Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

Self‐Assembly of a 3D DNA Crystal Structure with Rationally Designed Six‐Fold Symmetry

Programming self‐assembled designer DNA crystals with various lattices and functions is one of the most important goals for nanofabrication using nucleic acids. The resulting porous materials possess atomic precision for several potential applications that rely on crystalline lattices and cavities. Herein, we present a rationally designed and self‐assembled 3D DNA crystal lattice with hexagonal symmetry. In our design, two 21‐base oligonucleotides are used to form a duplex motif that further assembles into a 3D array. The interactions between the strands are programmed using Watson–Crick base‐pairing. The six‐fold symmetry, as well as the chirality, is directed by the Holliday junctions formed between the duplex motifs. The rationally designed DNA crystal provides a new avenue that could create self‐assembled macromolecular 3D crystalline lattices with atomic precision. In addition, the structure contains a highly organized array of well‐defined cavities that are suitable for future applications with immobilized guests.     

Electrochemical Genoassay Design for Allele‐Specific Detection of Toll‐Like Receptor‐2 Gene Polymorphism

An allele‐specific voltammetric genoassay for the detection of allele‐specific toll‐like receptor‐2 gene arg753gln polymorphism (TLR‐2) from polymerase chain reaction (PCR) amplified real samples was described in this study. Meldola blue (MDB), an intercalator molecule, was used as hybridization label. The wild‐type and mutant type oligonucleotide probes were immobilized onto disposable graphite electrode surfaces by covalent attachment method. The extent of hybridization between probe and target sequences was determined by using differential pulse voltammetry (DPV). As a result of the interaction between MDB and DNA at electrode surface, the MDB signal observed from probe sequence before hybridization and after hybridization with MM sequence is lower than that observed after hybridization with complementary sequence. The differences between the MDB reduction peaks obtained from probe modified, hybrid modified and MM modified electrode were used to detect TLR‐2 from PCR amplified real samples. The discrimination of homozygous and heterozygous alleles was also established by comparing the peak currents of MDB reduction signals. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

Surface‐Assisted Large‐Scale Ordering of DNA Origami Tiles

The arrangement of DNA‐based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close‐packing of symmetric, non‐interacting DNA origami structures, or by utilizing blunt‐end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.     

Quartic lattice interactions,soliton‐like excitations,and electron pairing in one‐dimensional anharmonic crystals

In this study, it is shown that two added, excess electrons with opposite spins in one‐dimensional crystal lattices with quartic anharmonicity may form a bisolectron, which is a localized bound state of the paired electrons to a soliton‐like lattice deformation. It is also shown that when the Coulomb repulsion is included, the wave function of the bisolectron has two maxima, and such a state is stable in lattices with strong enough electron (phonon/soliton)–lattice coupling. Furthermore, the energy of the bisolectron is shown to be lower than the energy of the state with two separate, independent electrons, as even with account of the Coulomb repulsion the bisolectron binding energy is positive. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Preparation of a Novel Polymer‐Modified Si Surface for DNA Immobilization

The design of a novel polymer‐modified overlayer composed of PPAPE and GPMS on a silicon wafer for immobilization of DNA molecules is described. After hydroxylation of Si(100) surfaces, GPMS molecules were self‐assembled onto these surfaces. PPAPE molecules were then covalently attached to the epoxy‐terminated surfaces. The incubation time and concentration of PPAPE was found to effect both layer thickness and water CA. The type of organic solvent and the pH were found to change the nature of the PPAPE‐modified surface for DNA immobilization. It is concluded that PPAPE‐modified surfaces show advantages for DNA immobilization by electrostatic interactions between DNA molecules and positively charged free amino groups of the PPAPE‐modified surfaces at the appropriate pH values.

     

Assembly of Two‐Dimensional DNA Crystals Carrying N4‐[2‐(tert‐Butyldisulfanyl)ethyl]cytosine Residues

DNA Lattices carrying two cytosine residues bearing the N⁴‐[(tert‐butyldisulfanyl)ethyl] group at the apex of hairpin topological markers are prepared for first time. We show that these residues are important for the deposition of DNA lattices on gold surfaces.     

Design and construction of double-decker tile as a route to three-dimensional periodic assembly of DNA

DNA is a useful material for nanoscale construction. Due to highly specific Watson-Crick base pairing, the DNA sequences can be designed to form small tiles or origami. Adjacent helices in such nanostructures are connected via Holliday junction-like crossovers. DNA tiles can have sticky ends which can then be programmed to form large one-dimensional and two-dimensional periodic lattices. Recently, a three-dimensional DNA lattice has also been constructed. Here we report the design and construction of a novel DNA cross tile, called the double-decker tile. Its arms are symmetric and have four double helices each. Using its sticky ends, large two-dimensional square lattices have been constructed which are on the order of tens of micrometers. Furthermore, it is proposed that the sticky ends of the double-decker tile can be programmed to form a three-dimensional periodic lattice with large cavities that could be used as a scaffold for precise positioning of molecules in space.     

Hydrogen‐Bond‐Regulated Distinct Functional‐Group Display at the Inner and Outer Wall of Vesicles

A unique supramolecular strategy enables the unidirectional assembly of two bola‐shaped unsymmetric π‐amphiphiles, NDI‐1 and NDI‐2, which feature a naphthalene–diimide chromophore connected to nonionic and anionic head groups on opposite arms. The amphiphiles differ only in the location of a hydrazide group, which is placed either on the nonionic or on the anionic arm of NDI‐1 and NDI‐2, respectively. The formation of hydrogen bonds between the hydrazides, which compensates for electrostatic and steric factors, promotes unidirectional alignment and the formation of monolayer vesicles. The zeta potentials and cation‐assisted quantitative precipitation reveal negatively charged and nonionic outer surfaces for NDI‐1 and NDI‐2, respectively, indicating that hydrogen bonding also dictates the directionality of the monolayer curvature, ensuring that in both cases, the hydrazides remain at the inner wall to benefit from stronger hydrogen bonding where they are in closer proximity. This is reflected in their different abilities to inhibit α‐chymotrypsin, which possesses a positively charged surface: NDI‐1 induced an inhibition of 80 % whereas hardly any inhibition was observed with NDI‐2.     

Shape asymmetry of star-branched random walks and nonreversal random walks

Star-branched random walks with 3, 4, 6, 8 and 12 arms (the total chain-length ranging from N = 49 to 1925) have been produced and analysed with respect to their instantaneous shape. The short-chain behaviour of nonreversal random walk stars (NRRWs) embedded in various lattices is compared to that of star-branched freely jointed (off-lattice) chains (RWs). While for all types of NRRW-stars examined as well as for RW-stars with bonds of constant length shape-asymmetry increases with increasing chain-lengths, the opposite behaviour is found for RW-stars with Gaussian-distributed bond-lengths. The amount of short-chain effects is strongly dependent on the number of arms and on the lattice type used. In the limit of infinitely large molecules, however, quantities characteristic of the shape converge to common values for all types of RWs and NRRWs examined.     

Impact of DNA Sequence and Oligonucleotide Length on a Polythiophene‐Based Fluorescent DNA Biosensor

DNA hybridization is a universal and specific mechanism for the recognition of biological targets. Some cationic polythiophene transducers sensitive to DNA structure have been previously utilized to detect such biomolecules. Further characterization of these systems indicates that both DNA sequence composition and length modulate the biosensor performance. It appears that different repeated sequence patterns cause different conformational changes of the polythiophene, from a more relaxed form to an extremely rigid one. A length difference between the DNA oligonucleotide probe and target has a detrimental effect on the fluorescent signal, but it can be attenuated by changing the sequence composition of the protruding target sequence. This demonstrates that the nature of DNA can be critical for hybridization‐based detection systems.

