Enhanced PCR Amplification of GC-Rich DNA Templates by Gold Nanoparticles

Gold nanoparticles (AuNPs) have been reported to facilitate double-stranded DNA dissociation and improve performance of several PCR systems. Here we investigated AuNPs’ effect on GC-rich DNA amplification. We found that AuNPs could enhance PCR amplification of the <i>GNAS1</i> promoter region (∼84% GC) mediated by <i>Pfu</i> or <i>Taq</i> DNA polymerase. However, under optimal concentrations of AuNPs, higher amounts of <i>Taq</i> were required. Furthermore, the GC-rich <i>FMR1</i> (80.4% GC) gene of <i>Homo sapiens</i> as well as <i>exoT</i> (67.3% GC), <i>exsE</i> (71% GC) and <i>pqqF</i> genes (74% GC) of <i>Pseudomonas aeruginosa</i> were also efficiently amplified. AuNPs can become an effective additive in GC-rich PCR and facilitate analysis of challenging genomic sequence in basic and clinical research

Heteroepitaxial Streptavidin Nanocrystals Reveal Critical Role of Proton “Fingers” and Subsurface Atoms in Determining Adsorbed Protein Orientation

Characterization of noncovalent interactions between nanometer-sized structures, such as proteins, and solid surfaces is a subject of intense interest of late owing to the rapid development of numerous solid materials for medical and technological applications. Yet the rational design of these surfaces to promote the adsorption of specific nanoscale complexes is hindered by a lack of an understanding of the noncovalent interactions between nanostructures and solid surfaces. Here we take advantage of the unexpected observation of two-dimensional nanocrystals of streptavidin on muscovite mica to provide details of the streptavidin–mica interface. Analysis of atomic force microscopic images together with structural modeling identifies six positively charged residues whose terminal amine locations match the positions of the single atom-sized anionic cavities in the basal mica surface to within 1 Å. Moreover, we find that the streptavidin crystallites are oriented only along a single direction on this surface and not in either of three different directions as they must be if the protein interacted solely with the 3-fold symmetric basal surface atoms. Hence, this broken symmetry indicates that the terminal amine protons must also interact directly with the subsurface hydroxide atoms that line the bottom of these anionic cavities and generate only a single axis of symmetry. Thus, in total, these results reveal that subsurface atoms can have a significant influence on protein adsorption and orientation and identify the insertion of proton “fingers” as a means by which proteins may generally interact with solid surfaces

Super-resolution Imaging of Individual Human Subchromosomal Regions <i>in Situ</i> Reveals Nanoscopic Building Blocks of Higher-Order Structure

It is widely recognized that the higher-order spatial organization of the genome, beyond the nucleosome, plays an important role in many biological processes. However, to date, direct information on even such fundamental structural details as the typical sizes and DNA content of these higher-order structures <i>in situ</i> is poorly characterized. Here, we examine the nanoscopic DNA organization within human nuclei using super-resolution direct stochastic optical reconstruction microscopy (dSTORM) imaging and 5-ethynyl-2′-deoxyuridine click chemistry, studying single fully labeled chromosomes within an otherwise unlabeled nuclei to improve the attainable resolution. We find that, regardless of nuclear position, individual subchromosomal regions consist of three different levels of DNA compaction: (i) dispersed chromatin; (ii) nanodomains of sizes ranging tens of nanometers containing a few kilobases (kb) of DNA; and (iii) clusters of nanodomains. Interestingly, the sizes and DNA content of the nanodomains are approximately the same at the nuclear periphery, nucleolar proximity, and nuclear interior, suggesting that these nanodomains share a roughly common higher-order architecture. Overall, these results suggest that DNA compaction within the eukaryote nucleus occurs <i>via</i> the condensation of DNA into few-kb nanodomains of approximately similar structure, with further compaction occurring <i>via</i> the clustering of nanodomains

Genome-wide analysis of core promoter structures in <i>Schizosaccharomyces pombe</i> with DeepCAGE

<div><p>The core promoter, which immediately flanks the transcription start site (TSS), plays a critical role in transcriptional regulation of eukaryotes. Recent studies on higher eukaryotes have revealed an unprecedented complexity of core promoter structures that underscores diverse regulatory mechanisms of gene expression. For unicellular eukaryotes, however, the structures of core promoters have not been investigated in detail. As an important model organism, <i>Schizosaccharomyces pombe</i> still lacks the precise annotation for TSSs, thus hampering the analysis of core promoter structures and their relationship to higher eukaryotes. Here we used a deep sequencing-based approach (DeepCAGE) to generate 16 million uniquely mapped tags, corresponding to 93,736 positions in the <i>S. pombe</i> genome. The high-resolution TSS landscape enabled identification of over 8,000 core promoters, characterization of 4 promoter classes and observation of widespread alternative promoters. The landscape also allowed precise determination of the representative TSSs within core promoters, thus redefining the 5' UTR for 82.8% of <i>S. pombe</i> genes. We further identified the consensus initiator (Inr) sequence – PyPyPuN(A/C)(C/A), the TATA-enriched region (between position −25 and −37) and an Inr immediate downstream motif – CC(T/A)(T/C)(T/C/A)(A/G)CCA(A/T/C), all of which were associated with highly expressed promoters. In conclusion, the detailed analysis of core promoters not only significantly improves the genome annotation of <i>S. pombe</i>, but also reveals that this unicellular eukaryote shares a highly similar organization in the core promoters with higher eukaryotes. These findings lend additional evidence for the power of this model system in delineating complex regulatory processes in multicellular organisms, despite its perceived simplicity.</p></div

