Neurodegenerative disorders including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and stroke are rapidly increasing as population ages. The field of nanomedicine is rapidly expanding and promises revolutionary advances to the diagnosis and treatment of devastating human diseases. This paper provides an overview of novel nanomaterials that have potential to improve diagnosis and therapy of neurodegenerative disorders. Examples include liposomes, nanoparticles, polymeric micelles, block ionomer complexes, nanogels, and dendrimers that have been tested clinically or in experimental models for delivery of drugs, genes, and imaging agents. More recently discovered nanotubes and nanofibers are evaluated as promising scaffolds for neuroregeneration. Novel experimental neuroprotective strategies also include nanomaterials, such as fullerenes, which have antioxidant properties to eliminate reactive oxygen species in the brain to mitigate oxidative stress. Novel technologies to enable these materials to cross the blood brain barrier will allow efficient systemic delivery of therapeutic and diagnostic agents to the brain. Furthermore, by combining such nanomaterials with cell-based delivery strategies, the outcomes of neurodegenerative disorders can be greatly improved.

The study of protein interactions with DNA is important to gain a fundamental understanding of how numerous biological processes occur, including recombination, transcription, repair, etc. In this study, we use the EcoRII restriction enzyme, which employs a three-site binding mechanism in order to catalyze cleavage of a single recognition site. Using high-speed atomic force microscopy (HS-AFM) to image single-molecule interactions in real time, we were able to observe binding, translocation, and dissociation mechanisms of the EcoRII protein. The results show that the protein can translocate along DNA to search for the specific binding site. Also, once specifically bound at a single site, the protein is capable of translocating along the DNA to locate the second specific binding site. Furthermore, two alternative modes of dissociation of the EcoRII protein from the loop structure were observed, which result in the protein stably bound as monomers to two sites or bound to a single site as a dimer. From these observations, we propose a model in which this pathway is involved in the formation and dynamics of a catalytically active three-site complex.The formation of synaptic protein-DNA complexes is central to many biological processes which require communication between two or more DNA regions, including recombination (1,2), replication (3), transcriptional regulation (4), repair (5), transposition (6), and restriction (7,8). Restriction enzymes serve as useful models to study mechanisms by which the intracellular protein machinery functions on DNA, including synapsis. Restriction enzymes (REases), which require binding to two or more cognate recognition sites in order to be catalytically active are widely spread (9). A multi-site mechanism suggests that restriction enzymes serve as evolutionary precursors to many DNA regulatory factors in the cell (10,11). Such a mechanism could also serve an inhibitory function to prevent rare unmethylated recognition sites in the host genome from undergoing restriction (12). In addition to systems involving interactions of two DNA helices, interactions of three or more DNA molecules may occur (13-17).EcoRII is a dimer which recognizes the sequence 5′-CCWGG-3′. It is generally known as a type IIE restriction enzyme. In general, the definition of typeIIE REases is that they bind two DNA recognition sites in order to cleave one of the sites (18). However, recent evidence suggests that the EcoRII protein actually requires three sites to concertedly cleave both strands For this study, we used high speed atomic force microscopy (HS-AFM) to directly image single molecule dynamics of the protein-DNA complexes formed by EcoRII restriction enzyme. It has been used previously to visualize looping and translocation mechanisms of the type III restriction enzyme EcoP15I (31). This HS-AFM relies on a small cantilever design based on the original design by T. Ando (32). This technique has the ability to observe molecular dynamics on a timescale that is 100 times faster than conventional...

Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide direct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed.