Electrochemistry using self-assembled DNA monolayers on highly oriented pyrolytic graphite

Duplex DNA functionalized with pyrene has been utilized to fabricate DNA-modified electrodes on highly oriented pyrolytic graphite (HOPG). Films have been characterized using AFM and radioactive labeling as well as electrochemically. The data obtained are consistent with a close-packed structure in the film with helices oriented in a nearly upright orientation, as seen earlier with the fabrication of thiol-tethered duplexes on gold. Also as on gold, we observe the reduction of DNA-bound intercalators in a DNA-mediated reaction. The reduction of the intercalator is attenuated in the presence of the single-base mismatches, CA and GT, independent of the sequence composition of the oligonucleotide. This sensitivity to single-base mismatches is enhanced when methylene blue reduction is coupled in an electrocatalytic cycle with ferricyanide. The extended potential range afforded by the HOPG surface has allowed us also to investigate the electrochemistry of previously inaccessible metallointercalators, Ru(bpy)2dppz2+ and Os(phen)2dppz2+, at the DNA-modified HOPG surface. These results support the application of DNA-modified HOPG as a convenient and reproducible surface for electrochemical DNA sensors using DNA-mediated charge transport.     

Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

In situ electrochemical distance tunneling spectroscopy of ds-DNA molecules

The electrical conductance of ds-DNA duplexes containing 8-14 base pairs modified at both ends with a -(CH(2))(6)-SH linker was measured in a buffered aqueous solution using electrochemically controlled distance tunneling spectroscopy. The tunneling experiment with self-complementary 5'-(GC)(n)()-3'-(CH(2))(6)-SH (n = 4-7) duplexes attached covalently to a gold STM tip and a Au(111) electrode shows a wide distribution of currents independent of the ds-DNA length. The voltage-induced horizontal orientation of ds-DNA within the junction results in decreased electrical conductance. The lower currents are also observed for ds-DNA molecules containing a single CA base mismatch.     

Ordered self-assembled locked nucleic acid (LNA) structures on gold(111) surface with enhanced single base mismatch recognition capability

Locked nucleic acid (LNA) is a conformationally restricted nucleic acid analogue, which is potentially a better alternative than DNA for application in the nucleic acid based biosensor technologies, due to its efficient and sequence-specific DNA/RNA detection capability and lack of molecule-surface interaction on solid surfaces, compared to DNA. We report, for the first time, a straightforward way (based on simple immersion method) of generating an ordered self-assembled LNA monolayer, which is bioactive, onto a gold(111) surface. This layer is capable of giving rise to a stronger DNA recognition signal (4-4.5 times) than its DNA counterpart, and importantly, it can differentiate between a fully complementary DNA target and that having a single base mismatch, where the mismatch discrimination ratio is almost two times compared to the ratio relevant in case of DNA-based detection. We have presented high-resolution atomic force microscopy (AFM) topographs of the well-defined one-dimensional LNA molecular ordering (few hundred nanometers long) and of the two-dimensional ordered assembly formed over a large area (7 μm × 7 μm) due to parallel positioning of the one-dimensional ordered arrangements. The effects of different parameters such as LNA concentration and incubation time on LNA self-assembly have been investigated. Further, reflection absorption infrared (RAIR) spectroscopy has been applied to obtain information about the orientation of the surface-immobilized LNA molecules for the first time. It has been found that the LNA molecules undergo an orientational transition from the "lying down" to the "upright" configuration in a time scale of few hours.     

Rapid Non‐Crosslinking Aggregation of DNA‐Functionalized Gold Nanorods and Nanotriangles for Colorimetric Single‐Nucleotide Discrimination

Gold nanoparticles modified with DNA duplexes are rapidly and spontaneously aggregated at high ionic strength. In contrast, this aggregation is greatly suppressed when the DNA duplex has a single‐base mismatch or a single‐nucleotide overhang located at the outermost surface of the particle. These colloidal features emerge irrespective of the size and composition of the particle core; however, the effects of the shape remain unexplored. Using gold nanorods and nanotriangles (nanoplatelets), we show herein that both remarkable rapidity in colloidal aggregation and extreme susceptibility to DNA structural perturbations are preserved, regardless of the shape and aspect ratio of the core. It is also demonstrated that the DNA‐modified gold nanorods and nanotriangles are applicable to naked‐eye detection of a single‐base difference in a gene model. The current study corroborates the generality of the unique colloidal properties of DNA‐functionalized nanoparticles, and thus enhances the feasibility of their practical use.     

Detection of non-cross-linking interaction between DNA-modified gold nanoparticles and a DNA-modified flat gold surface using surface plasmon resonance imaging on a microchip

Gold nanoparticles (GNPs) with fully matched DNA duplexes on their surfaces aggregate together without molecular cross-linking at high salt concentrations. The mechanism of this non-cross-linking (NCL) interaction has been elusive. In this paper, NCL interaction between duplex-modified GNPs and a duplex-modified flat gold surface is presented for the first time. This new experimental platform has enabled us to study the NCL interaction between duplexes with different sequences. We immobilized 15-base single-stranded (ss) DNA onto the surfaces of GNPs with a diameter of 40nm and onto a flat gold substrate. The GNPs were hybridized with 15-base ssDNA at a low salt concentration. A microfluidic device was used for simultaneous delivery of the following three components onto the gold substrate: the duplex-modified GNPs, 15-base ssDNA to be hybridized onto the substrate, and NaCl at a high concentration. Adsorption of the GNPs onto the substrate was monitored using surface plasmon resonance imaging. When the GNPs and the substrate had an identical sequence, the adsorption behavior was analogous to the aggregation behavior of GNPs in test tubes. Furthermore, we investigated 12 cases in which the GNPs and the substrate had completely different sequences, and obtained results suggesting that the NCL attraction force primarily depends on the terminal base pairs of the duplexes. This means that the main mechanism of the NCL interaction is likely to be inter-duplex base stacking rather than formation of Holliday junctions.     

Ground-state recovery following UV excitation is much slower in G x C-DNA duplexes and hairpins than in mononucleotides

Excited states in double-stranded oligonucleotides containing G.C base pairs were studied by femtosecond transient absorption spectroscopy. Relaxation to the electronic ground state occurs about 10 times more slowly in the duplexes and hairpins studied on average than in the individual mononucleotides of G and C. Detection of long-lived excited states in G.C oligonucleotides complements the earlier observation of slow ground-state recovery in A.T DNA, showing that excited states with picosecond lifetimes are formed in DNAs containing either kind of base pair. The results show further that Watson-Crick G.C base pairs in these base-paired and base-stacked duplexes do not enable subpicosecond relaxation to the electronic ground state. A model is proposed in which fluorescent exciton states decay rapidly and irreversibly to dark exciplex states. This model explains the seemingly contradictory observations of femtosecond fluorescence and slower, picosecond recovery of the ground-state population.     

Asymmetric charge partitioning upon dissociation of DNA duplexes

Upon collisional activation, a series of DNA duplexes exhibited a significant degree of asymmetric dissociation with respect to charge partitioning among the single strands. That is, the charge states of the single strand product ions did not equal q/2 for even precursor charge states or (q + 1)/2 and (q − 1)/2 for odd precursor charge states (where q is the charge of the precursor). The factors that affect this asymmetric charge partitioning were assessed. The smaller, lower charged duplexes resulted in more symmetric dissociation compared with larger duplexes in higher charge states, which displayed a high degree of asymmetry upon dissociation. The composition of the duplexes influenced charge partitioning, with those containing a greater number of A/T base pairs showing more symmetric dissociation relative to the more G/C rich duplexes. The use of higher collisional energies resulted in significantly more asymmetric dissociation. Comparisons were made with the dissociation behavior previously studied for protein noncovalent complexes and past studies of the gas-phase conformations and dissociation of DNA complexes.     

Immobilization of amine-modified oligonucleotides on aldehyde-terminated alkanethiol monolayers on gold

Chemistry is described for the fabrication of DNA arrays on gold surfaces. Alkanethiols modified with terminal aldehyde groups are used to prepare a self-assembled monolayer (SAM). The aldehyde groups of the monolayer may be reacted with amine-modified oligonucleotides or other amine-bearing biomolecules to form a Schiff base, which may then be reduced to a stable secondary amine by treatment with sodium cyanoborohydride. The surface modifications and reactions are characterized by polarization modulation Fourier transform infrared reflection absorption spectroscopy (PM-FTIRRAS), and the accessibility, binding specificity, and stability of the DNA-modified surfaces are demonstrated in hybridization experiments.     

DNA electrochemistry with tethered methylene blue

Methylene blue (MB'), covalently attached to DNA through a flexible C(12) alkyl linker, provides a sensitive redox reporter in DNA electrochemistry measurements. Tethered, intercalated MB' is reduced through DNA-mediated charge transport; the incorporation of a single base mismatch at position 3, 10, or 14 of a 17-mer causes an attenuation of the signal to 62 ± 3% of the well-matched DNA, irrespective of position in the duplex. The redox signal intensity for MB'-DNA is found to be least 3-fold larger than that of Nile blue (NB)-DNA, indicating that MB' is even more strongly coupled to the π-stack. The signal attenuation due to an intervening mismatch does, however, depend on DNA film density and the backfilling agent used to passivate the surface. These results highlight two mechanisms for reduction of MB' on the DNA-modified electrode: reduction mediated by the DNA base pair stack and direct surface reduction of MB' at the electrode. These two mechanisms are distinguished by their rates of electron transfer that differ by 20-fold. The extent of direct reduction at the surface can be controlled by assembly and buffer conditions.     

