DNA Molecules In Solution Research Articles

Ultra-Scaled Si Nanowire Biosensors for Single DNA Molecule Detection

In this study, we use NEGF quantum transport simulations to study the fundamental detection limit of ultra-scaled Si nanowire FET (NWT) biosensors. A N-doped NWT is found to be more sensitive for negatively charged analytes as explained by the nature of the detection mechanism. Our results predict threshold voltage shifts due to a single-charge analyte of tens to hundreds of mV in air or low-ionic solutions. However, with typical ionic solutions and SAM conditions, the sensitivity rapidly drops to the mV/q range. Our results are then extended to the detection of a single 20-base-long DNA molecule in solution. The impact of front- and/or back-gate biasing on the sensitivity and limit of detection is studied and a signal-to-noise ratio of 10 is predicted. Opportunities and challenges to reach down to single-analyte detection in such systems are also discussed, including the ionic and oxide-solution interface-charge screening and ways to recover unscreened sensitivities.

Effects of alternating electric field on the imaging of DNA double-helix structure by atomic force microscope

The effects of alternating electric field on the imaging of DNA double-helix structure were explored by atomic force microscope (AFM). First, the DNA sample was located under an alternating electric field in a fixed direction and dried. Then, AFM was used to obtain the DNA images under different alternating electric fields with the voltage range from 0.5 to 6.0 V and the frequency of 50 kHz. Thus, the DNA double-helix structures with different extensions were observed when the DNA molecules were gradually stretched under different field intensities. The distributions of DNA molecules in solution were random if there were no external forces, and the curved DNA molecules were observed in the AFM image. With the increase in alternating electric voltage (0.5–4.0 V), the DNA structure was shifted from random to oriented conformation and the DNA grooves were further unfolded. While the higher voltage (5.0–6.0 V) resulted in the rupture of DNA chains due to the excessive stretching force. It showed that the optimal voltage was 1.0 V, and the double-helix structure was observed. This method provides an efficient way for monitoring and measuring bio-macromolecules. It may also enable the exploration of the DNA–protein binding and DNA molecular self-assembly processes.

Interactions between identical DNA double helices.

The molecular mechanism of specific interactions between double stranded DNA molecules has been investigated for many years. Problems remain in how confinement, ions, and condensing agents change the interactions. We consider how the orientational alignment of DNAs contributes to the interactions via free energy simulations. Here we report on the effective interactions between two parallel DNA double helices in 150-mM NaCl solution using all atom models. We calculate the potential of mean force (PMF) of DNA-DNA interactions as a function of two coordinates, interhelical separation of parallel double helices and relative rotation of a DNA molecule with respect to the other about the helical axis. We generate the two-dimensional PMF to better understand the effective interactions when a DNA molecule is in juxtaposition with another. The analysis of the ion and solvent distributions around the DNA and particularly in the interface region shows that certain alignments of the DNA pair enhance the interactions. At local free energy minima in distance and alignment, water molecules and Na^{+} ions form a hydrogen bonded network with the phosphates from each DNA. This network contributes an attractive energy component to the DNA-DNA interactions. Our results provide a molecular mechanism whereby local DNA-DNA interactions, depending on the helical orientation, give a potential mechanism for stabilizing pairing of much larger lengths of homologous DNA that have been seen experimentally. The study suggests an atomically detailed local picture of relevance to certain aspects of DNA condensation or aggregation.

Active Feedback 3D Single-Molecule Tracking

Single molecule tracking has the potential to revolutionize the study of biological systems. However, most current single molecule imaging and tracking methods are limited to two dimensions. Meanwhile, real-time 3D single molecule tracking methods, which use active feedback to "lock-on" to single molecules in solution have until now been limited to observation times of 500 msec or less, greatly limiting their utility. Recently we developed a real-time 3D single particle tracking method known as 3D Dynamic Photon Localization Tracking (3D-DyPLoT). 3D-DyPLoT dynamically scans a focused laser spot in 3D with a tunable acoustic gradient lens and a 2D electro-optic deflector and has demonstrated real-time tracking at high diffusive speeds (up to 20 µm2/s) and low photon count rates 10 kHz. Here we demonstrate that the high tracking speed, sensitivity, and robustness of 3D-DyPLoT make it ideally suited active feedback 3D single molecule tracking. By implementing optimized tracking parameters based on single molecule model simulation, 3D-DyPLoT can continuously track single fluorescent dye molecules for several minutes at a time, two orders of magnitudes longer than previously reported methods. We demonstrate the application of this new single molecule tracking microscopy to track the free, 3D diffusion of DNA, RNA and proteins in solution. With two-color observation channels, 3D-DyPLoT was also applied to observing the transcription of single DNA molecules in solution by monitoring the real-time production of mRNA. This real-time 3D single molecule tracking method promises to be powerful tool for capturing the dynamics of single biomolecules at high speeds and over 3D distances. (Supported by NIH R35GM124868 and NSF 1847899).

Interaction of Platinum Compounds PtenCl2 and [Pten Cl SO(CH3)2] NO3 with DNA Molecule in Solution

It was demonstrated that the replacement of chlorine ion on dimethyl sulfoxide (DMSO) in the first coordination sphere of platinum changes the result of compound interaction with DNA. DMSO molecule blocks one of the active centers of the platinum compound participating in the formation of coordination bonds with macromolecule, and DNA does not substitute DMSO molecule in the platinum first coordination sphere.

Universal DNA biosensing based on instantaneously electrostatic attraction between hexaammineruthenium (III) and DNA molecules

Despite rapid progress in DNA biosensors by employing various materials as well as techniques, most of the reported sensors are based on specific recognition of a DNA fragment, however can not perform universal measurement of DNA molecules (i.e. genomic DNA). In this work, we proposed a novel DNA biosensing method based on instantaneously electrostatic attraction (IEA) between hexaammineruthenium (III) and DNA molecules. The current variation of freely diffused Ru(NH3)63+ caused by its quick and strong static interaction with phosphate backbones was employed as a universal probe to detect DNA molecules in solution, with no need for immobilization of capture probes on the electrode. After optimization, 30 μL of 300 μM Ru(NH3)63+ solution was added onto the gold electrode with a working electrode diameter of 2 mm, and a detection limit of 3.8 ng/μL was achieved, which is equivalent to NanoDrop™ One spectrometer, the commonly used instrument for DNA quantification. Using reusable and inexpensive gold electrode, the approach provided an easy-operated sequence-independent DNA detection method, and was proved to be able to detect genomic and plasmid DNA directly.

Structural interpretation of DNA-protein hydroxyl-radical footprinting experiments with high resolution using HYDROID.

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.

Conformational changes in the DNA molecule in solution caused by the binding of a light-sensitive cationic surfactant

In the work the properties of high-molecular DNA complexes with a photosensitive cationic azobenzene-containing surfactant capable of UV light induced trans–cis isomerization are studied. DNA compaction caused by its binding with surfactant cis- and trans-isomers is compared, the effect of the low-molecular NaCl salt concentration on the interaction process of the components is considered, and the effect of different surfactant concentrations on the volume and optical anisotropy of the DNA molecule is analyzed. The phase separation in solutions is examined.

Rheological Properties of DNA Molecules in Solution: Molecular Weight and Entanglement Influences.

Molecular weight, stiffness, temperature, and polymer and ionic concentrations are known to widely influence the viscosity of polymer solutions. Additionally, polymer molecular weight—which is related to its dimensions in solution—is one of its most important characteristics. In this communication, low molecular weight DNA from salmon sperm was purified and then studied in solutions in a wide concentration range (between 0.5 and 1600 mg/mL). The intrinsic viscosity of this low molecular weight DNA sample was firstly determined and the evidence of the overlap concentration was detected around the concentration of 125 mg/mL. The chain characteristics of these short molecules were studied in terms of the influence of their molecular weight on the solution viscosities and on the overlap parameter CDNA[η]. Furthermore, to complete previously reported experimental data, solutions of a large molecular weight DNA from calf-thymus were studied in a high concentration range (up to 40 mg/mL). The rheological behavior is discussed in terms of the generalized master curve obtained from the variation of the specific viscosity at zero shear rate (ηsp,0) as a function of CDNA[η].

Grand-canonical simulation of DNA condensation with two salts, effect of divalent counterion size.

The problem of DNA- DNA interaction mediated by divalent counterions is studied using a generalized grand-canonical Monte-Carlo simulation for a system of two salts. The effect of the divalent counterion size on the condensation behavior of the DNA bundle is investigated. Experimentally, it is known that multivalent counterions have strong effect on the DNA condensation phenomenon. While tri- and tetra-valent counterions are shown to easily condense free DNA molecules in solution into toroidal bundles, the situation with divalent counterions is not as clear cut. Some divalent counterions like Mg(+2) are not able to condense free DNA molecules in solution, while some like Mn(+2) can condense them into disorder bundles. In restricted environment such as in two dimensional system or inside viral capsid, Mg(+2) can have strong effect and able to condense them, but the condensation varies qualitatively with different system, different coions. It has been suggested that divalent counterions can induce attraction between DNA molecules but the strength of the attraction is not strong enough to condense free DNA in solution. However, if the configuration entropy of DNA is restricted, these attractions are enough to cause appreciable effects. The variations among different divalent salts might be due to the hydration effect of the divalent counterions. In this paper, we try to understand this variation using a very simple parameter, the size of the divalent counterions. We investigate how divalent counterions with different sizes can lead to varying qualitative behavior of DNA condensation in restricted environments. Additionally, a grand canonical Monte-Carlo method for simulation of systems with two different salts is presented in detail.

Design and Development of a Telomere Sensor Based on Fluorescence Energy Transfer

A 200 nm in-diameter single-bead sensor for detecting single unlabeled DNA molecules in solution based on fluorescence resonance energy transfer is presented. Both bulk and single molecule fluorescence experiments were carried out at room temperature. The G-rich telomere forming a G-quadruplex structure in the presence of ZnTCPP showed two strong donor and acceptor signals. DNA-bound Alexa 488 and crimson 625 loaded in a commercial bead serve as the donor and acceptor, respectively. When Binding of the target DNA to the single bead sensor induces G-quadruplex stretching, resulting in a decrease in fluorescence energy transfer. The limit of sensor's sensitivity can reach 1 fM. Under optimized conditions using a Polydimethylsiloxane (PDMS) microfluidic device direct bead-sensor counting is used to measure the number of beads by control of the flow rate through the probe volume and finish one experiment in 5 minutes. A new parameter based on the ratio of donor and acceptor fluorescence signal from 0.1 to 100 was proposed and used to define the sensitivity and selectivity of bio-sensor.

A single-bead telomere sensor based on fluorescence resonance energy transfer.

We present a 200 nm in-diameter single-bead sensor for the detection of single, unlabeled DNA molecules in solution using fluorescence resonance energy transfer technology. DNA-bound Alexa 488 and Crimson 625 loaded on commercial beads served as the donor and acceptor, respectively. Binding of the target DNA to the single bead sensor induces G-quadruplex stretching, resulting in a decrease in fluorescence energy transfer. G-rich telomere sequences formed a G-quadruplex structure in the presence of ZnTCPP, as demonstrated by the detection of two strong donor and acceptor signals. The sensitivity of the sensor was 1 fM. Under optimized conditions using a polydimethylsiloxane microfluidic device, we measured the number of sensor beads by direct counting. By controlling the flow rate via the probe volume, one sensing experiment can be completed in 5 minutes. Based on these results, we propose a new parameter-dependability (RS)-as a quantitative measure to judge the quality of a bio-sensor. This parameter is based on the ratio of the sensor and sensing sample fluorescence signals. This parameter can range from 0.1 to 100, where a value of 10 represents an optimized bio-sensor.

DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response

The rate of dissociation of a DNA–protein complex is often considered to be a property of that complex, without dependence on other nearby molecules in solution. We study the kinetics of dissociation of the abundant Escherichia coli nucleoid protein Fis from DNA, using a single-molecule mechanics assay. The rate of Fis dissociation from DNA is strongly dependent on the solution concentration of DNA. The off-rate (koff) of Fis from DNA shows an initially linear dependence on solution DNA concentration, characterized by an exchange rate of kex≈9×10−4(ng/μl)−1s−1 for 100mM univalent salt buffer, with a very small off-rate at zero DNA concentration. The off-rate saturates at approximately koff,max≈8×10−3s−1 for DNA concentrations above ≈20ng/μl. This exchange reaction depends mainly on DNA concentration with little dependence on the length of the DNA molecules in solution or on binding affinity, but this does increase with increasing salt concentration. We also show data for the yeast HMGB protein NHP6A showing a similar DNA-concentration-dependent dissociation effect, with faster rates suggesting generally weaker DNA binding by NHP6A relative to Fis. Our results are well described by a model with an intermediate partially dissociated state where the protein is susceptible to being captured by a second DNA segment, in the manner of “direct transfer” reactions studied for other DNA-binding proteins. This type of dissociation pathway may be important to protein–DNA binding kinetics in vivo where DNA concentrations are large.

Dielectrophoretic stretching of DNA tethered to a fiber tip

In this work, we studied the stretching of λ phage DNA molecules immobilized on an optical fiber tip attached to a force sensitive tuning fork under ac electric fields. We designed a two electrodes stretching system in a small chamber: one is a gold-coated optical fiber tip electrode, and the other is a gold-coated flat electrode. By applying a dielectrophoretic (DEP) force, the immobilized λ DNA molecules on the tip are stretched and the stretching process is monitored by a fluorescent microscope. The DNA stretching in three-dimensional space is optimized by varying electrode shape, electrode gap distance, ac frequency, and solution conductivity. By observing the vibrational amplitude change of a quartz tuning fork, we measured the effects due to Joule heating and the DEP force on the tethered DNA molecules in solution. This work demonstrates a method to manipulate and characterize immobilized λ DNA molecules on a probe tip for further study of single DNA molecules.

Size-fitting effect for hybridization of DNA/mercaptohexanol mixed monolayers on gold.

In this article we proposed a simple hexagonal model for exploring the hybridization of thiol-modified probe DNA self-assembled monolayers (SAMs) on gold with target DNA molecules in solution. The size-fitting coefficient d(c)/d(t) from the model was used to discuss the principle for DNA optimal hybridization, where d(c) was the channel diameter among three adjacent probe DNA molecules on gold and dt was the gyration diameter of the target DNA molecules in solution. Experimentally we investigated the hybridization effect (hybridization efficiency H(E) and hybridization density H(D)) of thiol-modified probe DNA (DNA base amount m = 15, 25 or 35)/mercaptohexanol (MCH) mixed SAMs on gold in 1 M electrolyte solution by chronocoulometry (CC) and electrochemical impedance spectroscopy (EIS). The surface coverage Γ(m) of the probe DNA on gold was adjusted by changing the mixed concentration ratio of probe DNA with MCH (C(DNA)/C(MCH)) in the assembly solution. Results indicated that with the increase of C(DNA)/C(MCH), H(E) decreased gradually; H(D) first increased and then decreased, which arrived at the biggest at C(DNA)/C(MCH) = 1 for all the probe DNA/MCH mixed SAMs (m = 15, 25, 35). The optimal Γ(m) for achieving the biggest H(D) in DNA hybridization decreased with the increase of m from 15 to 35. The experimental conclusions obtained by CC and EIS measurements verified each other. Combining the simple model with our experimental results, we ascertained that d(c)/d(t) decreased with the increase of m, which showed a good linear relationship. These conclusions provided an important reference and guidance for controllably constructing DNA sensors with optimal performance.