Dynamic Covalent Diblock Copolymers: Instructed Coupling, Micellation and Redox Responsiveness

Instructed by association units that allow reversible and unsymmetrical disulfide bond formation, hydrophilic (PEG) and hydrophobic (PLA) polymer chains are efficiently coupled into amphiphilic diblock copolymers. The desymmetrization of otherwise symmetrical reversible disulfide bond formation is achieved with amide association units that integrate both directional H-bonding and reversible disulfide bond formation, which ensure the connection of different polymer blocks while minimizing self-coupling. The resultant amphiphilic block copolymers self-assemble into long-lasting spherical micelles that are responsive to free thiols

Molecular Threading and Tunable Molecular Recognition on DNA Origami Nanostructures

The DNA origami technology holds great promise for the assembly of nanoscopic technological devices and studies of biochemical reactions at the single-molecule level. For these, it is essential to establish well controlled attachment of functional materials to predefined sites on the DNA origami nanostructures for reliable measurements and versatile applications. However, the two-sided nature of the origami scaffold has shown limitations in this regard. We hypothesized that holes of the commonly used two-dimensional DNA origami designs are large enough for the passage of single-stranded (ss)-DNA. Sufficiently long ssDNA initially located on one side of the origami should thus be able to “thread” to the other side through the holes in the origami sheet. By using an origami sheet attached with patterned biotinylated ssDNA spacers and monitoring streptavidin binding with atomic force microscopic (AFM) imaging, we provide unambiguous evidence that the biotin ligands positioned on one side have indeed threaded through to the other side. Our finding reveals a previously overlooked critical design feature that should provide new interpretations to previous experiments and new opportunities for the construction of origami structures with new functional capabilities

Additional file 6: Table S6. of Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells

Genes up- or down-regulated in Prmt5 kock-down FGSCs. (XLSX 733 kb

Enforced Tubular Assembly of Electronically Different Hexakis(<i>m</i>‑Phenylene Ethynylene) Macrocycles: Persistent Columnar Stacking Driven by Multiple Hydrogen-Bonding Interactions

Hexakis­(<i>m</i>-phenylene ethynylene) (<i>m</i>-PE) macrocycles <b>1</b>–<b>4</b>, sharing the same hydrogen-bonding side chains but having backbones of different electronic properties, are designed to probe the effectiveness of multiple H-bonding interactions in enforcing columnar assemblies. ¹H NMR, absorption, fluorescence, and circular dichroism (CD) spectroscopy indicate that, compared with analogous macrocycles that self-associate based on aromatic stacking which is highly sensitive to the electronic nature of the macrocyclic backbones, macrocycles <b>1</b>–<b>4</b> all exhibit strong aggregation down to the micromolar (μM) concentrations in nonpolar solvents. Increasing solvent polarity quickly weakens aggregation. In THF and DMF, the macrocycles exist as free molecules. The observed solvent effects, along with the behavior of <b>5-F</b>_<b>6</b> that cannot self-associate via H-bonding, confirm that H-bonding plays the dominating role in driving the self-association of <b>1</b>–<b>4</b>. The backbone electronic nature does not change the self-assembling pattern common to <b>1</b>–<b>4</b>. Fluorescence and CD spectra confirm that macrocycles <b>1</b>–<b>4</b> assemble anisotropically, forming helical stacks in which adjacent molecules undergo relative rotation to place individual benzene residues in the favorable offset fashion. Columnar alignment of <b>1</b>–<b>4</b> is confirmed by atomic force microscopy (AFM), which resolves single tubes consisting of stacked macrocycles. In addition, macrocycles with backbones of different electronic properties are found to undergo heteroassociation, forming hybrid nanotubes. This study has demonstrated the generality of enforcing the alignment of shape-persistent macrocycles, which represents an invaluable addition to the small number of known tubular stacks capable of accommodating structurally varied molecular components and provides self-assembling nanotubes with inner pores allowing ready structural and functional modification

Additional file 2: Table S2. of Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells

GO analysis of genes related to FGSC-specific active enhancers. (XLSX 10 kb

Additional file 5: Table S5. of Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells

List of genes methylated in FGSCs and/or MGSCs. (XLSX 180 kb