Solution structure of a DNA duplex containing a guanine-difluorotoluene pair: a wobble pair without hydrogen bonding?

The incorporation of synthetic nucleoside analogues into DNA duplexes provides a unique opportunity to probe both structure and function of nucleic acids. We used 1H and 19F NMR and molecular dynamics calculations to determine the solution structures of two similar DNA decamer duplexes, one containing a central G-T mismatched or "wobble" base pair, and one in which the thymine in this base pair is replaced by difluorotoluene (a thymine isostere) creating a G-F pair. Here, we show that the non-hydrogen-bonding G-F pair stacks relatively well into the helix and that the distortions caused by each non-Watson-Crick G-T or G-F base pair are quite localized to a three base pair site around the mismatch. A detailed structural analysis reveals that the absence of hydrogen bonding introduces more dynamic motion into the G-F pair relative to G-T and permits the G-F pair to exhibit stacking and conformational features characteristic of both a Watson-Crick base pair (on the guanine containing strand) and a wobble base pair (on the strand containing the difluorotoluene). We used these results to posit a rationale for recognition and repair of mismatch sites in DNA.     

Non-covalent monolayer-piercing anchoring of lipophilic nucleic acids: preparation, characterization, and sensing applications

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.     

Dynamic behavior of DNA base pairs containing 8-oxoguanine

The process by which DNA repair enzymes recognize and selectively excise damaged bases in duplex DNA is fundamental to our mechanistic understanding of these critical biological reactions. 8-Oxoguanine (8-oxoG) is the most common form of oxidative DNA damage; unrepaired, this lesion generates a G:C-->T:A mutation. Central to the recognition and repair of DNA damage is base extrusion, a process in which the damaged base lesion or, in some cases, its partner disengages from the helix and is bound to the enzyme's active site where base excision takes place. The conformation adopted by 8-oxoG in duplex DNA is affected by the base positioned opposite this lesion; conformational changes may also take place when the damaged base binds to its cognate repair enzyme. We performed unrestrained molecular dynamics simulations for several 13-mer DNA duplexes. Oligomers containing G:C and 8oxoG:C pairs adopted Watson-Crick geometries in stable B-form duplexes; 8oxoG showed increased local and global flexibility and a reduced barrier to base extrusion. Duplexes containing the G:A mismatch showed much larger structural fluctuations and failed to adopt a well-defined structure. For the 8oxoG:A mismatch that is recognized by the DNA glycosylase MutY, the damaged nucleoside underwent spontaneous and reproducible anti-->syn transitions. The syn conformation is thermodynamically preferred. Steric hindrance and unfavorable electrostatics associated with the 8oxoG O8 atom in the anti conformation were the major driving forces for this transition. Transition events follow two qualitatively different pathways. The overall anti-->syn transition rate and relative probability of the two transition paths were dependent on local sequence context. These simulations indicate that both the dynamic and equilibrium behavior of the duplex change as a result of oxidation; these differences may provide valuable new insight into the selective action of enzymes on damaged DNA.     

Ultrafast cooling of photoexcited electrons in gold nanoparticle-thiolated DNA conjugates involves the dissociation of the gold-thiol bond

Using UV-visible extinction spectroscopy and femtosecond pump-probe transient absorption spectroscopy, we have studied the effect of femtosecond laser heating on gold nanoparticles attached to DNA ligands via thiol groups. It is found that femtosecond pulse excitation of the DNA-modified nanoparticles at a wavelength of 400 nm leads to desorption of the thiolated DNA strands from the nanoparticle surface by the dissociation of the gold-sulfur bond. The laser-initiated gold-sulfur bond-breaking process is a new pathway for nonradiative relaxation of the optically excited electrons within the DNA-modified gold nanoparticles, as manifested by a faster decay rate of the excited electronic distribution at progressively higher laser pulse energies. The experimental results favor a bond dissociation mechanism involving the coupling between the photoexcited electrons of the nanoparticles and the gold-sulfur bond vibrations over one involving the conventional phonon-phonon thermal heating processes. The latter processes have been observed previously by our group to be effective in the selective photothermal destruction of cancer cells bound to anti-epidermal growth factor receptor-conjugated gold nanoparticles.     

