor for proteins48 and DNA49, and the possibility to engineer its size and properties50, we chose ClyA as the model nanopore to test our system. The encapsulated bilayer could be functionalized with ClyA pores by incorporating the oligomeric pore inside the aqueous droplet (Fig.…”

Modeling of Ion and Water Transport in the Biological Nanopore ClyA

“…Experimentally, it is often determined by placing the pore in a salt gradient (i.e., different salt concentrations in the cis and trans reservoirs) and measuring the potential at which the nanopore current is zero (reversal potential, V r ). 96,101 The Goldman-Hodkin-Katz (GHK) equation can then be used to convert V r into the permeability ratio P Na + = G Na + /G Cl − . Here, we represent the ClyA's ion selectivity (Figs.…”

“…Using the reversal potential method, Franceschini et al 101 found ClyA's ion selectivity to be t Na + = 0.66 (P Na + = 1.9). In our ePNP-NS simultation, this corresponds the selectivity at c s = 0.5 M (at V b = 0 mV), a value that lies in between the cis (1 M, t Na + = 0.57, concentrations.…”

“…and the solvent in terms of density, viscosity92 and relative permittivity 93 . To validate our new framework, we applied it directly to a 2D-axisymmetric model of Cytolysin A (ClyA), a large protein nanopore that typically contains 12 subunits 95 or more96 and has been extensively used in experimental studies of both proteins27,30,[96][97][98][99][100] and DNA 17,101. .…”

DNA capture into the ClyA nanopore: diffusion-limited versus reaction-limited processes

“…[7]. In this paper we analyze the capture of both ssDNA and dsDNA by Cytolysin A (ClyA), a biological nanopore which has been recently employed both for nucleic acid and protein analysis [3,9,4,11]. In experiments, DNA molecules are initially placed in the cis-side of the membrane.…”