Polynucleotide: Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose®. Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG

New insights for using self-assembly materials to improve the detection stability in label-free DNA-chip and immuno-sensors

This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene-glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene-glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8-11 nF/mm(2) for antibody layers and close to 6 nF/mm(2) for DNA probes. It is in the range of 10-12 nF/mm(2) and of 4-6 nF/mm(2) for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11-37% and of 0,23-0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene-glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene-glycol functional groups and to solution ions not entering into probe films is finally proposed. (C) 2008 Elsevier B.V. All rights reserved

The Interplay between Chemistry and Mechanics in the Transduction of a Mechanical Signal into a Biochemical Function

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single molecule methodologies, through their capability to introduce the force in studies of biological processes in which mechanical stresses are developed, are unveiling subtle intertwining mechanisms between chemistry and mechanics and in particular are revealing how chemistry can control mechanics. The possibility that chemistry interplays with mechanics should be always considered in biochemical studies.Comment: 50 pages, 18 figure

The dynamic properties of an intramolecular transition from DNA duplex to cytosine-thymine motif triplex.

We here report that the formation and breakdown of an intramolecular cytosine-thymine (CT) motif DNA triple-helix can be performed repeatedly, quickly and independently of its local concentration without performance reduction over successive cycles; as a consequence, we propose that this set of characteristics makes the DNA duplex-triplex transition an ideal candidate to power simple nanometer-scale devices capable of maintaining effective performance regardless of their local concentration

Inside the small length and energy scales of the world of the individual biological molecules.

Atomic force microscopy (AFM) has proved to be an essential tool of structural biology, being able not only to image but also to manipulate single biological molecules. These techniques make it possible to investigate the nanometer scale structure of single biological macromolecules and to study how an external force drives single biological molecules towards non-equilibrium conformations, by stretching and breaking bonds and interactions. This chapter focuses on the capabilities of the AFM-based single molecule methodologies to bring us into the nanometer-scale world of the single DNA molecules and into the pico-Newton force-scales of the interactions that sustain the protein folding

Immobilization of double functionnalized carbon nanotubes on glassy carbon electrodes for the electrochemical sensing of the biotin-avidin affinity

International audienceMulti-walled carbon nanotubes (MWCNTs) double functionalized with redox-active ferrocene and biotin (Fc-Biot-MWCNTs) were synthesized and used for the electrochemical detection of avidin. After dispersion in perfluorosulfonated polymer Nafion and immobilization on the electrode surfaces, the cyclic voltammetry response of the modified electrodes showed in aqueous medium a quasi-reversible one-electron system at 0.46 V vs. SCE, assigned to the bound ferrocene/ferrocenium redox couple. Upon the addition of avidin in the range 0.9-20 nM, a stepwise decrease of both anodic and cathodic peak currents ascribed to the ferrocene was observed. These electrochemical changes are specifically due to the formation of biotin-avidin complex and are explained by complexation-induced modifications in the environment of covalently bound ferrocene

Single Molecule Force Spectroscopy study of the coordination bond between a histidine tag and the nickel-nitrilotriacetate group.

Nowadays several methods used for the purification of recombinant proteins are based on the formation of a coordination bond between the nickel(II)-nitrilotriacetate (NTA) group present on a chromatographic matrix and a stretch of six consecutive histidine residues (6XHis-tag) appended to the primary sequence of the protein [1]. Measuring the force anchoring the His-tagged proteins on the Ni2+-NTA functionalized matrices at the single molecule level can provide an insight in the chemical details of the bond, which could then be used to design possible improvement strategies. This approach has been already explored by different groups which measured the force of the Ni2+-NTA-(His)6 bond obtaining highly variable results [2,3,4]. Probably, these differences derived from the different experimental setups used. We faced such a problem by using an internal force gauge constituted by a dsDNA linker tethering the nitrilotriacetate (NTA) group to the surface. The mechanical properties of dsDNA have been in fact thoroughly investigated by different groups at the single molecule level [5,6]. We constructed a DNA molecule presenting a Ni2+-NTA group at one end and a thiol group, for the binding onto a gold surface, at the other end. Force-distance curves between the AFM tip, previously functionalised with a CG6H6 peptide, and the DNA bound to the surface were collected. The formation of the desired coordination bond leads to a force curve with 3 phases: (1) entropic stretching of the DNA linker; (2) overstretching transition of the linker, generating a plateau whose length is equal to 70% of that of the employed DNA; (3) detachment of the probe, which corresponds to the breaking of the coordination bond. Preliminary results show the overstretching plateau at 50 pN, a value close to the earlier reported ones, followed by the two possible rupture force profiles: either a single entropic stretching represented by classical Worm Like Chain (WLC) or a more complex profile which seems to be the combination of two WLC. This means that there are two possible scenarios in our experiments. In the first case, after the overstretch, the DNA linker (in the S-DNA form) is extended till the force reaches the value of the rupture of the Ni2+-NTA-(His)6 bond; in the second case the stretching of the S-DNA is followed by a melting process leading to the partial separation of the DNA strands. A description of the experiments and the values of the forces measured for the Ni2+-NTA-(His)6 bonds, which seems in agreement with the one measured by the Hinterdorfer group [3], will be presented. [1] Hochuli E et al. (1987) J. Chromatogr., 411: 177. [2] Conti M et al. (2000) Angew. Chem. Int. Ed., 39: 215. [3] Kienberger F. et al (2000) Single Mol., 1: 59. [4] Schmitt L. et al. (2000) Biophys J., 78: 3275. [5] Smith, S. B. et al. (1996) Science. 271: 795. [6] Rief, M. et al. (1999) Nature Struct. Biol. 6: 346

Chip cleaning and regeneration for electrochemical sensor arrays

Sensing systems based on electrochemical detection have generated great interest because electronic readout may replace conventional optical readout in microarray. Moreover, they offer the possibility to avoid labelling for target molecules. A typical electrochemical array consists of many sensing sites. An ideal microfabricated sensor-chip should have the same measured values for all the equivalent sensing sites (or spots). To achieve high reliability in electrochemical measurements, high quality in functionalization of the electrodes surface is essential. Molecular probes are often immobilized by using alkanethiols onto gold electrodes. Applying effective cleaning methods on the chip is a fundamental requirement for the formation of densely-packed and stable self-assembly monolayers. However, the available well-known techniques for chip cleaning may not be so reliable. Furthermore, it could be necessary to recycle the chip for reuse. Also in this case, an effective recycling technique is required to re-obtain well cleaned sensing surfaces on the chip. This paper presents experimental results on the efficacy and efficiency of the available techniques for initial cleaning and further recycling of micro-fabricated chips. Piranha, plasma, reductive and oxidative cleaning methods were applied and the obtained results were critically compared. Some interesting results were attained by using commonly considered cleaning methodologies. This study outlines oxidative electrochemical cleaning and recycling as the more efficient cleaning procedure for electrochemical based sensor arrays. (C) 2009 Elsevier B.V. All rights reserved

Nanoscale film structure related to capacitive effects in ethylene-glycol monolayers

A large improvement of the electrochemical capacitive detection of ssDNA and antigens by using ethylene-glycol alkanethiol monolayers has been recently demonstrated. Alkanethiol monolayers without ethylene-glycol functionalization do not show the required electrochemical stability. In this paper, we demonstrate that both phenomena are related to their morphological structure at nano-scale. Ethylene-glycol monolayers were compared with similar films without ethylene-glycol function. Capacitance measurements were used to verify the improved capacitance stability of the electrochemical interface. AFM images were used to investigate films packing and morphology at the nano-scale. This letter demonstrates that films without ethylene-glycol segment present deep grooves which are related to electrochemical instability while the improvement in case of ethylene-glycols is due to the absence of such a feature. (C) 2009 Elsevier B.V. All rights reserved