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

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