Time-resolved total internal reflection fluorescence study on hybridization of complementary single-stranded DNAs at a water/oil interface

Hybridization of complementary single-stranded DNAs (ssDNA) at a water/CCl4 interface was studied on the basis of picosecond total internal reflection fluorescence spectroscopy. Complementary ssDNAs dissolved in water were shown to produce the relevant double-stranded DNA (dsDNA) at a water/CCl4 interface in the presence of octadecylamine (ODA) in the oil phase, while hybridization between ssDNAs did not proceed in the water phase, as demonstrated by the fluorescence dynamics of ethidium bromide as a probe for the DNA structure. The structures of dsDNA and the roles of ODA in hybridization of ssDNA at the interface were discussed.     

Non-equilibrium all-atom molecular dynamics simulations of free and tethered DNA molecules in nanochannel shear flows

In order to gain insight into the mechanical and dynamical behaviour of free and tethered short chains of ss/ds DNA molecules in flow, and in parallel to investigate the properties of long chain molecules in flow fields, we have developed a series of quantum and molecular methods to extend the well developed equilibrium software CHARMM to handle non-equilibrium dynamics. These methods have been applied to cases of DNA molecules in shear flows in nanochannels. Biomolecules, both free and wall-tethered, have been simulated in the all-atom style in solvent-filled nanochannels. The new methods were demonstrated by carrying out NEMD simulations of free single-stranded DNA (ssDNA) molecules of 21 bases as well as double-stranded DNA (dsDNA) molecules of 21 base pairs tethered on gold surfaces in an ionic water shear flow. The tethering of the linker molecule (6-mercapto-1-hexanol) to perfect Au(111) surfaces was parametrized based on density functional theory (DFT) calculations. Force field parameters were incorporated into the CHARMM database. Gold surfaces are simulated in a Lennard-Jones style model that was fitted to the Morse potential model of bulk gold. The bonding force of attachment of the DNA molecules to the gold substrate linker molecule was computed to be up to a few nN when the DNA molecules are fully stretched at high shear rates. For the first time, we calculated the relaxation time of DNA molecules in picoseconds (ps) and the hydrodynamic force up to a few nanoNewtons (nN) per base pair in a nanochannel flow. The velocity profiles in the solvent due to the presence of the tethered DNA molecules were found to be nonlinear only at high shear flow rates. Free ssDNA molecules in a shear flow were observed to behave differently from each other depending upon their initial orientation in the flow field. Both free and tethered DNA molecules are clearly observed to be stretching, rotating and relaxing. Methods developed in this initial work can be incorporated into multiscale simulations including quantum mechanical, molecular and the microfluidic continuum regimes. The results may also be useful in extending existing macroscopic empirical models of DNA response dynamics in shear flows.     

Dip pen nanolithography functionalized electrical gaps for multiplexed DNA detection

Nanoparticle-based, silver-enhanced DNA electrical detection shows great promise for point-of-care diagnostics. In this paper, we demonstrate that the dip pen nanolithography (DPN) method can be used to precisely functionalize multiple electrical gaps for multiplexed DNA detection. With the use of the DPN technique, capture ssDNAs are written inside 5 microm x 10 microm electrical gaps on substrates. The DPN functionalized electrical gaps can specifically hybridize to target ssDNAs in solution. Successful hybridization of the capture-target DNA complex is detected by the use of gold nanoparticles carrying ssDNA, which also hybridize to the target ssDNA, followed by silver enhancement. The drop of resistance across the gaps due to the formation of metal nanoparticle-DNA complexes is measured over time and compared against characteristics of control gaps, which are either left unfunctionalized or functionalized with noncomplementary capture ssDNA. This technique has potential for high-density multiplexed DNA assay chips. Multiplex detection of two different target ssDNAs in solution using DPN functionalized electrical gaps on the same chip is demonstrated. The lowest detection limit is 10 pM.     

Model systems for single molecule polymer dynamics

Double stranded DNA (dsDNA) has long served as a model system for single molecule polymer dynamics. However, dsDNA is a semiflexible polymer, and the structural rigidity of the DNA double helix gives rise to local molecular properties and chain dynamics that differ from flexible chains, including synthetic organic polymers. Recently, we developed single stranded DNA (ssDNA) as a new model system for single molecule studies of flexible polymer chains. In this work, we discuss model polymer systems in the context of "ideal" and "real" chain behavior considering thermal blobs, tension blobs, hydrodynamic drag and force-extension relations. In addition, we present monomer aspect ratio as a key parameter describing chain conformation and dynamics, and we derive dynamical scaling relations in terms of this molecular-level parameter. We show that asymmetric Kuhn segments can suppress monomer-monomer interactions, thereby altering global chain dynamics. Finally, we discuss ssDNA in the context of a new model system for single molecule polymer dynamics. Overall, we anticipate that future single polymer studies of flexible chains will reveal new insight into the dynamic behavior of "real" polymers, which will highlight the importance of molecular individualism and the prevalence of non-linear phenomena.     

Structure and dynamics of DNA-dendrimer complexation: role of counterions, water, and base pair sequence

We study sequence-dependent complexation between oligonucleotides (single-strand DNA) and various generation ethylene diamine (EDA) cored poly amido amide (PAMAM) dendrimers through atomistic molecular dynamics simulations accompanied by free energy calculations and inherent structure determination. Simulations reveal formation of a stable complex and provide a detailed molecular level understanding of the structure and dynamics of such a complexation. The reaction free energy surface in the initial stage is found to be funnel-like, with a significant barrier arising in the late stage due to the occurrence of misfolded states of DNA. Complexation shows surprisingly strong sensitivity to the ssDNA sequence, which is found to arise from a competition between enthalpic versus entropic rigidity of ssDNA.     

Direct observation of single flexible polymers using single stranded DNA()

Over the last 15 years, double stranded DNA (dsDNA) has been used as a model polymeric system for nearly all single polymer dynamics studies. However, dsDNA is a semiflexible polymer with markedly different molecular properties compared to flexible chains, including synthetic organic polymers. In this work, we report a new system for single polymer studies of flexible chains based on single stranded DNA (ssDNA). We developed a method to synthesize ssDNA for fluorescence microscopy based on rolling circle replication, which generates long strands (>65 kb) of ssDNA containing "designer" sequences, thereby preventing intramolecular base pair interactions. Polymers are synthesized to contain amine-modified bases randomly distributed along the backbone, which enables uniform labelling of polymer chains with a fluorescent dye to facilitate fluorescence microscopy and imaging. Using this approach, we synthesized ssDNA chains with long contour lengths (>30 μm) and relatively low dye loading ratios (~1 dye per 100 bases). In addition, we used epifluorescence microscopy to image single ssDNA polymer molecules stretching in flow in a microfluidic device. Overall, we anticipate that ssDNA will serve as a useful model system to probe the dynamics of polymeric materials at the molecular level.     

Probing the structure of DNA-carbon nanotube hybrids with molecular dynamics

DNA-carbon nanotube hybrids (DNA-CN) are novel nanoscale materials that consist of single-wall carbon nanotubes (SWCN) coated with a self-assembled monolayer of single-stranded DNA (ssDNA). Recent experiments on DNA-CN have shown that this material offers a remarkable set of technologically useful properties such as facilitation of SWCN sorting, chemical sensing, and detection of DNA hybridization. Despite the importance of DNA-CN, a detailed understanding of its microscopic structure and physical properties is lacking. To address this, we have performed classical all-atom molecular dynamics (MD) simulations exploring the self-assembly mechanisms, structure, and energetic properties of this nanomaterial. MD reveals that SWCN induces ssDNA to undergo a spontaneous conformational change that enables the hybrid to self-assemble via the pi-pi stacking interaction between ssDNA bases and SWCN sidewall. ssDNA is observed to spontaneously wrap about SWCN into compact right- or left-handed helices within a few nanoseconds. Helical wrapping is driven by electrostatic and torsional interactions within the sugar-phosphate backbone that result in ssDNA wrapping from the 3' end to the 5' end.     

