Amplified stretch of bottlebrush-coated DNA in nanofluidic channels.

The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

Effect of H-NS on the elongation and compaction of single DNA molecules in a nanospace.

The effect of the bacterial heat-stable nucleoid-structuring protein (H-NS) on the conformation of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. With increasing concentration of H-NS, the DNA molecules either elongate or contract. The conformational response is related to filamentation of H-NS on DNA through oligomerization and H-NS mediated bridging of distal DNA segments and is controlled by the concentration and ionic composition of the buffer. Confinement in a nanochannel also facilitates compaction of DNA into a condensed form for over-threshold concentrations of H-NS. Divalent ions such as magnesium facilitate but are not required for bridging nor condensation. The time scale of the collapse after exposure to H-NS was determined to be on the order of minutes, which is much shorter than the measured time required for filamentation of around one hour. We found that the effect of H-NS is not only related to its binding properties but also the confinement is of paramount importance. The interplay between confinement, H-NS-mediated attraction, and filamentation controls the conformation and compaction of DNA. This finding might have implications for gene silencing and chromosome organisation, because the cross-sectional dimensions of the channels are comparable to those of the bacterial nucleoid.

Conformation Model of Back-Folding and Looping of a Single DNA Molecule Confined Inside a Nanochannel.

Currently, no theory is available to describe the conformation of DNA confined in a channel when the nanochannel diameter is around the persistence length. Back-folded hairpins in the undulating wormlike chain conformation result in the formation of loops, which reduces the stretch of the molecule in the longitudinal direction of the channel. A cooperativity model is used to quantify the frequency and size of the loop domains. The predictions agree with results from the Monte Carlo simulation.

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.

Conformational response of supercoiled DNA to confinement in a nanochannel.

Monte Carlo simulations were done to study the conformation of supercoiled DNA confined in a nanochannel. The molecule has a superhelical density of around -0.05 and is bathed in a monovalent salt solution with an ionic strength of 2, 10, or 150 mM. The cross-sectional diameter of the circular shaped nanochannel was varied in the range of 10 to 80 nm. The conformational properties were characterized by the writhing number and the distribution in the distance between the two opposing strands of the superhelix. With increasing confinement, as set by a smaller tube diameter and/or decreased screening of the Coulomb interaction, the supercoil becomes more tightly interwound and long-range structural features such as branching and the formation of hairpins are progressively suppressed. Analysis of the energetics shows a concurrent increase in electrostatic energy and energy of interaction of the supercoil with the wall, but the elastic twisting energy decreases. Confinement in a nanochannel or otherwise hence results in a decrease in the absolute value of the twist exerted on the duplex. The bending energy remains approximately constant, which means that there are no significant deflections from the wall. The simulation results are interpreted with theory based on the wormlike chain model, including the effects of the wall, charge, elasticity, and configurational entropy. It was found that the theory is reasonably successful in predicting the structural response to the confinement at the local level of the diameter and pitch of the supercoil.