Atomic force microscopy characterization of synthetic pyrimidinic oligodeoxynucleotides adsorbed onto an HOPG electrode under applied potential

In order to better understand the adsorption mechanism of nucleic acids at electrode surface, MAC Mode atomic force microscopy (MAC Mode AFM) was used to investigate the adsorption of a 10-base synthetic oligodeoxynucleotide (ODN) with the sequence 5'-CTTTTTCTTT-3' containing only pyrimidinic bases, onto a highly oriented pyrolytic graphite (HOPG) electrode. Free adsorption and adsorption at applied potentials of +0.30 and +0.65 V versus AgQRE, were carried out. AFM images in air demonstrated that the molecules adsorb spontaneously on the electrode surface. The ODNs have the tendency to self-assemble from solution onto the solid support in a tight and well-spread two-dimensional lattice covering the entire surface, showing the existence of different molecular conformations and exposing parts of the HOPG surface. The degree of surface coverage and the adsorption pattern were directly dependent on ODN concentration and immobilization procedure. During free adsorption, the hydrophobic interactions of the ODNs with the HOPG represent the main adsorption mechanism. When a positive potential is applied to the HOPG, electrostatic interactions between the positively charged electrode surface and the negatively charged sugar-phosphate backbones of ODNs are predominant.http://www.sciencedirect.com/science/article/B6TG0-4JVSVMG-2/1/0c861dc43bb4382dd9c195a0ebd408b

Adsorption of synthetic homo- and hetero-oligodeoxynucleotides onto highly oriented pyrolytic graphite: Atomic force microscopy characterization

DNA adsorption on electrode surfaces is of fundamental interest for the development of DNA-based biosensors. The free adsorption of 10-mer synthetic oligodeoxynucleotides (ODNs) onto highly oriented pyrolytic graphite (HOPG) surfaces was studied using Magnetic AC mode atomic force microscopy (MAC Mode AFM). The mechanism of interaction of nucleic acids with carbon electrode surfaces was elucidated, using 10-mer synthetic homo- and hetero-ODNs sequences of known base sequences, because they allow clear interpretation of the experimental data. AFM images in air revealed different adsorption patterns and degree of HOPG surface coverage for the ODNs, and correlation with the individual structure and base sequence of each ODN molecule will be presented. The results demonstrated that the hydrophobic interactions with the HOPG hydrophobic surface explain the main adsorption mechanism, although other effects such as electrostatic and Van der Waals interactions may contribute to the free adsorption process. The ODNs interacted differently with the HOPG surface, according to the ODN sequence hydrophobic characteristics, being directly depending on the molecular mass, the hydrophobic character of the individual bases and on the secondary structure of the molecule. The importance of the type of base existent at the ODN chain extremities on the adsorption process was investigated and different adsorption patterns were obtained with ODN sequences composed by the same group of bases aligned in a different order.http://www.sciencedirect.com/science/article/B6TFB-4J6WR05-2/1/64ef57ec21856f05e10206d1d7b1033

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase

Type II restriction–modification (RM) systems are the most widespread bacterial antiviral defence mechanisms. DNA methyltransferase SsoII (M.SsoII) from a Type II RM system SsoII regulates transcription in its own RM system in addition to the methylation function. DNA with a so-called regulatory site inhibits the M.SsoII methylation activity. Using circular permutation assay, we show that M.SsoII monomer induces DNA bending of 31° at the methylation site and 46° at the regulatory site. In the M.SsoII dimer bound to the regulatory site, both protein subunits make equal contributions to the DNA bending, and both angles are in the same plane. Fluorescence of TAMRA, 2-aminopurine, and Trp was used to monitor conformational dynamics of DNA and M.SsoII under pre-steady-state conditions by stopped-flow technique. Kinetic data indicate that M.SsoII prefers the regulatory site to the methylation site at the step of initial protein–DNA complex formation. Nevertheless, in the presence of S-adenosyl-l-methionine, the induced fit is accelerated in the M.SsoII complex with the methylation site, ensuring efficient formation of the catalytically competent complex. The presence of S-adenosyl-l-methionine and large amount of the methylation sites promote efficient DNA methylation by M.SsoII despite the inhibitory effect of the regulatory site

A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.

Nicking endonucleases (NEases) selectively cleave single DNA strands in double-stranded DNAs at a specific site. They are widely used in bioanalytical applications and in genome editing; however, the peculiarities of DNA-protein interactions for most of them are still poorly studied. Previously, it has been shown that the large subunit of heterodimeric restriction endonuclease BspD6I (Nt.BstD6I) acts as a NEase. Here we present a study of interaction of restriction endonuclease BspD6I with modified DNA containing single non-nucleotide insertion with an azobenzene moiety in the enzyme cleavage sites or in positions of sugar-phosphate backbone nearby. According to these data, we designed a number of effective stimulus-responsive oligonucleotide inhibitors bearing azobenzene or triethylene glycol residues. These modified oligonucleotides modulated the functional activity of Nt.BspD6I after cooling or heating. We were able to block the cleavage of T7 phage DNA by this enzyme in the presence of such inhibitors at 20-25°C, whereas the Nt.BspD6I ability to hydrolyze DNA was completely restored after heating to 45°C. The observed effects can serve as a basis for the development of a platform for regulation of NEase activity in vitro or in vivo by external signals