Nuclease resistance of telomere-like oligonucleotides monitored in live cells by fluorescence anisotropy imaging

Telomeres carry important biological functions such as the protection of chromosomes. In this paper, we have developed a fluorescence anisotropy imaging system for monitoring DNA digestion inside live cells. The nuclease-resistant capability of telomere-like ssDNAs in nuclei of human breast cancer cells is studied. We found that those oligonucleotides were clearly more stable than regular DNA sequences during the time course of the experiments. We conclude that the G-quadruplex structure of the telomere-like ssDNA makes it inherently more stable in intracellular environments than non-G-quadruplex structures. This will help us understand why the G-quadruplex forming telomere sequences were adopted by almost all eukaryotic cells to protect the ends of chromosomes. This is the first time such a phenomenon was observed in live cells. Our fluorescence anisotropy imaging provides an efficient way to directly monitor DNA digestion in any region of live cells in real time, providing insights into many important and related intracellular processes.     

Fabricating RNA microarrays with RNA-DNA surface ligation chemistry

A novel surface attachment strategy that utilizes RNA-DNA surface ligation chemistry to create renewable RNA microarrays from single-stranded DNA (ssDNA) microarrays on gold surfaces is demonstrated. The enzyme T4 DNA ligase was used to catalyze the formation of a phosphodiester bond between 5'-phosphate-modified ssDNA attached to the surface and the 3'-hydroxyl group of unlabeled RNA molecules from solution in the presence of a complementary template DNA strand. Surface plasmon resonance imaging (SPRI) measurements were performed to characterize the ligation process as well as to verify the bioactivity of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array to form a surface RNA-DNA heteroduplex and (ii) the hydrolysis of the RNA microarrays with either ribonuclease S or ribonuclease H (RNase H). The hydrolysis of the surface-bound RNA with RNase H required the presence of a surface heteroduplex and, upon completion, regenerated the original 5'-phosphate-terminated ssDNA array elements. These ssDNA array elements could be ligated again to create a new RNA microarray. These RNA microarrays can be used in the study of RNA-protein/RNA/aptamer bioaffinity interactions and for the enzymatically amplified SPRI detection of DNA in the presence of RNase H.     

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.     

Hierarchy of DNA immobilization and hybridization on poly-L-lysine using an atomic force microscopy study

The atomic force microscopy has been used to analyze the immobilization of single stranded DNA on poly-L-lysine-coated glass and subsequent hybridization with complimentary DNA with the Z-threshold parameter and fractal analysis methods. The poly-L-lysine layer, which has a thickness of approximately 7 nm, presents nano-defects that could be critical for DNA immobilization by acting as a nucleation sites for ssDNA and subsequently for dsDNA aggregates. The Z-threshold for the dsDNA aggregates is much larger than for ssDNA, but the statistical fractal dimension is very similar, suggesting a conformal increase of the dimensions of the dsDNA aggregates mainly in the Z-direction, due to an effective ssDNA-ccDNA molecular recognition. This study demonstrates the use of fractal analysis in conjunction with the distribution of heights to evaluate the efficiency of DNA-DNA molecular recognition on surfaces and the impact of nanodefects.     

Two‐Dimensional Periodic Relief Gratings as a Versatile Platform for Label‐Free Specific DNA Detection

In this study, nanopillar arrays of silicon oxide are fabricated through a process involving very‐large‐scale integration, for use as two‐dimensional periodic relief gratings (2DPRGs) on silicon surfaces. Oligonucleotides are successively immobilized on the pillar surface, allowing the system to be used as an optical detector specific for the targeted single‐stranded DNAs (ssDNAs). The surfaces of the oligonucleotides‐modified 2DPRGs undergo insignificant structural changes, but upon hybridizing with target ssDNA, the 2DPRGs undergo dramatic changes in terms of their pillar scale. Binding of the oligonucleotides to the 2DPRG occurs in a way that allows them to retain their function and selectively bind the target ssDNA. The performance of the sensor is evaluated by capturing the target ssDNA on the 2DPRGs and measuring the effective refractive index (n_eff). The binding of the target ssDNA species to the 2DPRGs results in a color change from pure blue to red, observable by the naked eye along an angle of 15–20°. Moreover, effective medium theory is used to calculate the filling factors inside the 2DPRGs and, thereby, examine the values of n_eff during the structural changes of the 2DPRGs. Accordingly, these new films have potential applications as label‐free optical biosensors.     

Unimolecular beacons for the detection of DNA-binding proteins

A new methodology for detecting sequence-specific DNA-binding proteins has been recently developed (Heyduk, T.; Heyduk, E. Nat. Biotechnol. 2002, 20, 171). The core feature of this methodology is protein-dependent association of two fluorochrome-labeled DNA fragments, which allows generation of a fluorescence signal reporting the presence of the target protein. Previous kinetic experiments identified the association of the two DNA fragments as the rate-limiting step of the assay. Here we report on a variant of the assay, in which components of the assay--fluorescent DNA fragments--were covalently tethered by a non-DNA linker with the goal of increasing the rate of association of the two fragments. We investigated the effect of the tether on the performance of the assay under a variety of conditions using a model DNA-binding protein. Quantitative titrations and rapid kinetic stopped-flow experiments were conducted to validate the molecular model that describes the two linked equilibria: oscillation of the tethered construct between the open and closed states and the exclusive association of the protein with the closed state. Experiments were also performed to demonstrate the ability of these tethered constructs to signal when attached to a solid surface. The major advantage of this new assay format is the faster response time for the detection allowing the higher throughput of the analysis. Additionally, it will be possible to attach tethered beacons to other solid surfaces, thus allowing the preparation of arrays containing molecular beacons for many different DNA-binding proteins.     

Gating of Single‐Layer Graphene with Single‐Stranded Deoxyribonucleic Acids

Patterning of biomolecules on graphene layers could provide new avenues to modulate their electrical properties for novel electronic devices. Single‐stranded deoxyribonucleic acids (ssDNAs) are found to act as negative‐potential gating agents that increase the hole density in single‐layer graphene. Current–voltage measurements of the hybrid ssDNA/graphene system indicate a shift in the Dirac point and “intrinsic” conductance after ssDNA is patterned. The effect of ssDNA is to increase the hole density in the graphene layer, which is calculated to be on the order of 1.8 × 10¹² cm^?2. This increased density is consistent with the Raman frequency shifts in the G‐peak and 2D band positions and the corresponding changes in the G‐peak full width at half maximum. Ab initio calculations using density functional theory rule out significant charge transfer or modification of the graphene band structure in the presence of ssDNA fragments.     

Large-scale molecular dynamics simulation of DNA: implementation and validation of the AMBER98 force field in LAMMPS

Molecular modelling played a central role in the discovery of the structure of DNA by Watson and Crick. Today, such modelling is done on computers: the more powerful these computers are, the more detailed and extensive can be the study of the dynamics of such biological macromolecules. To fully harness the power of modern massively parallel computers, however, we need to develop and deploy algorithms which can exploit the structure of such hardware. The Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) is a scalable molecular dynamics code including long-range Coulomb interactions, which has been specifically designed to function efficiently on parallel platforms. Here we describe the implementation of the AMBER98 force field in LAMMPS and its validation for molecular dynamics investigations of DNA structure and flexibility against the benchmark of results obtained with the long-established code AMBER6 (Assisted Model Building with Energy Refinement, version 6). Extended molecular dynamics simulations on the hydrated DNA dodecamer d(CTTTTGCAAAAG)(2), which has previously been the subject of extensive dynamical analysis using AMBER6, show that it is possible to obtain excellent agreement in terms of static, dynamic and thermodynamic parameters between AMBER6 and LAMMPS. In comparison with AMBER6, LAMMPS shows greatly improved scalability in massively parallel environments, opening up the possibility of efficient simulations of order-of-magnitude larger systems and/or for order-of-magnitude greater simulation times.     

