Electrochemically Modified Carbon and Chromium Surfaces for AFM Imaging of Double‐Strand DNA Interaction with Transposase Protein

Abstract

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.

Kinetic Analysis of the Interaction of Mos1 Transposase with its Inverted Terminal Repeats Reveals New Insight into the Protein–DNA Complex Assembly

“…The Au-AT quartz crystal was modified as previously described by aryldiazonium salt electrografting. [14] The electrochemical measurements were realised on a VMP2 potentiostat/galvanostat (Bio-Logic). Briefly, a compact organic layer containing Ar-SO 3 H groups was made by electrografting an aryldiazonium salt formed in situ from sulfanilic acid.…”

“…DNA immobilisation was performed on a flat chromium surface functionalised by a previously described procedure. [14] b-MOS1 or e-MOS1 was then injected at 90 nm to ensure that the transposase concentration was greater than that of the DNA at the imaging surface. After washing, the surface was imaged by tapping mode AFM in air.…”

“…Previous studies have used the quartz crystal microbalance (QCM) method to record the formation of double-stranded DNA by DNA polymerase II, [11] the interaction between doublestrand DNA (dsDNA) and the endonuclease EcoRV [12] and the detection of PCR products [13] or transposase-ITR complexes. [14] Here, we report that QCM can be used to determine not only K d, but also the association and dissociation rates (k on and k off ) of the interaction between DNA immobilised on a quartz crystal and a transposase in solution. Surprisingly, the reactivity of the MOS1 protein was shown to depend significantly on the expression system (eukaryotic or prokaryotic).…”

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

“…[21] The development of HsRad51 inhibitors is, therefore, of pharmacological importance. [30] Herein, a similar method was used to modify a gold surface. Many of these previous studies are based on surface plasmon resonance (i.e.…”

“…[23][24][25][26][27][28][29] We have recently developed a surface modification with a ArSO 2 Cl (Ar = aryl) organic layer that allows the fixation of long dsDNA containing ssDNA extremities on a chromium surface. [30] Herein, a similar method was used to modify a gold surface. The gold quartz crystal and the gold plate for AFM were at first modified by an organic layer (ArSO 3 H) by using the electrografting of aryl radicals obtained by electroreduction of aryldiazonium salts, and then they were activated by a solution of PCl 5 in CH 2 Cl 2 to obtain the final organic ArSO 2 Cl layer.…”