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In a remote New Mexico dessert, Klaus Schulten met Amit Meller, who told him a story of two DNA hairpins. The first one, when threaded through the transmembrane pore of alpha-hemolysin blocks the ionic current to 12 pA and escapes from the pore about three times slower than the other one that blocks the current to only 9 pA. The amazing fact about these experiments was that the two DNA hairpins were identical in sequence. The only difference was in the global orientation of the single stranded part of the hairpin in the pore of alpha-hemolysin. Astonished by the outcome of their experiments, Amit Meller and Jérôme Mathé sought an explanation from the modelers (Klaus Schulten and Aleksei Aksimentiev). Cautious as the experimentalists are, they did not tell the modelers which of the DNA orientations produces the larger current blockade and which one escapes faster from the pore. Below we describe our quest for the solution of the puzzle. A formal report can be found here.

Deoxyribonucleic acid (DNA)

dsDNADouble stranded DNA
Double stranded DNA (dsDNA). In a living organism, a DNA molecule carriers genetic instructions for synthesis of all the proteins required for building new cells or sustaining the existing ones. DNA is usually drawn as a double helix, in which two complementary but disjoint strands intertwine with one another through a network of hydrogen bonds. DNA is a rather rigid molecule: at physiological conditions, DNA curves at the length scale of about 50 nm, which is 20 times the diameter of the double helix.

ssDNASingle stranded DNA
Single stranded DNA (ssDNA). For the sequence of DNA to be read, the double helix of DNA is split open, exposing single DNA strands to DNA binding proteins. Once bound to DNA, the proteins, in carrying out their functions, will crawl along the DNA strand in one of two directions, toward DNA's 3' or 5' end (see below). Single stranded DNA is much more flexible than double stranded DNA and often folds onto itself forming a complicated secondary structure.

DNA direction. Yes, a DNA strand has a direction. It is determined by the arrangement of the phosphate and deoxyribose sugar groups along the DNA backbone. One of the DNA ends terminates with the O3-H group (the 3' end), whereas the other one terminates with the O5'-H group (the 5' end). Note that there are N-1 phosphate groups in an N-nucleotide ssDNA. All sequences of DNA are usually written from 5' to 3' end. When forming a double helix, the complimentary DNA strands are oriented in opposite directions.

The alignment of the bases can indicate the global orientation of a DNA strand

Alignment of two
  poly(dA<sub>20</sub>) strands Alignment of two poly(dA20) strands that differ from each other only by their global orientations.

DNA strands are hard to destinguish in solution.

To reveal how the global orientation of a DNA strand determines its escape time from a nanopore, as well as the blockade of ionic current, we examined in atomic detail conformations of DNA in solution and inside alpha-hemolysin. Before running MD simulations, two identical poly(dA20) strands, different only by their global orientation, were aligned with each other, as shown in the right figure. The two strands appeared to have, overall, similar conformations, the only difference visible being the ordering of the backbone atoms: When moving along the strand from 5 to 3 end, a phosphate group follows the deoxyribose sugar ring, the C5 carbon of deoxyribose follows the phosphate. Reversing the order changes the orientation of deoxyribose in the backbone. Although this change is sufficient to disrupt the function of a dephosphorylative enzyme, when put into the perspective of the alpha-hemolysin structure, it is an unlikely reason for the observed directionality.
DNA permeates the alpha-hemolysin channel Normalized distributions of the angle between the base and the backbone of A, C, G, and T nucleotides in an unrestrained DNA strand. The average was taken over a 12.5-ns molecular dynamics trajectory.
First, the experiments showed no evidence of a chemical reaction between alpha-hemolysin and DNA; second, if one assumes that a DNA strand is mobile inside the alpha-hemolysine pore (and our MD simulations confirm this assumption), 12 sugars forming the backbone of a DNA fragment confined within the alpha-hemolysin pore should be able to sample the pore's interior regardless of the sugars' orientation.

Another possible explanation for the observed 3'- and 5'- directionality might be that an ensemble of DNA conformations, by itself, contains the information about the global orientation of a DNA strand. To determine whether this is the case, we computed the average angle between nucleotide bases and the backbone in a DNA strand submerged in water. Our results shown at the left indicate that for purine nucleotides (A and G) the most probable angle is ~88, whereas for pyrimidines (C and T) that angle is ~105. From this analysis, we conclude that local conformations can indeed indicate a global orientation preference of a DNA strand, although the conformational anisotropy in an unrestrained strand is small and depends on sequence.

Stretching DNA tilts its bases.