     

DNA binding to electropolymerized N‐substituted polypyrrole surfaces

This study explores the use of N‐substituted polypyrroles as a route for localizing DNA molecules onto conducting surfaces. N‐substituted pyrrole monomers containing N‐hydroxysuccinimidyl groups for DNA binding reactions were synthesized. These monomers were electro‐copolymerized under different conditions on platinum or gold working electrodes in a three‐electrode/single compartment cell. Subsequent DNA reactions were performed by incubating the resulting polymer conductive films with amino‐substituted DNA sequences. In addition, the electro‐copolymerization reactions of pyrrole monomers were conducted on preselected electrode positions of the Molecular Nanosystems (MNS) wafers and the formation of conductive films was demonstrated. In these experiments, it was determined also that by controlling the electro‐copolymerization reactions, the conductive films can be restricted to grow on specific locations of the MNS wafer. This was achieved by electrically passivating the chosen electrodes with self‐assembled multilayers (SAM)s of alkane thiols. Hexadecane thiol (HDT) was found the most efficient in forming SAMs and in preventing the pyrrole electropolymerization. Various analytical techniques including AFM, IR, and cyclic voltammetry (CV) were used to characterize the monomers, the electropolymerized polymers, and the attachment of amine‐terminated DNA to polypyrrole copolymers. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6014–6024, 2009     

Synthesis and Spectroscopic Characterization of Fluorophore‐Labeled Oligospiroketal Rods

Fluorescence probes consisting of well‐established fluorophores in combination with rigid molecular rods based on spirane‐type structures were investigated with respect to their fluorescence properties under different solvent conditions. The attachment of the dyes was accomplished by 1,3‐dipolar cycloaddition between alkynes and azides (‘click’ reaction) and is a prime example for a novel class of sensor constructs. Especially, the attachment of two (different) fluorophores on opposite sides of the molecular rods paves the way to new sensor systems with less bulky (compared to the conventional DNA‐ or protein‐based concepts), nevertheless rigid spacer constructs, e.g., for FRET‐based sensing applications. A detailed photophysical characterization was performed in MeOH (and in basic H₂O/MeOH mixtures) for i) rod constructs containing carboxyfluorescein, ii) rod constructs containing carboxyrhodamine, iii) rod constructs containing both carboxyfluorescein and carboxyrhodamine, and iv) rod constructs containing both pyrene and perylene parts. For each dye (pair), two rod lengths with different numbers of spirane units were synthesized and investigated. The rod constructs were characterized in ensemble as well as single‐molecule fluorescence experiments with respect to i) specific rod? dye and ii) dye? dye interactions. In addition to MeOH and MeOH/NaOH, the rod constructs were also investigated in micellar systems, which were chosen as a simplified model for membranes.     

“Clickable” and Antifouling Block Copolymer Brushes as a Versatile Platform for Peptide‐Specific Cell Attachment

To tailor cell–surface interactions, precise and controlled attachment of cell‐adhesive motifs is required, while any background non‐specific cell and protein adhesion has to be blocked effectively. Herein, a versatile and highly reproducible antifouling surface modification based on “clickable” groups and hierarchically structured diblock copolymer brushes for the controlled attachment of cells is reported. The polymer brush architecture combines an antifouling bottom block of poly(2‐hydroxyethyl methacrylate) poly(HEMA) and an ultrathin azide‐bearing top block, which can participate in well‐established “click” reactions including the highly selective copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) reaction under mild conditions. This straightforward approach allows the rapid conjugation of a cell‐adhesive, alkyne‐bearing cyclic RGD peptide motif, enabling subsequent specific attachment of NIH 3T3 fibroblasts, their extensive proliferation and confluent cell sheet formation after 48 h of incubation. The generally applicable strategy presented in this report can be employed for surface functionalization with diverse alkyne‐bearing biological moieties via CuAAC or copper‐free alkyne‐azide cycloaddition protocols, making it a versatile functionalization approach and a promising tool for tissue engineering, biomaterial implant design, and other applications that require surfaces supporting highly specific cell attachment.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Computer simulation of relaxation phenomena in star‐branched polymers. Temperature dependence

