Probing transient protein-mediated DNA linkages using nanoconfinement.

We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNApolymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches.

Chromatin modification mapping in nanochannels.

We report the simultaneous mapping of multiple histone tail modifications on chromatin that has been confined to nanofluidic channels. In these channels, chromatin is elongated, and histone modification can be detected using fluorescently tagged monoclonal antibodies. Using reconstituted chromatin with three distinct histone sources and two histone tail modification probes (H3K4me3 and H3K9ac), we were able to distinguish chromatin from the different sources. Determined ratios of the two modifications were consistent with the bulk composition of histone mixtures. We determined that the major difficulty in transitioning the mapping method to site-specific profiling within single genomic molecules is the interference of naturally aggregating, off-the shelf antibodies with the internal structure of chromatin.

Fluctuation modes of nanoconfined DNA.

We report an experimental investigation of the magnitude of length and density fluctuations in DNA that has been stretched in nanofluidic channels. We find that the experimental data can be described using a one-dimensional overdamped oscillator chain with nonzero equilibrium spring length and that a chain of discrete oscillators yields a better description than a continuous chain. We speculate that the scale of these discrete oscillators coincides with the scale at which the finite extensibility of the polymer manifests itself. We discuss how the measurement process influences the apparent measured dynamic properties, and outline requirements for the recovery of true physical quantities.

DNA methylation profiling in nanochannels.

We report the profiling of the 5-methyl cytosine distribution within single genomic-sized DNA molecules at a gene-relevant resolution. This method linearizes and stretches DNA molecules by confinement to channels with a dimension of about 250×200 nm(2). The methylation state is detected using fluorescently labeled methyl-CpG binding domain proteins (MBD), with high signal contrast and low background. DNA barcodes consisting of methylated and non-methylated segments are generated, with both short and long concatemers demonstrating spatially resolved MBD binding. The resolution of the technique is better than 10 kbp, and single-molecule read-lengths exceeding 140 kbp have been achieved.

Density fluctuations dispersion relationship for a polymer confined to a nanotube.

DNA confined to rigid nanotubes shows density fluctuations around its stretched equilibrium conformation. We report an experimental investigation of the length-scale dependent dynamics of these density fluctuations. We find that for highly elongated molecules a Rouse description is consistent with observations at sufficiently large length scales. We further find that for strongly fluctuating molecules, or short length scales, such Rouse modes cannot be detected due to strong mixing of fluctuation modes.