Differences in gene expression may play a major role in speciation and phenotypic diversity. We examined genome-wide differences in transcription factor (TF) binding in several humans and a single chimpanzee using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). The binding sites of RNA Polymerase II (PolII) and a key regulator of immune responses, NFκB (p65), were mapped in ten lymphoblastoid cell lines and 25% and 7.5% of the respective binding regions were found to differ between individuals. Binding differences were frequently associated with SNPs and genomic structural variants (SVs) and were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, comparing PolII binding between human and chimpanzee suggests extensive divergence in TF binding. Our results indicate that many differences in individuals and species occur at the level of TF binding and provide insight into the genetic events responsible for these differences. TextDifferences in gene expression have been observed in a variety of species (1-3). However, the extent to which TF binding differences occur both within individuals and closely related species and the global relationship between TF binding and genetic variation are largely unexplored (4). We used ChIP-Seq to map NFκB and PolII binding sites in ten humans: five are of European ancestry (including a parent-offspring trio), two of eastern Asian ancestry, and three of Nigerian

An inhibition assay method was developed based on the modulation in the FRET efficiency between quantum dots (QDs) and gold nanoparticles (AuNPs) in the presence of the molecules which inhibit the interactions between QD- and AuNP-conjugated biomolecules. For the functionalization, AuNPs were first stabilized by chemisorption of n-alkanethiols and then capped with the first generation polyamidoamine (G1 PAMAM) dendrimers. By employing a streptavidin-biotin couple as a model system, avidin was quantitatively analyzed as an inhibitor by sensing the change in photoluminescence (PL) quenching of SA-QDs by biotin-AuNPs. The detection limit for avidin was about 10 nM. It is anticipated that the PL quenching-based sensing system can be used for the quantitative analysis and high throughput screening of molecules which inhibit the specific biomolecular interactions.

Poly(amidoamine) dendrimers having various degrees of modification with the redox-active ferrocenyls were prepared by controlling the molar ratio of ferrocenecarboxaldehyde to amine groups of dendrimers. By alternate layer-by-layer depositions of partial ferrocenyl-tethered dendrimers (Fc-D) with periodate-oxidized glucose oxidase (GOx) on a Au surface, an electrochemically and enzymatically active multilayered assembly of enzyme was constructed. The resulting GOx/Fc-D multilayer-associated electrodes were electrochemically analyzed, and the surface concentration of ferrocenyl groups, active enzyme coverage, and sensitivity were estimated. A 32% dendrimer modification level of surface amines to ferrocenyls was found to be an optimum in terms of enzyme-dendrimer network formation, electrochemical interconnectivity of ferrocenyls, and electrode sensitivity. With the prepared Fc(32%)-tethered dendrimers, mono- and multilayered GOx/Fc-D electrodes were constructed, and their electrochemical and catalytic properties were characterized. The bioelectrocatalytic signals from the multilayered GOx/Fc-D electrodes were shown to be directly correlated to the number of deposited bilayers. From this result, it seems that the electrode sensitivity is directly controllable, and the multilayer-forming strategy with partial ferrocenyl-tethered dendrimers is useful for the construction of reagentless biosensors.

Avidin-biotin interactions as a typical protein-ligand model were investigated on the monolayers of a fourth-generation poly(amidoamine) dendrimer that were constructed on the self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) on gold. Surface plasmon resonance (SPR) spectroscopic analysis revealed a resonance angle shift of 0.34°( 0.03°for the formation of dendrimer monolayers on reactive SAMs, which indicates that about 89% of the gold surface is covered with dendrimer molecules. The dendrimer monolayers were functionalized with biotin, and the efficacy of dendrimer monolayers as a biomolecular interface was evaluated in terms of the surface density of biotin ligands and the avidin binding level. For comparisons, the mixed SAMs and polymeric layers of poly-L-lysine (PLL) on MUA SAMs were prepared and examined by a similar procedure. The specific binding of avidin to the biotinylated dendrimer monolayers approached a surface density of 5.0 ( 0.2 ng‚mm -2 , which corresponds to about 88% surface coverage by avidin, showing a much higher level than those from mixed SAMs (2.3 ( 0.1 ng‚mm -2 ) and PLL layers (3.2 ( 0.2 ng‚mm -2 ). Interestingly, the fully biotinylated dendrimer monolayers gave rise to efficient avidin-biotin interactions, resulting in about 80% of the maximum avidin binding level, even under the condition that a serious steric hindrance would occur due to densely packed biotin ligands. These results strongly imply that efficient avidin-biotin interaction originates from a structural feature of dendrimer monolayers such as a surface exposure of derivatized biotin ligands and a corrugated surface.

This unit describes ChIP-Seq methodology, which involves chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (Seq), and enables the genome-wide identification of binding sites of transcription factors (TFs) and other DNA-binding proteins. The process is initiated by cross-linking DNA and DNA-bound proteins. Subsequently, chromatin is isolated from nuclei and subjected to sonication. An antibody against a specific TF or DNA-binding protein is then used to immunoprecipitate specific DNA-TF complexes. ChIP DNA is purified, sequencing adapters are ligated, and 30- to 35-nucleotide (nt) sequence reads are generated. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding sites.

We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOF-SIMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface-immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface-immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips.

No abstract

The interaction of streptavidin (SA) with a biotinylated surface has been of great interest in the development of an interfacial layer for protein immobilization based on self-assembled monolayers (SAMs) and polymeric layers. Here, we demonstrate the unique characteristics of protein-ligand interactions on dendrimer monolayers based on kinetic and equilibrium binding analyses. With amine-ended poly(amidoamine) dendrimers from the first (G1) to fourth (G4) generation, the formation of even, compact dendrimer monolayers on gold was confirmed using FT-IR spectroscopy and ellipsometry. For the SA-biotin interaction, quantitative analysis of bound SA using surface plasmon resonance showed that the saturation binding level of SA was fairly higher in all dendrimer layers when compared to other tested systems of 11-mercaptoundecylamine SAMs and a poly(L-lysine) layer. Kinetic studies revealed that the initial binding rate of SA up to the saturation level was 2-fold higher in all dendrimer layers than in the SAMs regardless of the surface density of functionalized biotin. Concurrently, the dendrimer layers led to much higher values of sticking probability, which is defined as the probability that the SA molecule adsorbs upon collision with a biotinylated surface, at a fixed SA coverage, and prolonged the significant levels around the maximum probability with increasing SA coverage. Plots of the saturation coverage of SA versus the SA concentration in solution showed that SA binding onto the biotinylated G1 and G3 layers fit to a Langmuir isotherm model. Taken together, faster binding of SA and highly ordered packing of the molecules seems to be achieved through typical properties of the dendrimer monolayers such as surface distribution of functionalized biotin, surface corrugation, and flexibility of highly branched larger dendrimers, which provides a guideline for the construction and analysis of an interfacial layer in biosensing applications.