Nonlinear elasticity of microsphere heaps

Thermal fluctuations, geometric exclusion, and external driving all govern the mechanical response of dense particulate suspensions. Here, we measure the stress-strain response of quasi-two-dimensional flow-stabilized microsphere heaps in a regime in which all three effects are present using a microfluidic device. We observe that the elastic modulus and the mean interparticle separation of the heaps are tunable via the confining stress provided by the fluid flow. Furthermore, the measured stress-strain curves exhibit a universal nonlinear shape, which can be predicted from a thermal van der Waals equation of state with excluded volume. This analysis indicates that many-body interactions contribute a significant fraction of the stress supported by the heap

CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level

The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2. Single-molecule fluorescence tracking on DNA tightropes containing regions with CpG-rich and CpG-free regions reveals that MBD2 carries out unbiased 1D diffusion on CpG-rich DNA but subdiffusion on CpG-free DNA. In contrast, the protein stably and statically binds to methylated CpG (mCpG) regions. The intrinsically disordered region (IDR) on MBD2 both reduces exchange between mCpG sites along the DNA as well as the dissociation from DNA, acting like an anchor that restricts the dynamic mobility of the MBD domain. Unexpectedly, MBD2 binding to methylated CpGs induces DNA bending that is augmented by the IDR region of the protein. These results suggest that MBD2 targets NuRD to unmethylated or methylated CpG islands where its distinct dynamic binding modes help maintain open or closed chromatin, respectively

Statics and dynamics of single DNA molecules confined in nanochannels

The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics

Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2

Shelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding

The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic