Electrodes for Membrane Surface Science. Bilayer Lipid Membranes Tethered by Commercial Surfactants on Electrochemical Sensors.

Commercial surfactants, which are inexpensive and abundant, were covalently grafted to flat and transparent electrodes, and it appears to be a simple functionalization route to design biomembrane sensors at large-scale production. Sparsely tethered bilayer lipid membranes (stBLM) were stabilized using such molecular coatings composed of diluted anchor-harpoon surfactants that grab the membrane with an alkyl chain out of a PEGylated-hydrogel layer, which acts as a soft hydration cushion. The goal of avoiding the synthesis of complex organic molecules to scale up sensors was achieved here by grafting nonionic diblock oligomers (Brij58 = C xH2 x+1(OCH2CH2) nOH with x = 16 and n = 23) and PEO short chains ((OCH2CH2) nOH with n = 9 and n = 23) from their hydroxyl (-OH) end-moiety to a monolayer of -Ar-SO2Cl groups, which are easy to form on electrodes (metals, semiconducting materials, ...) from aryl-diazonium salt reduction. A hybrid molecular coating on gold, with scarce Ar-SO2-Brij58 and PEO oligomers, was used to monitor immobilization and fusion kinetics of DOPC small unilamellar vesicles (SUV) by both quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) techniques. Using flat and transparent thin chromium film electrodes, we designed biosensors to couple surface sensitive techniques for membranes, including X-ray reflectivity (XRR), atomic force microscopy (AFM) with subnanometer resolution, and optical microscopy, such as fluorescence recovery after photobleaching measurements (FRAP), in addition to electrochemistry techniques (cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS)). The advantages of this biomembrane-sensing platform are discussed for research and applications.

The epigenetic regulation of HsMar1, a human DNA transposon.

BACKGROUND: Both classes of transposable elements (DNA and RNA) are tightly regulated at the transcriptional level leading to the inactivation of transposition via epigenetic mechanisms. Due to the high copies number of these elements, the hypothesis has emerged that their regulation can coordinate a regulatory network of genes. Herein, we investigated whether transposition regulation of HsMar1, a human DNA transposon, differs in presence or absence of endogenous HsMar1 copies. In the case where HsMar1 transposition is regulated, the number of repetitive DNA sequences issued by HsMar1 and distributed in the human genome makes HsMar1 a good candidate to regulate neighboring gene expression by epigenetic mechanisms. RESULTS: A recombinant active HsMar1 copy was inserted in HeLa (human) and CHO (hamster) cells and its genomic excision monitored. We show that HsMar1 excision is blocked in HeLa cells, whereas CHO cells are competent to promote HsMar1 excision. We demonstrate that de novo HsMar1 insertions in HeLa cells (human) undergo rapid silencing by cytosine methylation and apposition of H3K9me3 marks, whereas de novo HsMar1 insertions in CHO cells (hamster) are not repressed and enriched in H3K4me3 modifications. The overall analysis of HsMar1 endogenous copies in HeLa cells indicates that neither full-length endogenous inactive copies nor their Inverted Terminal Repeats seem to be specifically silenced, and are, in contrast, devoid of epigenetic marks. Finally, the setmar gene, derived from HsMar1, presents H3K4me3 modifications as expected for a human housekeeping gene. CONCLUSIONS: Our work highlights that de novo and old HsMar1 are not similarly regulated by epigenetic mechanisms. Old HsMar1 are generally detected as lacking epigenetic marks, irrespective their localisation relative to the genes. Considering the putative existence of a network associating HsMar1 old copies and SETMAR, two non-mutually exclusive hypotheses are proposed: active and inactive HsMar1 copies are not similarly regulated or/and regulations concern only few loci (and few genes) that cannot be detected at the whole genome level.

Grafting Commercial Surfactants (Brij, CiEj) and PEG to Electrodes via Aryldiazonium Salts.

Grafting commercial surfactants appears to be a simple way to modify electrodes and conducting interfaces, avoiding the synthesis of complex organic molecules. A new surface functionalization route is presented to build surfactant coatings with monolayer thickness grafting molecules considered as nonreactive. A monolayer of -SO2Cl functions (from a p-benzenesulfonyl chloride) was first electrografted. It showed a high reactivity toward weak nucleophiles commonly found on surfactant end-moieties such as hydroxyl groups (-OH), and it was used to covalently graft the following: (1) nonionic diblock oligomers (Brij or CiEj, CxH2x + (OCH2CH2)nOH with x = 16 and n = 23 for Brij58, x = 16 and n = 10 for Brij C10, and x = 16 and n = 2 for Brij52); (2) poly(ethylene glycol) (PEG) short chains (PEO9 for (OCH2CH2)nOH with n = 9) and mixed formula. The surface modification due to these molecular coatings was investigated in terms of wetting properties and interfacial electrochemistry characteristics (charge transfer resistivity, capacity, and ions dynamics). Built on flat and transparent thin chromium films, Brij and PEO mixed coatings have been proven to be promising coatings for electrochemical biosensor application such as for stabilizing a partially tethered supported biomimetic membrane.

Kinetic analysis of the interaction of Mos1 transposase with its inverted terminal repeats reveals new insight into the protein-DNA complex assembly.

Transposases are specific DNA-binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic-expressed MOS1 showed no cooperativity and displayed a Kd of about 300 nM. In contrast, the eukaryotic-expressed MOS1 generated a cooperative system, with a lower Kd (∼ 2 nm). The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic- and eukaryotic-expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition.

Assessment of DNA binding to human Rad51 protein by using quartz crystal microbalance and atomic force microscopy: effects of ADP and BRC4-28 peptide inhibitor.

The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors.

One-pot in situ mixed film formation by azo coupling and diazonium salt electrografting.

So simple: The in situ synthesis of an aryldiazonium salt and an azo-aryldiazonium salt by azo coupling from sulfanilic acid and aniline is reported. Formation of a mixed organic layer is monitored by cyclic voltammetry and atomic force microscopy. A compact mixed layer is obtained with a global roughness of 0.4 nm and 10-15 % vertical extension in the range 1.5-6 nm.

Electrochemically modified carbon and chromium surfaces for AFM imaging of double-strand DNA interaction with transposase protein.

Carbon and chromium surfaces were modified by electrochemical reduction of a diazonium salt formed in situ from the sulfanilic acid. The organic layer formed was activated by phosphorus pentachloride (PCl(5)) to form a benzene sulfonil chloride (Ar-SO(2)Cl). An electrochemical study of the blocking effect and the activity of this surface was carried out on a carbon electrode. The chromium surface study was completed by X-ray photoelectron spectroscopy and atomic force microscopy to characterize the formation of a compact monolayer (0.8 nm height and roughness 0.2-0.3 nm). The compactness and the activity of this organic monolayer allowed us to affix a length dsDNA with the aim of analyzing the formation of a complex between dsDNA and a protein. The interaction of a transposase protein with its target dsDNA was investigated. The direct imaging of the nucleoproteic complex considered herein gives new insights in the comprehension of transposase-DNA interaction in agreement with biochemical data.

Electrochemistry and reactivity of surface-confined catechol groups derived from diazonium reduction. Bias-assisted Michael addition at the solid/liquid interface.

We have designed a novel catechol-modified electrode that could be used for bias-assisted Michael addition at the solid/liquid interface. The glassy carbon electrode was modified by the electrochemical reduction of a catechol para-substituted phenyldiazonium salt. The electrochemistry of surface-confined catechol moieties was investigated by cyclic voltammetry. The transfer coefficient and apparent surface standard electron-transfer rate constant were obtained using Laviron's theory. We demonstrate that o-quinone moieties linked to the surface remain quite reactive with nucleophilic species by Michael addition at the solid/liquid interface. To demonstrate the versatility of this procedure, 4-nitrobenzyl alcohol, (4-nitrobenzyl)amine, and a ferrocenealkylamine were chosen as nucleophile models due to their well-known redox properties. Electrochemically triggered Michael addition was validated, leading to redox headgroup-tethered surfaces.