DNA imaged on a HOPG electrode surface by AFM with controlled potential

Single-molecule AFM imaging of single-stranded and double-stranded DNA molecules self-assembled from solution onto a HOPG electrode surface is reported. The interaction of DNA with the hydrophobic surface induced DNA aggregation, overlapping, intra- and intermolecular interactions. Controlling the electrode potential and using the phase images as a control method, to confirm the correct topographical characterization, offers the possibility to enlarge the capability of AFM imaging of DNA immobilized onto conducting substrates, such as HOPG. The application of a potential of +300 mV (versus AgQRE) to the HOPG enhanced the robustness and stability of the adsorbed DNA molecules, increasing the electrostatic interaction between the positively charged electrode surface and the negatively charged DNA sugar-phosphate backbone.http://www.sciencedirect.com/science/article/B6W72-4D5X9NS-1/1/d1528546598a5fb98abb8331a760556

Synthetic oligonucleotides: AFM characterisation and electroanalytical studies

One of the most important steps in designing more sensitive and stable DNA based biosensors is the immobilisation procedure of the nucleic acid probes on the transducer surface, while maintaining their conformational flexibility. MAC Mode AFM images in air demonstrated that the oligonucleotide sequences adsorb spontaneously on the electrode surface, showing the existence of pores in the adsorbed layer that reveal big parts of the electrode surface, which enables non-specific adsorption of other molecules on the uncovered areas. The electrostatic immobilisation onto a glassy carbon electrode followed by hybridisation with a complementary sequence and control with a non-complementary sequence was studied using differential pulse voltammetry and electrochemical impedance spectroscopy. Changes in the oxidation currents of guanosine and adenosine were observed after hybridisation events as well as after control experiments. Modification of the double layer capacitance that took place after hybridisation or control experiments showed that non-specific adsorption of complementary or non-complementary sequences occur allowing the formation of a mixed multilayer.http://www.sciencedirect.com/science/article/B6W72-4GP1VK1-1/1/c44e3d2a4c722d5cec59a4d09d0a744

AFM and electroanalytical studies of synthetic oligonucleotide hybridization

The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.http://www.sciencedirect.com/science/article/B6TFC-4CYNVTG-7/1/9dd6257bfec8e07f1a15905be07dbd1

Graphite–castor oil polyurethane composite electrode surfaces – AFM morphological and electrochemical characterisation

Graphite–castor oil polyurethane composite electrodes with different graphite weight percentages, 30–70% graphite–polyurethane w w−1, were morphologically studied by atomic force microscopy (AFM) and voltammetry. AFM images and r.m.s. roughness measurements demonstrated that the polyurethane roughness decreased with increasing the graphite content, composites of 50% and 60% graphite–polyurethane w w−1 showing the smother electrode surface. The electrochemical characterisation was performed in solutions of K4Fe(CN)6 by cyclic voltammetry and impedance spectroscopy. For compositions of 60% and 70% graphite–polyurethane w w−1, the cyclic voltammetry results showed the K4Fe(CN)6 system reversibility. The charge transfer resistance, determined from the EIS spectra, decreased significantly with increasing the graphite/polyurethane ratio, and the capacitance increased for higher graphite percentages. AFM and voltammetric results enable to conclude that 60% graphite–polyurethane w w−1 was the optimal composition for the preparation of the graphite–polyurethane composite electrodes

DNA Interaction with Palladium Chelates of Biogenic Polyamines Using Atomic Force Microscopy and Voltammetric Characterization

The interaction of double-stranded DNA with two polynuclear Pd(II) chelates with the biogenic polyamines spermidine (Spd) and spermine (Spm), Pd(II)-Spd and Pd(II)-Spm, as well as with the free ligands Spd and Spm, was studied using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite (HOPG) surface, voltammetry at a glassy carbon (GC) electrode, and gel electrophoresis. The AFM and voltammetric results showed that the interaction of Spd and Spm with DNA occurred even for a low concentration of polyamines and caused no oxidative damage to DNA. The Pd(II)-Spd and Pd(II)-Spm complexes were found to induce greater morphological changes in the dsDNA conformation, when compared with their ligands. The interaction was specific, inducing distortion and local denaturation of the B-DNA structure with release of some guanine bases. The DNA strands partially opened give rise to palladium intra- and interstrand cross-links, leading to the formation of DNA adducts and aggregates, particularly in the case of the Pd(II)-Spd complex

Atomic Force Microscopy and Voltammetric Investigation of Quadruplex Formation between a Triazole-Acridine Conjugate and Guanine-Containing Repeat DNA Sequences

The interactions of the Tetrahymena telomeric repeat sequence d(TG4T) and the polyguanylic acid (poly(G)) sequence with the quadruplex-targeting triazole-linked acridine ligand GL15 were investigated using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite and voltammetry at a glassy carbon electrode. GL15 interacted with both sequences, in a time dependent manner, and G-quadruplex formation was detected. AFM showed the adsorption of quadruplexes as small d(TG4T) and poly(G) spherical aggregates and large quadruplex-based poly(G) assemblies, and voltammetry showed the decrease and disappearance of GL15 and guanine oxidation peak currents and appearance of the G-quadruplex oxidation peak. The GL15 molecule strongly stabilized and accelerated G-quadruplex formation in both Na+ and K+ ion-containing solution, although only K+ promoted the formation of perfectly aligned tetra-molecular G-quadruplexes. The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the G-quadruplex complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string.Financial support from Fundação para a Ciência e Tecnologia (FCT), Grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim), Project Grant (A.D.R. Pontinha), projects PTDC/SAU-BMA/118531/2010, PTDC/QEQ-MED/0586/2012, PEst-C/EME /UI0285/2013 and CENTRO-07-0224-FEDER-002001 (MT4MOBI) (co-financed by the European Community Fund FEDER), FEDER funds through the program COMPETE - Programa Operacional Factores de Competitividade is gratefully acknowledged. Work in the S.N. laboratory was supported by Programme Grant No. C129/A4489, from Cancer Research UK, and by the FP6 framework grant “Molecular Cancer Medicine” from the EU. S.S. was a Maplethorpe Fellow of The University of London.Peer reviewe

AFM and electroanalytical studies of synthetic oligonucleotide hybridization

Abstract The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN

Competing Interactions in DNA Assembly on Graphene

We study the patterns that short strands of single-stranded DNA form on the top graphene surface of graphite. We find that the DNA assembles into two distinct patterns, small spherical particles and elongated networks. Known interaction models based on DNA-graphene binding, hydrophobic interactions, or models based on the purine/pyrimidine nature of the bases do not explain our observed crossover in pattern formation. We argue that the observed assembly behavior is caused by a crossover in the competition between base-base pi stacking and base-graphene pi stacking and we infer a critical crossover energy of eV. The experiments therefore provide a projective measurement of the base-base interaction strength

A role for hydrophobicity in a Diels–Alder reaction catalyzed by pyridyl-modified RNA

New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels–Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu2+, as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification's role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism

Microscopia de varrimento de sonda de DNA e de outras moléculas biológicas absorvidas na superfície de eléctrodos

Tese de doutoramento em Bioquímica (Técnicas Bioquímicas) apresentada à Fac. de Ciências e Tecnologia de CoimbraO objectivo deste trabalho foi o estudo dos processos de adsorção da guanina, da desoxiguanosina, de vários tipos de ácidos nucleicos e do fármaco adriamicina, na superfície de eléctrodos de ouro e grafite pirolítica altamente orientada (HOPG) por microscopia de túnel de varrimento (STM) e microscopia de força atómica (AFM) com vista à caracterização morfológica da superfície de eléctrodos modificados com DNA, os biossensores electroquímicos com DNA. A adsorção espontânea na superfície de HOPG, a partir de soluções saturadas de guanina, determinou a formação de múltiplas camadas moleculares estáveis e resistentes e a adsorção com potencial aplicado permitiu a condensação da guanina, conjuntamente com dímeros, trímeros e outros produtos de oxidação da guanina, formando filmes espessos, heterogéneos, menos compactos e resistentes. A desoxiguanosina adsorveu espontaneamente na superfície de ouro, por intermédio de processos de nucleação e crescimento, sendo observados por STM núcleos de desoxiguanosina, alinhados em filas. As características da adsorção da desoxiguanosina foram estudadas em função do potencial aplicado ao eléctrodo de ouro e de HOPG. As imagens de AFM de DNA hélice simples, de DNA hélice dupla, do ácido poliadenílico e de oligonucleotídeos sintéticos, imobilizados na superfície de HOPG, mostraram a tendência das moléculas para uma auto–organização espontânea na superfície. Os ácidos nucleicos condensaram-se em redes bidimensionais densas, extensas e cobrindo uniformemente toda a superfície. A interacção com a superfície hidrofóbica de HOPG induziu sobreposições de moléculas e interacções intramoleculares e intermoleculares. A aplicação dum potencial positivo ao eléctrodo de HOPG durante a adsorção aumentou a robustez e a estabilidade mecânica dos filmes de DNA, devido a múltiplas interacções electrostáticas estabelecidas entre o esqueleto de açúcar–fosfato do DNA carregado negativamente e a superfície carregada positivamente. O controlo do pH da solução de DNA foi determinante para a cobertura da superfície. A adsorção espontânea de moléculas de adriamicina na superfície de HOPG e a formação de monocamadas estáveis para baixas concentrações de adriamicina e tempos curtos de adsorção foi investigada, e é determinante na detecção da interacção DNA-adriamicina utilizando um biossensor electroquímico com DNA