Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (ϕ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5′s orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, ϕ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins.     

Numerical study on electrophoretical stretching dynamics of DNA in microcontraction

Brownian dynamics simulations are used to characterize the electrophoretical stretching process of long T4 DNA in microchannels. When DNA is forced to move through the microchannels, the pure elongational flow generated by electric field gradients in hyperbolic contraction will unravel the molecules of DNA. The effects of hydrodynamic interactions, the strain rate, the Brownian fluctuation, and the initial states of molecules on the stretching dynamics are analyzed in this paper. The computational results show us the weak dependence of polymer dynamics on hydrodynamic interactions in microcontractions. In the case of low Deborah number, the stretching process of a molecule depends on the Brownian fluctuation. However, in the case of high Deborah number, the individualistic stretching behavior can be traced to variations in the starting conformation.     

A Nanochannel Platform for Single DNA Studies: From Crowding,Protein DNA Interaction,to Sequencing of Genomic Information

The study of nanochannel-confined DNA is important from biotechnological and biophysical points of view. We produce nanochannels in elastomer with soft lithography and proton beam writing. Issues concerning DNA confined in such quasi one-dimensional channels are discussed. We describe DNA stretching via the control of channel diameter and buffer conditions and how the extension can be interpreted with theory and computer simulation. We then discuss the conformation of nano-confined DNA crowded by neutral polymers and like-charged proteins. As an example of a protein that has an affinity to DNA, the effect of heat-stable nucleoid-structuring protein, H-NS, on the folding and compaction of DNA is reviewed. Compaction of DNA by eukaryotic protamine and unpacking of pre-compacted DNA through an increase in salt concentration are discussed. We review results obtained with a novel, cross-channel device that allows the monitoring of the dynamic, conformational response of DNA after exposure to a ligand or protein and/or a change in buffer conditions in situ. As a biotechnological application, linearization of DNA by bottlebrush coating with a polypeptide copolymer is discussed. It is demonstrated that large-scale genomic organization can be sequenced using single DNA molecules on an array of elastomeric nanochannels. Overall, our results show that the effects of ligands and proteins on the conformation, folding, and condensation of DNA are not only related to classical controlling factors, such as osmotic pressure, charge, and binding, but that the interplay with confinement in a nanospace is of paramount importance.     

Electrophoretical stretching of DNA in a hybrid microchannel

The electrophoretical stretching of DNA in a hybrid microchannel is analyzed in this paper. The channel comprises a large insulating cylinder and a hyperbolic contraction that can cause the DNA deformation. Brownian dynamics simulation is used to characterize the dynamical stretching process of a long T4 DNA in hybrid microchannels. The computational results show us the larger average extension of DNA in cylinder-hybridized microchannels than that in the single microcontractions due to the prestretched effect of cylinder on DNA. Moreover, the location and the radius of the insulating cylinder in hybrid microchannels have great effects on the stretching behavior of DNA.     

Nanopillar,Nanowall, and Nanowire Devices for Fast Separation of Biomolecules

Nanostructures based on nanotechnologies have opened up a novel research field for the fast analysis of biomolecules with ultrahigh resolution, including the analysis of single biomolecules. Nanostructures for electrophoretic separation, especially, are an exciting topic among researchers in many areas, and their designs are widely expected to contribute to the goal of developing a single separation tool for a wide range of biomolecules. In this review, nanopillar, nanowall, and nanowire devices are introduced for fast separation of DNA molecules and protein samples, and the numerous advantages of these devices are described. This review also outlines the fabrication processes for nanostructures, including “top-down” and “bottom-up” nanofabrication approaches. Besides describing the fast separation of biomolecules, the electroosmotic flow (EOF) suppression effect, and its related online concentration technique in nanopillar devices, is reviewed. The nanowall devices have the unique feature that longer DNA molecules migrate faster than shorter ones, and that is completely different from the separation behavior of DNA molecules based on nanopillar devices. The feasibility is shown for self-assembly of the nanowire structure embedded in a microchannel on a fused silica substrate, as a means to separate DNA molecules. Applications of a newly-fabricated 3D network structure with spatial density control for the fast separation of a wide range of DNA molecules are also given.     

Chemical Approaches to DNA Nanotechnology

Due to its self‐assembling nature, DNA is undoubtedly an excellent molecule for the creation of various multidimensional nanostructures and the placement of functional molecules and materials. DNA molecules behave according to the programs of their sequences. Mixtures of numbers of DNA molecules can be placed precisely and organized into single structures to form nanoarchitectures. Once the appropriate sequences for the target nanostructure are established, the predesigned structure can be built up by self‐assembly of the designed DNA strands. DNA nanotechnology has already reached the stage at which the organization of desired functional molecules and nanomaterials can be programmed on a defined DNA scaffold. In this review, we will focus on DNA nanotechnology and describe the potential of synthetic chemistry to contribute to the further development of DNA nanomaterials.     

Probing the basicity of zeolite frameworks with N2O4: A DFT approach

The basicity of alkali exchanged faujasite type zeolites is estimated on the basis of DFT calculations of the NO⁺ stretching frequencies of adsorbed N₂O₄. N₂O₄ is allowed to interact with the alkali cations and a range of molecules covering a wide basicity scale. An alkali nitrate salt is formed and NO⁺ interacts with the basic molecules. The resulting NO⁺ stretching frequencies decrease with increasing proton affinity and basicity of the basic molecules for each alkali cation. If experimental proton affinities and basicities of the model molecules are used, the data fall on two straight lines, which are almost parallel. The zeolitic basicities and proton affinities are obtained by interpolation of the experimental NO⁺ frequencies of alkali exchanged faujasites on the linear plots of NO⁺ frequencies against the experimental proton affinities and basicities. With theoretical proton affinities and basicities two non-parallel straight lines are obtained and three molecules are offset. The reason for this is unclear and needs more investigation. The NO⁺ stretching frequency is linearly correlated with the hardness of the alkali cations in zeolite X. For zeolite Y the same trend is observed, but the scatter of the data is too large to define it as linear.     

Evidence for molecular interactions in drag reduction in turbulent pipe flows

Experiments which test the concentration and molecular weight dependence of turbulent pipe flow drag reduction for random coiling polymers in dilute solutions show correlations with concentration to the one-half power and molecular weight to the 0.8 power for good solvents. This result is not consistent with a model of extension of single1 molecules, but could be related to the increase in bulk viscosity of interacting molecules after some extension. In this work, measurements for very low amounts of drag reduction for rigid rod molecules arc reported, and the effect of tube diameter on the amount of drag reduction is examined for fiexible rod molecules. No diameter effect is observed for the rigid rods, but an increase in drag reduction with increase in pipe diameter is found for the flexible polyeleetrolytes. In all cases, the volume occupied by spheres which circumscribe the molecules is greater than the actual volume when drag reduction is found. The results indicate that combined effects of individual molecule stretching and molecular interactions are present in drag reduction for random coiling or flexible rod molecules.     

Biofunctionalization of zinc oxide nanowires for DNA sensory applications

We report on the biofunctionalization of zinc oxide nanowires for the attachment of DNA target molecules on the nanowire surface. With the organosilane glycidyloxypropyltrimethoxysilane acting as a bifunctional linker, amino-modified capture molecule oligonucleotides have been immobilized on the nanowire surface. The dye-marked DNA molecules were detected via fluorescence microscopy, and our results reveal a successful attachment of DNA capture molecules onto the nanowire surface. The electrical field effect induced by the negatively charged attached DNA molecules should be able to control the electrical properties of the nanowires and gives way to a ZnO nanowire-based biosensing device.     

Scanning force microscopy study on a single-stranded DNA: the genome of parvovirus B19

The genome of parvovirus B19 is a 5600-base-long single-stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single-stranded DNA are contained in distinct virions and they hybridize intermolecularly to double-stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double-stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single-stranded and double-stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single-stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms.     

Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces

To study enzyme–DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrogen bond dynamics in liquid water: Ab initio molecular dynamics simulation

The effect of intermolecular interaction on the distribution of the harmonic vibrational frequencies of water molecules was investigated through ab initio molecular dynamics simulations based on the Born-Oppenheimer approach. For single water, the effect of the dynamics of the oxygen atom in single water and the simulation time step on the frequency distribution were examined. The distributions of the OH stretching and HOH bending vibrational frequencies of liquid water were compared to those of single water. The probability distributions of the change in OH bond length and the lifetime of the dangling OH bond were also obtained. The distribution of the frequencies was strongly affected by the long lifetime of the dangling OH bond, resulting in the formation of hydrogen bonds between water molecules.     

Stabilization Mechanism of Micropore in High‐Density Polyethylene: A Comparison between Thermal and Mechanical Pathways

Aiming to reveal the stabilization mechanism of micropore embryos formed during cold stretching in high‐density polyethylene films, samples are subsequently subjected to temperature elevation and strain holding at 25 °C, respectively. The corresponding structure evolution is tracked. It is found that after strain holding at 25 °C and subsequent strain recovery, inhomogeneously distributed cavities are produced, most of which can be healed as temperature is elevated to 110 °C. Consequently, only a small number of nonevenly distributed micropores are formed during the subsequent hot stretching. While for thermal pathway, micropores and fibrils can be formed as temperature is elevated. The hot stretching membrane exhibits uniformly distributed micropores and the micropores are well interconnected, indicating that micropores stabilized via temperature elevation are permanent and homogeneous. The results reveal different stabilization mechanisms of micropores via the thermal and mechanical pathways with regard to the distribution as well as the amount of permanent micropores.     

Synthesis and characteristics of NH<Subscript>2</Subscript>-functionalized polymer films to align and immobilize DNA molecules

We developed a method to use NH₂-functionalized polymer films to align and immobilize DNA molecules on a Si substrate. The plasma-polymerized cyclohexane film was deposited on the Si substrate according to the radio frequency plasma-enhanced chemical vapor deposition method using a single molecular precursor, and it was then treated by the dielectric barrier discharge method in a nitrogen environment under atmospheric pressure. Changes in the chemistry of the surface functional groups were studied using X-ray photoelectron spectroscopy and Fourier transformed infrared spectroscopy. The wettability of the surfaces was examined using dynamic contact angle measurements, and the surface morphology was evaluated using atomic force microscopy.     

Natural Molecules and Neuroprotection: Kynurenic Acid,Pantethine and α-Lipoic Acid

The incidence of neurodegenerative diseases has increased greatly worldwide due to the rise in life expectancy. In spite of notable development in the understanding of these disorders, there has been limited success in the development of neuroprotective agents that can slow the progression of the disease and prevent neuronal death. Some natural products and molecules are very promising neuroprotective agents because of their structural diversity and wide variety of biological activities. In addition to their neuroprotective effect, they are known for their antioxidant, anti-inflammatory and antiapoptotic effects and often serve as a starting point for drug discovery. In this review, the following natural molecules are discussed: firstly, kynurenic acid, the main neuroprotective agent formed via the kynurenine pathway of tryptophan metabolism, as it is known mainly for its role in glutamate excitotoxicity, secondly, the dietary supplement pantethine, that is many sided, well tolerated and safe, and the third molecule, α-lipoic acid is a universal antioxidant. As a conclusion, because of their beneficial properties, these molecules are potential candidates for neuroprotective therapies suitable in managing neurodegenerative diseases.     