Stable immobilization of an oligonucleotide probe on a gold substrate using tripodal thiol derivatives

We proposed an interface molecule for immobilization of DNA probes on solid substrates of DNA chips. We have designed and synthesized tripodal thiol derivatives for stable immobilization of oligonucleotide probes on a gold surface. On the basis of the tetrahedral structure of tripod, the tripodal thiol derivatives were bonded upright to the gold substrate, which would control the orientation of oligonucleotide probes. When the gold substrate with oligonucleotide probes tethered using the thiol derivatives was exposed to deionized water at higher temperatures, the tripodal interface molecules were attached to the gold surface more stably than the single contact molecules. The DNA chip platform combined with the functional interface molecule is suitable for a reproducible, inexpensive, and high-throughput detection system for genetic analyses in clinical diagnostics.     

Sharp melting transitions in DNA hybrids without aggregate dissolution: proof of neighboring-duplex cooperativity

Similar to DNA-modified gold nanoparticles, comb polymer-DNA hybrids exhibit very sharp melting transitions that can be utilized in highly selective DNA detection systems. Current theories suggest that such sharp melting results from either a phase transition caused by the macroscopic dissolution of the aggregate or neighboring-duplex interactions in the close-packed environment between adjacent DNA duplexes. To delineate the contributions of each of these effects, an aggregate system based on polymer-DNA hybrids was designed to include both polymer-linked and partially untethered duplexes. When this hybridized system was subjected to thermal analysis, both types of duplexes exhibited sharp melting transitions. The very sharp melting transition displayed by the partially untethered DNA duplexes offers proof that neighboring-duplex interactions can indeed induce cooperativity. Contributions of this neighboring-duplex effect, as well as the enhanced stabilization observed in polymer-DNA:polymer-DNA aggregates, can be quantitatively assessed using a simple thermodynamic model. While neighboring-duplex interactions alone can lead to cooperative melting, the enhanced stabilization observed in polymer-DNA aggregates is a function of both neighboring-duplex interactions and multivalent or aggregate properties.     

Preparation and structure of a low-density, flat-lying decanethiol monolayer from the densely packed, upright monolayer on gold

This study investigates the formation of low-density, flat-lying decanethiol chemisorbed on Au prepared by heating the surface covered with a densely packed, upright monolayer to a surface temperature above that of the onset of desorption. We determined conditions for preparing the low-density phase by observing the evolution of the photoemission spectrum as a function of the surface temperature using polarized ultraviolet light and by utilizing scanning tunneling microscopy. The preparation conditions were similar for single- and polycrystalline gold surfaces. Once the low-density decanethiol phase was formed, reflection absorption infrared spectroscopy was employed to determine the orientation of the carbon chain backbone with respect to the Au surface. The nature of the valance electronic structure for flat-lying decanethiol is described.     

Impact of Single Basepair Mismatches on Electron‐Transfer Processes at Fc‐PNA⋅DNA Modified Gold Surfaces

Gold‐surface grafted peptide nucleic acid (PNA) strands, which carry a redox‐active ferrocene tag, present unique tools to electrochemically investigate their mechanical bending elasticity based on the kinetics of electron‐transfer (ET) processes. A comparative study of the mechanical bending properties and the thermodynamic stability of a series of 12‐mer Fc‐PNA?DNA duplexes was carried out. A single basepair mismatch was integrated at all possible strand positions to provide nanoscopic insights into the physicochemical changes provoked by the presence of a single basepair mismatch with regard to its position within the strand. The ET processes at single mismatch Fc‐PNA?DNA modified surfaces were found to proceed with increasing diffusion limitation and decreasing standard ET rate constants k⁰ when the single basepair mismatch was dislocated along the strand towards its free‐dangling Fc‐modified end. The observed ET characteristics are considered to be due to a punctual increase in the strand elasticity at the mismatch position. The kinetic mismatch discrimination with respect to the fully‐complementary duplex presents a basis for an electrochemical DNA sensing strategy based on the Fc‐PNA?DNA bending dynamics for loosely packed monolayers. In a general sense, the strand elasticity presents a further physicochemical property which is affected by a single basepair mismatch which may possibly be used as a basis for future DNA sensing concepts for the specific detection of single basepair mismatches.     

DNA hybridization "turns on" electro-catalysis at gold electrodes

The efficiency of electro-catalysis occurring at DNA-modified gold electrodes is highly dependently on the density of DNA monolayers, as a result, DNA hybridization can "turn on" electro-catalysis by increasing the DNA surface density.     

Influence of intervening mismatches on long-range guanine oxidation in DNA duplexes

A systematic investigation of the efficiency of oxidative damage at guanine residues through long-range charge transport was carried out as a function of intervening base mismatches. A series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC approximately GG approximately GT approximately GA > AA > CC approximately TT approximately CA approximately CT. For all assemblies, damage found with the Delta-Ru diastereomer was found to be greater than with the Lambda-diastereomer. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from 1H NMR measurements of the imino proton exchange rates. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, damage ratios correlate most closely with base-pair lifetimes. Competitive hole trapping at the mismatch site does not appear to be a key factor governing the efficiency of transport through the mismatch. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.