Two-dimensional enzyme diffusion in laterally confined DNA monolayers

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon

spectroscopic ellipsometry meets afm nanolithography about hydration of bio inert oligo ethylene glycol terminated self assembled monolayers on gold

An accurate thickness determination provides insight into the complex vertical morphology of OEG-terminated SAMs

Electrophysiological and Neuropsychological Indices of Cognitive Dysfunction in Patients with Chronic Insomnia and Severe Benzodiazepine Use Disorder.

Benzodiazepine (BDZ) misuse is a growing health problem, with 1-2% of patients under BDZ treatment meeting the criteria for use disorder or dependence. Although BDZ addiction potential has been known for decades, much remains unknown its effects on brain functions. The aim of this study was to assess the neuropsychological and neurophysiological profile of a group of chronic insomniacs taking long-term high doses of benzodiazepine. We recruited 17 consecutive patients admitted to our third-level Sleep Medicine Unit for drug discontinuation (7 males, mean age 49.2 ± 11.2 years, mean education 13.7 ± 3.9 years, mean daily diazepam-equivalent BDZ: 238.1 ± 84.5 mg) and 17 gender/age-matched healthy controls (7 males, mean age 46.8 ± 14.1 years, mean education 13.5 ± 4.5 years). We performed a full neuropsychological evaluation of all subjects and recorded their scalp event-related potentials (Mismatch-Passive Oddball-Paradigm and Active Oddball P300 Paradigm). Patients with chronic insomnia and BDZ use disorder showed a profound frontal lobe executive dysfunction with significant impairment in the cognitive flexibility domain, in face of a preserved working, short and long-term memory. In patients, P300 amplitude tended to be smaller, mainly over the frontal regions, compared to controls. BDZ use disorder has a severe cognitive impact on chronic insomnia patients. Long-term high-dose BDZ intake should be carefully evaluated and managed by clinicians in this specific patient population, especially in relation to risky activities

Crowding effects on biochemical reactions of surface-bound DNA.

2006/2007Next-generation DNA detection arrays are expected to achieve unprecedented sensitivity, reducing the minimum amount of genetic material that can be directly (PCR-free and label-free) and quantitatively detected, up to the single cell limit. To realize these goals, we propose a new method for the miniaturization of DNA arrays to the nano-scale, which has the unique capability of controlling the packing quality of the deposited bio- molecules. We used NanoGrafting, a nano-lithography technique based on atomic force microscopy (AFM), to fabricate well ordered thiolated single stranded (ss)-DNA nano-patches within a self-assembled monolayer (SAM) of inert thiols on gold surfaces. By varying the “writing” parameters, in particular the number of scan lines, we were able to vary the density of the supported DNA molecules inside the nano-patches in a controlled manner. Our findings can be resumed in two parts: 1) Combining accurate height and compressibility measurements, before and after hybridization, we demonstrate that high-density ss-DNA nanografted patches hybridize with high efficiency, and that, contrary to current understanding, is not the density of probe molecules to be responsible for the lack of hybridization observed in high density ss-DNA SAMs, but the poor quality of their structure. 2) Dpn II enzymatic reactions were carried out over nanopatches with different molecular density and different geometries. Using nanopatch height measurements we are able to show that the capability of the Dpn II enzyme to reach and react at the recognition site significantly depends on the molecular density in the nanopatches. In particular the inhibition of the reaction follows a step-wise fashion at relatively low DNA densities. These findings suggest that, due to the enzyme size, it is possible to tune the efficiency of an enzymatic reaction within surface-bound DNA nanostructures by changing only the crowding of DNA on the surface and without introducing any further physical or chemical variable.XIX Ciclo197

The atomic force microscopy as a lithographic tool: nanografting of DNA nanostructures for biosensing applications.

Current in vitro techniques cannot accurately identify small differences in concentration in samples containing few molecules in single or few cells. Nanotechnology overcomes these limitations with the possibility of measuring protein amounts down to a hundred molecules and subnanomolar concentrations and in nanoliter to picoliter volumes. The nanoscale approach, therefore, permits measurements in samples consisting of single or few cells. Atomic force microscopy (AFM) nanografting can be utilized to prepare DNA nanopatches of different sizes (from few hundreds of nanometers to few microns in size) onto which DNA-antibody conjugates can be anchored through DNA-directed immobilization. AFM height measurements are used to assess the binding of the proteins as well as their subsequent interaction with other components, such as specific proteins from the serum. Recent results have contributed to demonstrate that nanografted patch arrays are well suited for application in biosensing and could enable the fabrication of multifeature protein nanoarrays

Enzymatic Reactions in Nano-Confined DNA Monolayers

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On the Fractionation and Physicochemical Characterisation of Self-Assembled Chitosan–DNA Polyelectrolyte Complexes

Chitosan is extensively studied as a carrier for gene delivery and is an attractive non-viral gene vector owing to its polycationic, biodegradable, and biocompatible nature. Thus, it is essential to understand the chemistry of self-assembled chitosan–DNA complexation and their structural and functional properties, enabling the formation of an effective non-viral gene delivery system. In this study, two parent chitosans (samples NAS-032 and NAS-075; Mw range ~118–164 kDa) and their depolymerised derivatives (deploy nas-032 and deploy nas-075; Mw range 6–14 kDa) with degrees of acetylation 43.4 and 4.7%, respectively, were used to form polyelectrolyte complexes (PECs) with DNA at varying [–NH3+]/[–PO4−] (N/P) molar charge ratios. We investigated the formation of the PECs using ζ-potential, asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS), refractive index (RI), ultraviolet (UV) and dynamic light scattering (DLS) detectors, and TEM imaging. PEC formation was confirmed by ζ-potential measurements that shifted from negative to positive values at N/P ratio ~2. The radius of gyration (Rg) was determined for the eluting fractions by AF4-MALS-RI-UV, while the corresponding hydrodynamic radius (Rh), by the DLS data. We studied the influence of different cross-flow rates on AF4 elution patterns for PECs obtained at N/P ratios 5, 10, and 20. The determined rho shape factor (ρ = Rg/Rh) values for the various PECs corresponded with a sphere morphology (ρ ~0.77–0.85), which was consistent with TEM images. The results of this study represent a further step towards the characterisation of chitosan–DNA PECs by the use of multi-detection AF4 as an important tool to fractionate and infer aspects of their morphology