Dynamic Monte Carlo simulations of simple models of star‐branched polymers were conducted. A model star macromolecule consisted of f = 3 arms of equal length with a total number of polymer segments up to 800. The chain was confined to a simple cubic lattice with simple nearest neighbor attractive interactions. The relaxation phenomena were studied by means of autocorrelation functions in wide ranges of temperatures. Short‐time‐scale dynamic processes in the entire star‐branched chain were examined. It was found that under good solvent conditions the longest relaxation time of the end‐to‐center vector decreases with decreasing temperature. For low temperatures (below the Θ‐point) where the chain is collapsed, the dependence of the relaxation time on the temperature is opposite.     

Sequence‐specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid‐DNA) in capillary and microfluidic electrophoretic separations

Reversible noncovalent but sequence‐dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite‐modified oligonucleotides has enabled sequence‐specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self‐assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid‐DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence‐dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information‐processing systems.     

Three‐Dimensional Tissue Models Constructed by Cells with Nanometer‐ or Micrometer‐Sized Films on the Surfaces

Living tissues or organ modules consist of different types of highly organized cells and extracellular matrices (ECMs) in a hierarchical manner, such as the multilayered structure of blood vessels and the radial structures of hepatic lobules. Due to animal examinations being banned in the EU since 2013 and a shortage in the demand for tissue repair or organ transplantation, the creation of artificial 3D tissues possessing specific structures and functions similar to natural tissues are key challenges in tissue engineering. To date, we have developed a simple but unique bottom‐up approach, a hierarchical cell manipulation technique, with a nanometer‐sized ECM matrix consisting of fibronectin (FN) and gelatin (G) on cell surfaces. About 10 nm thick FN/G ECM films on cell surfaces were coated successfully by using layer‐by‐layer coating methodology. Various 3D constructs with higher cell density with different types of cells were successfully constructed. In addition to the construction of tissues with higher cell densities, other tissues, such as cartilage or skin tissues, with different cell densities are also important tissue models for tissue engineering and pharmaceutical industries. Thus, we recently developed other methodologies, the collagen coating method and multiple coating method, to fabricate micrometer‐sized level ECM layers on cell surfaces. Various micro‐ or millimeter‐sized 3D constructs with lower cell densities were constructed successfully. By using these two methods, cell distances in 2D or 3D views can be controlled by different thicknesses of ECM layers on cell surfaces at the single‐cell level. Both FN/G and the collagen coating method resulted in homogenous 3D tissues with a controlled layer numbers, cell type, cell location, and properties; these will be promising to achieve different goals in tissue engineering.

     

DNA‐Directed Attachment of Carbon Nanotubes for Enhanced Label‐Free Electrochemical Detection of DNA Hybridization

Multi‐walled carbon nanotubes (MWNTs) were used as nanowires, which combined DNA molecules to a carbon paste electrode (CPE). The attachment of MWNT on the electrode surface was controlled by a hybridization assay between adenine and thymine containing oligonucleotides. The appearance of guanine oxidation signal after hybridization with target DNA greatly simplified the specific sequence DNA detection mechanism. Combination of sidewall‐ and end‐functionalization of MWNT provided a significant enhancement in the voltammetric signal of guanine oxidation in comparison with the signals obtained from only end‐oxidized MWNT modified CPE and a bare CPE. A control experiment involving adenine containing polynucleotide (poly(A)) instead of adenine probe modified MWNT was performed. The effect of target and noncomplementary DNA concentration on the guanine signal was also monitored. Discrimination against single‐base mismatch and noncomplementary DNA was achieved by surfactant containing washing solution. The promising conductivity of carbon nanotubes, and the creation of a larger surface area for DNA immobilization by sidewall‐ and end‐oxidation of MWNT provided a detection limit down to 10 pg/mL, which is compatible with the demand of the genetic tests.