Single Double-stranded DNA Molecule Research Articles

Biological Characteristics and Pathogenesis of Monkeypox Virus: An Overview.

Although the smallpox virus has been eradicated worldwide, the World Health Organization (WHO) has issued a warning about the virus's potential to propagate globally. The WHO labeled monkeypox a world public health emergency in July 2022, requiring urgent prevention and treatment. The monkeypox virus is a part of the Poxviridae family, Orthopoxvirus genus, and is accountable for smallpox, which has killed over a million people in the past. Natural hosts of the virus include squirrels, Gambian rodents, chimpanzees, and other monkeys. The monkeypox virus has transmitted to humans through primary vectors (various animal species) and secondary vectors, including direct touch with lesions, breathing particles from body fluids, and infected bedding. The viral particles are ovoid or brick-shaped, 200-250 nm in diameter, contain a single double-stranded DNA molecule, and reproduce only in the cytoplasm of infected cells. Monkeypox causes fever, cold, muscle pains, headache, fatigue, and backache. The phylogenetic investigation distinguished between two genetic clades of monkeypox: the more pathogenic Congo Basin clade and the West Africa clade. In recent years, the geographical spread of the human monkeypox virus has accelerated despite a paucity of information regarding the disease's emergence, ecology, and epidemiology. Using lesion samples and polymerase chain reaction (PCR), the monkeypox virus was diagnosed. In the USA, the improved Ankara vaccine can now be used to protect people who are at a higher risk of getting monkeypox. Antivirals that we have now work well against smallpox and may stop the spread of monkeypox, but there is no particular therapy for monkeypox.

Measuring DNA folding by the SARS-CoV-2 nucleocapsid protein.

The SARS-CoV-2 virus is a deadly virus responsible for COVID-19. Here we are interested in the mechanics of how the nucleocapsid protein (N-protein) in SARS-CoV-2 folds the viral RNA into the capsid. This process could be a target for antiviral research which looks to prevent further proliferation of the virus. Our goal is to understand the role that electrostatic interactions play in the folding of the negatively charged RNA by the positively charged N-protein. To isolate these interactions from specific interactions with the RNA bases or conformation, we measure N-protein folding single molecules of double-stranded DNA. We take structural images of the folding using an atomic force microscope (AFM) or look at real-time folding using tethered particle motion (TPM) assays. We hypothesize that N-proteins might fold double-stranded DNA using a method similar to positively charged protamine proteins which act to package the DNA in sperm cells. These protamine proteins “bind and bend” the DNA in multiple locations using discrete, long-lived (∼100 s), reversible bending steps. We observe similar folding characteristics for N-protein, but also see some important differences. We will report on progress toward this goal.

Stretching and Heating Single DNA Molecules with Optically Trapped Gold–Silica Janus Particles

Self-propelled micro- and nanoscale motors are capable of autonomous motion typically by inducing local concentration gradients or thermal gradients in their surrounding medium. This is a result of the heterogeneous surface of the self-propelled structures that consist of materials with different chemical or physical properties. Here we present a self-thermophoretically driven Au–silica Janus particle that can simultaneously stretch and partially melt a single double-stranded DNA molecule. We show that the effective force acting on the DNA molecule is in the ∼pN range, well suited to probe the entropic stretching regime of DNA, and we demonstrate that the local temperature enhancement around the gold side of the particle produces partial DNA dehybridization.

Stability, resolution, and ultra-low wear amplitude modulation atomic force microscopy of DNA: Small amplitude small set-point imaging

A way to operate fundamental mode amplitude modulation atomic force microscopy is introduced which optimizes stability and resolution for a given tip size and shows negligible tip wear over extended time periods (∼24 h). In small amplitude small set-point (SASS) imaging, the cantilever oscillates with sub-nanometer amplitudes in the proximity of the sample, without the requirement of using large drive forces, as the dynamics smoothly lead the tip to the surface through the water layer. SASS is demonstrated on single molecules of double-stranded DNA in ambient conditions where sharp silicon tips (R ∼ 2–5 nm) can resolve the right-handed double helix.

Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A

Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In our experiments, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously.

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA

The cyanine dye, YOYO-1, has frequently been used in single DNA molecule imaging work to stain double-stranded DNA as it fluoresces strongly when bound. The binding of YOYO-1 lengthens the DNA due to bis-intercalation. We have investigated the kinetics of binding, via this increase in DNA length, for single, hydrodynamically-stretched molecules of lambda DNA observed via Total Internal Reflection Fluorescence (TIRF) microscopy. The rate and degree of lengthening in 40mM NaHCO3 (pH 8.0) buffer depend upon the free dye concentration with the reaction taking several minutes to reach completion even in relatively high, 40nM, concentrations of YOYO-1. In the absence of overstretching of the DNA molecule, we determine the second order rate constant to be 3.8±0.7×105s−1M−1, the dissociation constant to be 12.1±3.4nM and the maximum DNA molecule extension to be 36±4%. The intercalation time constant (inverse of the pseudo-first order rate constant), τ, decreased from 309 to 62s as YOYO-1 levels increased from 10 to 40nM. The kinetics of binding help with interpretation of the behavior of DNA–YOYO-1 complexes when overstretched and establish defined conditions for the preparation of DNA–YOYO-1 complexes.

Conductance of DNA molecules: Effects of decoherence and bonding

The influence of decoherence and bonding on the linear conductance of single double-stranded DNA molecules is examined by fitting a phenomenological statistical model developed recently (EPJB {\bf 68}, 237 (2009)) to experimental results. The DNA molecule itself is described by a tight binding ladder model with parameters obtained from published ab initio calculations (J.Am.Chem.Soc. {\bf 127}, 14894 (2005)). The good agreement with the experiments on sequence and length dependence gives a hint on the nature of conduction in DNA and at the same time provides a crucial test of the model.

Slowing the translocation of double-stranded DNA using a nanopore smaller than thedouble helix

It is now possible to slow and trap a single molecule of double-stranded DNA (dsDNA), bystretching it using a nanopore, smaller in diameter than the double helix, in a solid-statemembrane. By applying an electric force larger than the threshold for stretching, dsDNAcan be impelled through the pore. Once a current blockade associated with a translocatingmolecule is detected, the electric field in the pore is switched in an interval less than thetranslocation time to a value below the threshold for stretching. According to moleculardynamics (MD) simulations, this leaves the dsDNA stretched in the pore constriction withthe base-pairs tilted, while the B-form canonical structure is preserved outside the pore.In this configuration, the translocation velocity is substantially reduced from1 bp/10 ns to ∼ 1 bp/2 ms in the extreme, potentially facilitating high fidelity reads for sequencing, precise sorting,and high resolution (force) spectroscopy.

Electrically induced bonding of DNA to gold

The development of single-molecule techniques has afforded many new methods for the observation and assembly of supramolecular structures and biomolecular networks. We previously reported a method, known as the single-molecule cut-and-paste approach, to pick up and deposit individual DNA strands on a surface. This, however, required pre-functionalization of the surface with DNA strands complementary to those that were to be picked up and then deposited. Here we show that single molecules of double-stranded DNA, bound to the tip of an atomic force microscope, can be deposited on a bare gold electrode using an electrical trigger (surface potential cycling). The interactions between the DNA and the electrode were investigated and we found that double-stranded DNA chemisorbs to the gold electrode exclusively at its end through primary amine groups. We corroborated this finding in experiments in which only a single adenosine nucleotide on a polyethylene glycol spacer was 'electrosorbed' to the gold electrode.

Stretching single DNA molecules to demonstrate high‐force capabilities of holographic optical tweezers

The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 mum particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies.

Discriminating Bases by Stretching Double-Stranded DNA in a Nanopore

We report a new method for trapping a single molecule of double-stranded DNA (dsDNA) in a solid-state nanopore in SiNx membrane and describe the prospects for sequencing it. It is possible to trap a single dsDNA molecule in a nanopore <3nm in diameter by first applying a voltage larger than this threshold and forcing the molecule to translocate through the pore. If the electric field is then rapidly switched to a value below threshold, the DNA becomes trapped for seconds in the pore, compared to a sub-millisecond translocation if the field is maintained above threshold. Moreover, if the duration in the trap is commensurate with the bandwidth we can discriminate distinct signatures of C-G and A-T base-pairs by simply measuring the pore current. Molecular dynamics simulations of these experiments reveal that, when trapped, the dsDNA is stretched in the pore in a specific tilted orientation, depending on the orientation of the leading nucleotides, while the B-form canonical structure is preserved outside the pore. Finally, we show using streptavidin bound biotinylated dsDNA (Fig. 1a) that it's possible to discriminate stretched basepairs in the trapped configuration (Fig. 1b,c) between A-T and C-G.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Binding of DNA to Zwitterionic Lipid Layers Mediated by Divalent Cations

