No abstract

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.

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.

An easy method is introduced allowing fast polydimethylsiloxane (PDMS) replication of nanofluidic lab-on-chip devices using accurately fabricated molds featuring cross-sections down to 60 nm. A high quality master is obtained through proton beam writing and UV lithography. This master can be used more than 200 times to replicate nanofluidic devices capable of handling single DNA molecules. This method allows to fabricate nanofluidic devices through simple PDMS casting. The extensions of YOYO-1 stained bacteriophage T4 and kÀDNA inside these nanochannels have been investigated using fluorescence microscopy and follow the scaling prediction of a large, locally coiled polymer chain confined in nanochannels.