Imparting chemical specificity to nanometer-spaced electrodes

In this paper we are demonstrating an electrochemically driven self-assembling approach to achieve the space-resolved chemical functionalization of nanoelectrodes. After forming a self-assembled monolayer of electroactive quinones on a pair of nano-spaced (<100?nm) electrodes, we enabled the binding of ssDNA exclusively on a single nanoelectrode by controlling the oxidation state at each modified electrode. This procedure attained the chemical differentiation of otherwise identical nanoelectrodes as the immobilized ssDNA retained its hybridization ability. Furthermore, we established that Kelvin probe force microscopy is a suitable space-resolved analytical technique for detecting this chemical functionalization at the nanoscale. The reported approach, enabling the space-selective patterning of (bio)molecules on nanoelectrode surfaces, can find application in complex nanosensor structure and molecular electronics implementations.     

Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.     

Detection of DNA targets hybridized to solid surfaces using optical images of liquid crystals

In this paper, we report a method of detecting DNA targets hybridized to a solid surface by using liquid crystals (LC). The detection principle is based on different interference colors of LC supported on surfaces decorated with single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). However, the contrast between the ssDNA and dsDNA is not obvious, unless DNA-streptavidin complexes are introduced to the dsDNA to increase the surface mass density. Two different approaches of introducing streptavidin to the system are studied and compared. We find that by premixing the biotin-labeled DNA targets with streptavidin prior to the DNA hybridization, branched-streptavidin complexes are formed and clear LC signal can be observed. This LC-based DNA detection principle represents an important step toward the development of a simple, instrument- and fluorophore-free DNA detection method.     

Detection of human papilloma viruses using nanostructurated silicon support in microarray technology

In a typical microarray experiment, DNA is arrayed on a solid substrate as spots, the array being probed with a sample or a capture molecule of interest and the interaction monitored through different detection methods. The present study evaluates the possibility to use micro-array technology to genotype samples with Human Papilloma Viruses (HPV). The performance of DNA microarrays strongly depend on their surface properties. The efficiency of DNA immobilization in terms of sensitivity and specificity is one of the most important step in obtaining a microarray chip for diagnosis of HPV family viruses. Here we report the preparation and evaluation of nano-porous silicon surfaces for HPV detection based on DNA micro-array technique. Two different surfaces based on similar porous structure chemically modified in order to efficiently immobilize ss-DNA specific for HPV viruses were investigate.     

Detection of Single DNA Molecule Hybridization on a Surface by Atomic Force Microscopy

Improving the detection of DNA hybridization is a critical issue for several challenging applications encountered in microarray and biosensor domains. Herein, it is demonstrated that hybridization between complementary single‐stranded DNA (ssDNA) molecules loosely adsorbed on a mica surface can be achieved thanks to fine‐tuning of the composition of the hybridization buffer. Single‐molecule DNA hybridization occurs in only a few minutes upon encounters of freely diffusing complementary strands on the mica surface. Interestingly, the specific hybridization between complementary ssDNA is not altered in the presence of large amounts of nonrelated DNA. The detection of single‐molecule DNA hybridization events is performed by measuring the contour length of DNA in atomic force microscopy images. Besides the advantage provided by facilitated diffusion, which promotes hybridization between probes and targets on mica, the present approach also allows the detection of single isolated DNA duplexes and thus requires a very low amount of both probe and target molecules.