Note: FixSol solvation model and FIXPVA2 tessellation scheme

The FixSol conductorlike solvation model and the fixed points with variable areas (FIXPVA2) surface tessellation scheme are described. In the FixSol model, the singularity of the surface charge interaction is avoided by introducing a soft-core potential. The use of soft-core potential allows for the use of the FIXPVA2 scheme to partition the molecular surface into tesserae with desirable areas. The FixSol model is used together with biological force field to perform molecular dynamics simulation for proteins and DNA molecules in solution.

Probing the structure of long DNA molecules in solution using synchrotron radiation linear dichroism

Linear dichroism (LD), a spectroscopic method for aligned samples, has been used with a synchrotron radiation source to reveal insights into the structure and stability of DNA with increasing salt concentrations (thus stabilizing the base pairing) and increasing temperature while remaining below the melting point (thus destabilizing the base pairing). Measurements have been made from 350 nm to 182 nm, and the spectral changes observed quantified using a Bayesian Markov chain Monte Carlo (MCMC) algorithm, which uses statistical methods to fit to experimental data. Based on literature H-D exchange experiments, we surmise that the cause of the spectral variations is the induction of transient single stranding of tracts in the DNA polymer, particularly those with significant content of the weaker AT base pairs. More detailed analysis of the LD data will require better nucleotide transition polarization assignments.

Investigation of the radioprotective properties of some tea polyphenols

The influence of tea and coffee flavonoids (−)-epicatechin (EC) and (+)-catechin (C) on the effect of γ-irradiation of DNA molecules in solutions was investigated. The concentrations of these and other components in different samples of black and green tea were analyzed by the capillary electrophoresis. It was determined from the experimental data obtained by different methods (circular dichroism and UV spectra, viscosimetry and optical investigations) that C and EC do not form complexes with DNA in solutions. It was shown that C and EC have radioprotective properties, being scavengers of the water radiolysis products.

Catch, Hold, and Release: Manipulating Single DNA Molecules in Solution

The ability to manipulate matter on submicron length scales has revolutionized biophysical research and fueled important scientific and technological advances in past decades. For example, larger dielectric particles can be trapped free in solution by steep electromagnetic field gradients produced by a strongly focused laser beam (optical tweezers).Pushing the limits to the nanometer level, however, has proven challenging. The only known method for trapping fluorescent nanoparticles such as single molecules uses a time-varying DC field in a feedback loop to counteract Brownian motion. This trap, however, can only operate when the particle can be located through fluorescence detection, which is problematic in the case of single molecules where intermittent fluorescence emission is often observed, and requires a complex hardware and software setup.In this contribution, we will discuss a novel and elegant approach for the trapping and manipulation of single molecules and other particles over extended periods of time: the electrostatic corral. The proposed trapping scheme has distinct characteristics which set it apart from other trapping techniques, such as a trapping efficiency that scales favorably with particle size (down to the single molecule level), a stable potential well that does not require any imaging for particle trapping, and multi-particle trapping capabilities. The feasibility of the corral trap approach will be demonstrated with experiments on micro- and nanoscale particles, with particular emphasis on the trapping of single-stranded DNA molecules.

Heuristic Correlation of Rheological Measurements and Electrochemical Impedance Spectroscopy (EIS) of the DNA/Water System

In this work, it is reported viscosity, density, sound velocity and electrochemical impedance spectroscopy (EIS) measurements of calf thymus DNA in aqueous solutions, in order to investigate the overlapping C* and entanglement concentrations Ce. These critical concentrations demark the conditions at which start appearing interactions and entanglements between DNA molecules in solution. EIS has been proposed as new alternative analytical techniques to obtain C* and Ce in polyelectrolyte solutions. A new parameter the characteristic time (τc) was defined, and it is obtained from a new type of log-log Bode plot using the crossover frequency (τc=1/ωc) of the real (Z') and imaginary (Z") components when they are plotted as a function of the frequency. Preliminary results show similar values C* and Ce by EIS to those obtained from rheological, densimetry and sound velocity measurements.