DNA permeates the
alpha-hemolysin channel
To investigate the influence of the restricted pore geometry on the ensemble of conformations adapted by a DNA strand, a poly(dA11) strand, submerged in a 1 M KCl solution, was confined by a mathematical surface representing the shape of a cylindrical pore. Initially, this pore was wide enough to accommodate the entire ssDNA strand the backbone of which assumed a Watson-Crick helical form. During a 1.2-ns simulation, the diameter of the pore was reduced from 3.0 to 1.0 nm. DNA atoms that lay outside of this surface were subject to a 10-pN force directed toward the center of the pore. These forces adjusted conformations of DNA to the shape of the pore with the DNA backbone straightened; water and ions were not subject to the restraining force in this simulation. In the left figure, we plot the resulting average base-backbone angle as a function of the pore diameter. Similar results were obtained with poly(dC11) and dC7dAdC3 strands. We noticed that when the pore becomes 1.5 nm in diameter, the DNA bases of ssDNA start to tilt collectively toward the 5' end of the strand. DNA bases were observed to tilt toward the 5' end of the strand also when a 58-nt strand was confined inside a phantom pore shaped like the alpha-hemolysin channel. The same tilt was observed without confinement when the DNA backbone was stretched (see the movies below).

  • Click here for a movie (mpeg, 4.2M) illustrating a molecular dynamics simulation of poly(dA11) strand in a shrinking pore.
  • Click here for a movie (mpeg, 4.4M) illustrating the stretching of a poly(dA11) strand by a constant force applied to its 5' (bottom) end.
  • 360,000-atom MD simulation reveals the mechanism of the direction-specific interaction between DNA and alpha-hemolysin

    DNA permeates the alpha-hemolysin channel Molecular dynamics simulation of DNA permeation through alpha-hemolysin.

    What consequence does the uniform tilting of the bases have on DNA translocation kinetics and on current blockades? Intuitively, one could equate driving ssDNA with an electric field through the narrow constriction of alpha-hemolysin to bringing a tree through a door: The tree moves more easily trunk first than tip first (because branches tend to grow toward the tip of the tree). Naturally, to determine the molecular mechanism underlying DNA directionality, one has to visualize the translocation event in atomic detail. Currently available computer resources only allow MD simulations of a few tens of nanoseconds. However, one can start a simulation with the DNA strand threaded halfway through the pore and inspect over a few nanoseconds in how far switching on an electrical field drives the strand for the next few nanoseconds through the pore.

    We have adopted this procedure to test the directionality dependence of the field-induced transport of ssDNA. Two systems, each comprising one alpha-hemolysin channel, a DPPC membrane, a poly(dA58) strand, water, and ions, were subject to an external electric field equivalent to a 1.2-V bias. In the first system, the 5' end of the strand was located at the trans side of the membrane; the system is shown in the figure. In the second system, the 5' end was located at the cis side. The applied bias forced DNA strands toward the trans compartment.

    DNA displacement through alpha-hemolysin
    In the left figure we present how the DNA center of mass shifted through the pore over the duration of 25 ns. One clearly observes faster translocation for the DNA strand entering the pore 3 end first from the cis side, in agreement with the experimental results discussed earlier. Visual inspection of the DNA translocation events with VMD revealed the molecular mechanism for the directionality of the translocation kinetics. In the case of a field forcing ssDNA 3' end first through the pore, the bases remain tilted in the same (3-to-5) direction; in the opposite orientation the bases have to reorient to fit through the pore at its narrow parts.

    Ionic current
  through alpha-hemolysin blocked by DNA strands (cis)5-dA58-3(trans) data are plotted as squares; (cis)3-dA58-5(trans) data are plotted as circles.
    We further computed the currents of K and Cl ions flowing through the alpha-hemolysin during polynucleotide translocation according to the procedure described earlier. In the right figurew we plot, in unitary charge units (e =1.6·10-19 C), the resulting cumulative currents. A linear regression fit yields the average total currents of 330 pA for (cis)3-dA58-5(trans) and 280 pA for (cis)5-dA58-3(trans), which are 24% and 20% of the open pore current, respectively. Although we could not directly compare these values to measured currents, the ratio of the currents, which is 1.2, is close to the experimental value (1.3). We found that the total current is mostly generated by the flow of K ions; the ratio of K to Cl current at a 1.2-V bias is 5:1. Inside the alpha-hemolysin pore, the ions remain hydrated; their trajectories pass through volumes free of DNA. The total charge carried by K and Cl ions inside the alpha-hemolysin stem remained constant (15±1) in both simulations.

    In conclusion, through large-scale molecular dynamics simulations (and a lot of creative thinking) we were able to reproduce the experiments results in silico and uncover the molecular mechanism of the observed directionality. To our delight, simulations and experiments converged!

    Publications

    Publications Database Orientation discrimination of single stranded DNA inside the a-hemolysin membrane channel. Jerome Mathé, Aleksei Aksimentiev, David R. Nelson, Klaus Schulten, and Amit Meller. Proceedings of the National Academy of Sciences, USA, 102:12377-12382, 2005. Publications Database Imaging alpha-hemolysin with molecular dynamics: Ionic conductance, osmotic permeability and the electrostatic potential map. Aleksij Aksimentiev and Klaus Schulten. Biophysical Journal, 88:3745-3761, 2005.

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