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.

Nanouidic compaction of DNA by like-charged protein.

The effects of the like-charged proteins bovine serum albumin and hemoglobin on the conformation and compaction of single DNA molecules confined in rectangular nanochannels were investigated with fluorescence microscopy. The channels have lengths of 50 µm and cross-sectional diameters in the range of 80-300 nm. In the wider channels, the DNA molecules are compressed and eventually condense into a compact form with increasing concentration of protein. In the narrow channels, no condensation was observed. The threshold concentration for condensation depends on the channel cross-sectional diameter as well as the ionic strength of the supporting medium. The critical values for full compaction are typically less than one-tenth of a millimolar. In the bulk phase and in the same environmental conditions, no condensation was observed. Anisotropic nanoconfinement hence facilitates compaction of DNA by negatively charged protein. We tentatively interpret this behavior in terms of enhanced depletion interaction between segments of the DNA molecule due to orientation order imposed by the channel walls.