A Nanochannel Platform for Single DNA Studies: From Crowding, Protein DNA Interaction, to Sequencing of Genomic Information

Advances in nanolithography have made it possible to fabricate nanofluidic devices with at least one dimension of around a few tens of nanometers. Nano-devices featuring entropic trap arrays, pillar arrays, pores, or channels have been designed and used for the analysis of the properties of long biopolymers, including DNA. Here, we discuss long and straight quasi one-dimensional channel devices with cross-sectional diameters of tens to hundreds of nanometers. Within such a nanochannel, a single DNA molecule or nucleoprotein complex can be confined and visualized with fluorescence microscopy. The nanofluidic devices are hence complementary to other single-molecule manipulation techniques, such as those based on tweezers. An important difference is that the confinement experiment does not require chemical modification to attach the biomolecule to molecular pincers. The molecules can hence be investigated closer to their native state. Confinement is important in certain biological processes, such as DNA packaging in viruses, or segregation of DNA in bacteria. Accordingly, besides single-molecule manipulation, nanochannels serve as a model system for the investigation of the compaction of DNA in a nanospace, in conjunction with other important compaction modes, such as macromolecular crowding and/or binding of condensing proteins or ligands. The opportunity to investigate single molecules inside nanochannels has stimulated fundamental research as to the effects of channel diameter, and salt concentration, on the conformation of DNA. Other studies have focused on the dynamics, to elucidate the variation in the extension due to thermal fluctuation, or the response of the molecular motion to entropic, electrophoretic, and frictional forces. The conformation and dynamics of DNA in confinement have also been investigated with computer simulation. These simulations contribute to our understanding of the different physical mechanisms at play. Besides fundamental studies, nanochannels are important for biotechnological applications, Abstract : The study of nanochannel-confined DNA is important from biotechnological and biophysical points of view. We produce nanochannels in elastomer with soft lithography and proton beam writing. Issues concerning DNA confined in such quasi one-dimensional channels are discussed. We describe DNA stretching via the control of channel diameter and buffer conditions and how the extension can be interpreted with theory and computer simulation. We then discuss the conformation of nano-confined DNA crowded by neutral polymers and like-charged proteins. As an example of a protein that has an affinity to DNA, the effect of heatstable nucleoid-structuring protein, H-NS, on the folding and compaction of DNA is reviewed. Compaction of DNA by eukaryotic protamine and unpacking of pre-compacted DNA through an increase in salt concentration are discussed. We review results obtained with a novel, cross-channel device that allows the monitoring of the dynamic, conformational response of DNA after exposure to a ligand or protein and/or a change in buffer conditions in situ. As a biotechnological application, linearization of DNA by bottlebrush coating with a polypeptide copolymer is discussed. It is demonstrated that large-scale genomic organization can be sequenced using single DNA molecules on an array of elastomeric nanochannels. Overall, our results show that the effects of ligands and proteins on the conformation, folding, and condensation of DNA are not only related to classical controlling factors, such as osmotic pressure, charge, and binding, but that the interplay with confinement in a nanospace is of paramount importance.