Effect of crowding on the conformation of interwound DNA strands from neutron scattering measurements and Monte Carlo simulations

With a view to determining the distance between the two opposing duplexes in supercoiled DNA, we have measured small angle neutron scattering from pHSG298 plasmid (2675 base pairs) dispersed in saline solutions. Experiments were carried out under full and zero average DNA neutron scattering contrast using hydrogenated plasmid and a 1:1 mixture of hydrogenated and perdeuterated plasmid, respectively. In the condition of zero average contrast, the scattering intensity is directly proportional to the single DNA molecule scattering function (form factor), irrespective of the DNA concentration and without complications from intermolecular interference. The form factors are interpreted with Monte Carlo computer simulation. For this purpose, the many body problem of a dense DNA solution was reduced to the one of a single DNA molecule in a congested state by confinement in a cylindrical potential. It was observed that the interduplex distance decreases with increasing concentration of salt as well as plasmid. Therefore, besides ionic strength, DNA crowding is shown to be important in controlling the interwound structure and site juxtaposition of distal segments of supercoiled DNA. This first study exploiting zero average DNA contrast has been made possible by the availability of perdeuterated plasmid.

Figure 1

Gel electrophoresis with 80 mg/L chloroquine phosphate. The lanes are relaxed ${\text{DNA}}^{\text{H}}$ (L1), ${\text{DNA}}^{\text{H}}$ (L2), ${\text{DNA}}^{\text{D}}$ (L3 and L4), and a 1:1 mixture of ${\text{DNA}}^{\text{H}}$ and ${\text{DNA}}^{\text{D}}$ (L5). Note that the migration distance increases with increasing value of $\Delta Lk$. The open circular and relaxed $\left(\Delta Lk=0\right)$ states are indicated by oc and 0, respectively.

Figure 2

Form factor $P$ (open symbols) and structure factor $S$ (closed symbols) versus momentum transfer $q$. The DNA concentrations are 6 $\left(A\right)$ and 24 g/L $\left(B\right)$. The NaCl concentrations are indicated in mM. The solid curves represent the form factor $P_{\text{d}}$ pertaining to the duplex with a cross-sectional radius of gyration $r_{\text{c}}=0.8\text{nm}$. To avoid overlap, the data are shifted along the $y$ axis with a multiplicative constant.

Figure 3

Normalized form factor $P/P_{\text{d}}$ versus momentum transfer $q$. The NaCl concentrations $c_{\text{s}}=4$ $\left(A\right)$, 20 $\left(B\right)$, and 100 mM $\left(C\right)$. The DNA concentrations are indicated in g/L. The curves are the simulated normalized form factors ${\overline{P}}_{\text{sc}}/P_{\text{v}}$ with interduplex distances $D_{\text{sc}}$ as shown in Fig. 6. To avoid overlap, the data are shifted along the $y$ axis with an incremental constant.

Figure 4

(Color online) Distribution function $p_{\text{v}}$ versus the intervertex distance $r_{\text{ij}}$ for $D_{\text{cyl}}=20\text{nm}$ and $c_{\text{s}}=20\text{mM}$. The most probable distance $D_{\text{sc}}=12\text{nm}$. The inset is a snapshot of an equilibrated conformation after $16\times {10}^6\text{cycles}$.

Figure 5

Cylinder diameter $D_{\text{cyl}}$ versus DNA concentration $c_{\text{DNA}}$. The bars indicate the variance. The NaCl concentrations are 4 (○), 20 (◻), and 100 (△) mM. The solid curve is the lateral intermolecular distance $D_{\text{lat}}$ for closely packed wormlike cylinders.

Figure 6

Interduplex distance $D_{\text{sc}}$ versus DNA concentration $c_{\text{DNA}}$. The bars indicate the variance. The NaCl concentrations are 4 (○), 20 (◻), and 100 (△) mM. The dashed curves are guides to the eyes.