Recent developments in top-down lithography have made possible the use of nanoconfined environments for the separation and analysis of DNA molecules. Separation systems are based an energy barrier caused primarily by the lower entropy confined state. The propensity of DNA to pass through this entropic barrier varies for different molecule sizes. In contrast, genomic analytical systems, which present individual DNA molecules rely on the low entropy environment to effectively decrease the number of possible conformations of DNA and ease its observation. Until recently, most fabricated devices were aimed at separations; however, engineering concerns for directing the movement of DNA within a device designed for DNA presentation requires vastly different considerations. As such, it is desirable to finely control the motion of DNA within these devices for optimizing measurements reporting biochemical states reflecting sequence composition, protein or drug binding events within the context of developing systems for genome analysis.
Towards this goal, I am studying the diffusion, relaxation, and local melting behavior of DNA within nanochannels whose dimensions are on the order of that of DNA’s persistence length. The external and internal diffusion and conformation will be measured as functions of channel dimensions and solution conditions. Two fluorophores will be attached to the DNA molecule; one will be intercalated into the DNA backbone while the other will be attached to specific loci of the DNA. The two dyes will interact through a process known as FRET. Using fluorescence microscopy, the intercalated dye will allow for the visualization of the molecule while the second dye will allow for the measurement of the position of specific locations of the DNA chain in space. Under the influence of an electric field, DNA will diffuse into the nanochannels. After the electric field has been switched off, the motion of the center of mass as well as the motion of the marked locations along the DNA backbone will be measured through time-lapse imaging. In addition, local melting behavior will be studied using a double stranded molecule of DNA whose composition consists of a poly-A region surrounded by two poly-G regions. The poly-A region is expected to melt under milder conditions than the poly-G region forming a bubble within the molecule. By correlating the internal and external motion of DNA in a nanochannel with channel dimensions and different solution conditions, a better understanding of the effects of its confinement will be gained and this new knowledge applied for design of next generation platforms that will leverage nanoconfinement effects.