α-Synuclein and DJ-1 as Potential Biological Fluid Biomarkers for Parkinson's Disease

Parkinson's disease (PD) is the most common form of movement disorder and affects approximately 4% of the population aged over 80 years old. Currently, PD cannot be prevented or cured, and no single diagnostic biomarkers are available. Notably, recent studies suggest that two familial PD-linked molecules, α-synuclein and DJ-1, are present in cerebrospinal fluid (CSF) and that their levels may be altered during the progression of PD. In this regard, sensitive and accurate methods for evaluation of α-synuclein and DJ-1 levels in the CSF and blood have been developed, and the results suggest that the levels of both molecules are significantly decreased in the CSF in patients with PD compared with age-matched controls. Furthermore, specific detection and quantification of neurotoxic oligometric forms of α-synuclein in the blood using enzyme-linked immunosorbent assays might be expected as potential peripheral biomarkers for PD, although further validation is required. Currently, neither α-synuclein nor DJ-1 is satisfactory as a single biomarker for PD, but combinatory evaluation of these biological fluid molecules with other biomarkers and imaging techniques may provide reliable information for diagnosis of PD.     

In-Silico Selection of Aptamer Targeting SARS-CoV-2 Spike Protein

Aptamers are single-stranded, short DNA or RNA oligonucleotides that can specifically bind to various target molecules. To diagnose the infected cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in time, numerous conventional methods are applied for viral detection via the amplification and quantification of DNA or antibodies specific to antigens on the virus. Herein, we generated a large number of mutated aptamer sequences, derived from a known sequence of receptor-binding domain (RBD)-1C aptamer, specific to the RBD of SARS-CoV-2 spike protein (S protein). Structural similarity, molecular docking, and molecular dynamics (MD) were utilized to screen aptamers and characterize the detailed interactions between the selected aptamers and the S protein. We identified two mutated aptamers, namely, RBD-1CM1 and RBD-1CM2, which presented better docking results against the S protein compared with the RBD-1C aptamer. Through the MD simulation, we further confirmed that the RBD-1CM1 aptamer can form the most stable complex with the S protein based on the number of hydrogen bonds formed between the two biomolecules. Based on the experimental data of quartz crystal microbalance (QCM), the RBD-1CM1 aptamer could produce larger signals in mass change and exhibit an improved binding affinity to the S protein. Therefore, the RBD-1CM1 aptamer, which was selected from 1431 mutants, was the best potential candidate for the detection of SARS-CoV-2. The RBD-1CM1 aptamer can be an alternative biological element for the development of SARS-CoV-2 diagnostic testing.     

Sequence‐Specific Molecular Lithography Towards DNA‐Templated Electronics

Recent advances in the realization of individual molecular‐scale devices [1,2] highlight the integration of individual devices into large‐scale functional circuits as the major challenge. DNA‐programmed assembly is a promising avenue in that direction due to the large amount of information that can be coded into the molecules and the ability to translate that information into physical constructs [3]. Large‐scale DNA‐templated electronics require, however, complex manipulation of double‐stranded DNA (dsDNA) molecules, as well as patterning of the electrical properties instilled to them by, e.g., metallization. To that end, sequence‐specific molecular lithography on single DNA molecules has been developed [4]. This was achieved by harnessing the exquisite homologous recombination process of the RecA protein. Sequence‐specific patterning of the metal coating of DNA molecules, localization of arbitrary labeled molecular objects at any desired dsDNA address without prior modifications, and generation of molecularly accurate stable dsDNA‐dsDNA junctions are demonstrated. The information encoded in the DNA molecules directs the lithographic process in analogy to the masks used in conventional microelectronics. The RecA protein provides the assembling capabilities, as well as the resist function.     

蛭石矿物材料层间水的赋存状态研究

利用红外光谱研究了蛭石矿物材料层间水的赋存状态。结果表明：蛭石晶层的层间距主要受层间水分子层数的影响,而后者又主要由层间阳离子的离子势大小决定。在层间都为2层水的情况下,蛭石层间距与层间阳离子的离子半径成正相关,这是由于蛭石层间为强静电场的缘故。利用Fourier自消卷积分峰技术,得到蛭石层间水的反对称伸缩振动、对称伸缩振动和弯曲振动的泛音等红外吸收谱,其中对称伸缩频率与层间水脱失温度成负相关,弯曲振动频率及其泛音与层间水脱失温度成正相关。