Divalent cations, i.e., calcium, magnesium, and others, are able to enhance the ability of DNA to interact with membranes that are composed of zwitterionic lipids such as phosphatidylcholine. The resulting condensed complexes offer potential applications as nontoxic gene delivery vehicles. The present study suggests a generic theoretical model to describe the energetics and structural features of a zwitterionic lipid-DNA complex in the presence of divalent cations. Specifically, we consider the adsorption of a single molecule of double-stranded DNA onto a planar zwitterionic lipid layer. Our theoretical model is based on the continuum Poisson-Boltzmann formalisms, which we modified so as to account for the two opposite charges and orientational freedom of the zwitterionic lipid headgroups. We find a substantially more favorable adsorption free energy of the DNA if divalent cations are present. In addition, our model predicts the divalent cations to preferentially interact with the phosphate groups of the zwitterionic lipids, given these lipids are located in close vicinity to the DNA. This is accompanied by a small but notable reorientation of the zwitterionic headgroups toward the DNA. We demonstrate that the binding of DNA onto a zwitterionic lipid layer is not driven by the release of counterions. Instead, the binding leads to a partial redistribution of the divalent cations, from the phosphate groups of the DNA (prior to the binding) to the phosphate groups of the zwitterionic lipids (after the binding). Our results thus suggest a general physical mechanism underlying complex formation between DNA and zwitterionic lipids in terms of mean-field electrostatics, i.e., neither involving correlations nor specific interactions of the divalent cations.

Charge-Coupled Device Operated in a Time-Delayed Integration Mode as an Approach to High-Throughput Flow-Based Single Molecule Analysis

Single molecule detection (SMD) readouts are particularly attractive for assays geared toward high-throughput processing, because they can potentially reduce assay time by eliminating various processing steps. Unfortunately, most flow-based SMD experiments have generated low throughputs due primarily to the fact that they are configured in single assay formats. The use of a charge-coupled device (CCD) with flow-based SMD can image multiple single molecule assays simultaneously to realize high-throughput processing capabilities. We present, for the first time, the ability to simultaneously track and detect single molecules in multiple microfluidic channels by employing a CCD camera operated in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement. As an example of the technology, we have configured a CCD to operate in a TDI mode to detect single double-stranded DNA molecules (lambda and pBR322) labeled with an intercalating dye (TOTO-3) in a series of microfluidic channels poised on a poly(methyl methacrylate), PMMA, chip. A laser beam was launched into the side of the chip, which irradiated a series of fluidic channels (eight) with the resulting fluorescence imaged onto a CCD. Using this system, we were able to identify single DNA molecules based on the fluorescence burst intensity arising from differences in the extent of dye labeling associated with the DNA molecule length. The CCD/TDI approach allowed increasing sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s (-1) per channel and 2208 molecules s (-1) for an eight channel microfluidic system was demonstrated. Operated in its full capacity, this multichannel format was projected to yield a sample throughput of 1.7 x 10 (7) molecules s (-1), which represents a 170-fold improvement over previously reported single molecule sampling rates.

Surface-Directed and Ethanol-Induced DNA Condensation on Mica

The adsorption of lambda-phage DNA onto mica was investigated with atomic force microscopy. We found that the morphologies depended on the solvent conditions in the sample preparation procedure. Flat-lying networks of hybridized single-stranded DNA were obtained if ultrapure water was used. If buffered conditions are maintained during the whole of the preparation procedure, single double-stranded DNA molecules are adsorbed. The adsorbed double-stranded DNA molecules subsequently can be condensed in situ on the surface by a brief rinse with anhydrous ethanol in the presence of divalent magnesium cations. The majority of these surface-directed and ethanol-induced condensed structures are toroids, but a small fraction of rods also has been observed. Analysis of the height and lateral dimensions shows that the toroids are single-molecular and disk-like with a height of one to two DNA diameters. The thin toroid morphology appears to be a general phenomenon of surface-directed condensation, irrespective of the nature of the condensing ligands and the specific surface interaction.

Visualizing the Assembly of Human Rad51 Filaments on Double-stranded DNA

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study the dynamic behavior of Rad51 we have developed a single-molecule assay that relies on a combination of hydrodynamic force and microscale diffusion barriers to align individual DNA molecules on the surface of a microfluidic sample chamber that is coated with a lipid bilayer. When visualized with total internal reflection fluorescence microscopy (TIRFM), these “molecular curtains” allow for the direct visualization of hundreds of individual DNA molecules. Using this approach, we have analyzed the binding of human Rad51 to single molecules of double-stranded DNA under a variety of different reaction conditions by monitoring the extension of the fluorescently labeled DNA, which coincides with assembly of the nucleoprotein filament. We have also generated several mutants in conserved regions of Rad51 implicated in DNA binding, and tested them for their ability to assemble into extended filaments. We show that proteins with mutations within the DNA-binding surface located on the N-terminal domain still retain the ability to form extended nucleoprotein filaments. Mutations in the L1 loop, which projects towards the central axis of the filament, completely abolish assembly of extended filaments. In contrast, most mutations within or near the L2 DNA-binding loop, which is also located near the central axis of the filament, do not affect the ability of the protein to assemble into extended filaments on double-stranded (ds)DNA. Taken together, these results demonstrate that the L1-loop plays a crucial role in the assembly of extended nucleoprotein filaments on dsDNA, but the N-terminal domain and the L2 DNA-binding loop have significantly less impact on this process. The results presented here also provide an important initial framework for beginning to study the biochemical behaviors of Rad51 nucleoprotein filaments using our novel experimental system.

Relaxation dynamics of a single DNA molecule

The relaxation of a single DNA molecule is studied. The experimental system consists of optical tweezers and a micron-sized bead that is tethered to the bottom of the sample by a single double-stranded DNA molecule. The bead slows down the DNA relaxation from a strongly stretched configuration such that it is passing through stretched equilibrium states. This allows for a theoretical description of the relaxation trajectory, which is in good agreement with experiment.

Stretching of single DNA molecules complexed with restriction endonuclease by Langmuir–Blodgett method

We have proposed a new technique for stretching single double-stranded DNA molecules on solid substrates by the Langmuir–Blodgett (LB) method. The polyion complex monolayer of a cationic amphiphile and DNA molecules formed at the air–water interface was transferred on a clean glass substrate. Vertical lifting up of the glass substrate provided the transferred monolayer consisting the stretched individual DNA molecules aligned parallel to the lifting direction on the glass. The DNA molecules complexed with the restriction endonuclease ( EcoRI) were employed for stretching by using this method. Fluorescence images of the transferred monolayer showed that the EcoRI-binding DNA molecules could be stretched and immobilized on the glass substrate. A specific sequence of DNA recognized by EcoRI was detected as spatial positions of the stretched DNA molecules.

Combinatorics of peptide sextets encoded by a single microgene

Genetic information stored in DNA sequences is translated into protein by linking a triplet nucleotide sequence and an amino acid. Because the frames of the triplets can be configured in three ways, a total of six polypeptides, each with a different sequence, can be produced from a single double-stranded DNA molecule. We recently developed the MolCraft system [reviewed in K. Shiba, J. Mol. Catal. B 18 (2004) xxx], which enables us to make combinatorial polymers of three peptides translated from one strand of a double-stranded DNA molecule. To explore all the information that a single double-stranded DNA molecule encodes, we have now developed a new system, La-MolCraft, in which all six reading frames encoded by both strands are combinatorially polymerized using loop-mediated isothermal amplification of DNA (LAMP) [Nucl. Acids Res. 28 (2000) E63].

Combinatorics of peptide sextets encoded by a single microgene

Genetic information stored in DNA sequences is translated into protein by linking a triplet nucleotide sequence and an amino acid. Because the frames of the triplets can be configured in three ways, a total of six polypeptides, each with a different sequence, can be produced from a single double-stranded DNA molecule. We recently developed the MolCraft system [reviewed in K. Shiba, J. Mol. Catal. B 18 (2004) xxx], which enables us to make combinatorial polymers of three peptides translated from one strand of a double-stranded DNA molecule. To explore all the information that a single double-stranded DNA molecule encodes, we have now developed a new system, La-MolCraft, in which all six reading frames encoded by both strands are combinatorially polymerized using loop-mediated isothermal amplification of DNA (LAMP) [Nucl. Acids Res. 28 (2000) E63].

Sticker DNA computer model — Part II: Application

Sticker model is one of the basic models in the DNA computer models. This model is coded with single-double stranded DNA molecules. It has the following advantages that the operations require no strands extension and use no enzymes; What’s more, the materials are reusable. Therefore, it arouses attention and interest of scientists in many fields. In this paper, we extend and improve the sticker model, which will be definitely beneficial to the construction of DNA computer. This paper is the second part of our series paper, which mainly focuses on the application of sticker model. It mainly consists of the following three sections: the matrix representation of sticker model is first presented; then a brief review of the past research on graph and combinatorial optimization, such as the minimal set covering problem, the vertex covering problem, Hamiltonian path or cycle problem, the maximal clique problem, the maximal independent problem and the Steiner spanning tree problem, is described; Finally a DNA algorithm for the graph isomorphic problem based on the sticker model is given.