Effect of Noise on DNA Sequencing via Transverse Electronic Transport

Previous theoretical studies have shown that measuring the transverse current across DNA strands while they translocate through a nanopore or channel may provide a statistically distinguishable signature of the DNA bases, and may thus allow for rapid DNA sequencing. However, fluctuations of the environment, such as ionic and DNA motion, introduce important scattering processes that may affect the viability of this approach to sequencing. To understand this issue, we have analyzed a simple model that captures the role of this complex environment in electronic dephasing and its ability to remove charge carriers from current-carrying states. We find that these effects do not strongly influence the current distributions due to the off-resonant nature of tunneling through the nucleotides—a result we expect to be a common feature of transport in molecular junctions. In particular, only large scattering strengths, as compared to the energetic gap between the molecular states and the Fermi level, significantly alter the form of the current distributions. Since this gap itself is quite large, the current distributions remain protected from this type of noise, further supporting the possibility of using transverse electronic transport measurements for DNA sequencing.     

Intrinsic fluorescence from DNA can be enhanced by metallic particles

High sensitivity detection of DNA is essential for genomics. The intrinsic fluorescence from DNA is very weak and almost all methods for detecting DNA rely on the use of extrinsic fluorescent probes. We show that the intrinsic emission from DNA can be enhanced many-fold by spatial proximity to silver island films. Silver islands are subwavelength size patches of metallic silver on an inert substrate. Time-resolved measurements show a decreased lifetime for the intrinsic DNA emission near the silver islands. These results of increased intensity and decreased lifetime indicate a metal-induced increase in the radiative rate decay of the DNA bases. The possibility of increased radiative decay rates for DNA bases and other fluorophores suggest a wide variety of DNA measurements and other biomedical assays based on metal-induced increases in the fluorescence quantum yield of weakly fluorescent substances.     

Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore

Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and Hel308 helicase are capable of controlling the motion of DNA containing dNaM and d5SICS through the pore and that single reads are sufficient to detect the presence and location of dNaM and d5SICS within single molecules.     

Contribution of the intrinsic curvature to measured DNA persistence length

The persistence length of DNA, a, depends both on the intrinsic curvature of the double helix and on the thermal fluctuations of the angles between adjacent base-pairs. We have evaluated two contributions to the value of a by comparing measured values of a for DNA containing a generic sequence and for an "intrinsically straight" DNA. In each 10 bp segment of the intrinsically straight DNA an initial sequence of five bases is repeated in the sequence of the second five bases, so any bends in the first half of the segment are compensated by bends in the opposite direction in the second half. The value of a for the latter DNA depends, to a good approximation, on thermal fluctuations only; there is no intrinsic curvature. The values of a were obtained from measurements of the cyclization efficiency for short DNA fragments, about 200 bp in length. This method determines the persistence length of DNA with exceptional accuracy, due to the very strong dependence of the cyclization efficiency of short fragments on the value of a. We find that the values of a for the two types of DNA fragment are very close and conclude that the contribution of the intrinsic curvature to a is at least 20 times smaller than the contribution of thermal fluctuations. The relationship between this result and the angles between adjacent base-pairs, which specify the intrinsic curvature, is analyzed.     

Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination

Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation.     

Probing distance and electrical potential within a protein pore with tethered DNA

DNA molecules tethered inside a protein pore can be used as a tool to probe distance and electrical potential. The approach and its limitations were tested with alpha-hemolysin, a pore of known structure. A single oligonucleotide was attached to an engineered cysteine to allow the binding of complementary DNA strands inside the wide internal cavity of the extramembranous domain of the pore. The reversible binding of individual oligonucleotides produced transient current blockades in single channel current recordings. To probe the internal structure of the pore, oligonucleotides with 5' overhangs of deoxyadenosines and deoxythymidines up to nine bases in length were used. The characteristics of the blockades produced by the oligonucleotides indicated that single-stranded overhangs of increasing length first approach and then thread into the transmembrane beta-barrel. The distance from the point at which the DNA was attached and the internal entrance to the barrel is 43 A, consistent with the lengths of the DNA probes and the signals produced by them. In addition, the tethered DNAs were used to probe the electrical potential within the protein pore. Binding events of oligonucleotides with an overhang of five bases or more, which threaded into the beta-barrel, exhibited shorter residence times at higher applied potentials. This finding is consistent with the idea that the main potential drop is across the alpha-hemolysin transmembrane beta-barrel, rather than the entire length of the lumen of the pore. It therefore explains why the kinetics and thermodynamics of formation of short duplexes within the extramembranous cavity of the pore are similar to those measured in solution, and bolsters the idea that a "DNA nanopore" provides a useful means for examining duplex formation at the single molecule level.     

Gene Transfer: How Can the Biological Barriers Be Overcome?

Physical methods represent a promising approach for the safe delivery of therapeutic plasmid DNA in genetic and acquired human diseases. However, their development in clinics is limited by their low efficacy. At the cellular level, efficient gene transfer is dependent on several factors including extracellular matrix, plasmid DNA uptake and nucleocytoplasmic transport. We review the main barriers that plasmid DNA encounters from the extracellular environment toward the interior of the cell and the different strategies developed to overcome these biological barriers. Diffusional and metabolic fences of the extracellular matrix and the cytoplasm affect plasmid DNA uptake. These barriers reduce the number of intact plasmids that reach the nucleus. Nuclear uptake of plasmid DNA further requires either an increase of nuclear permeability or an active nuclear transport via the nuclear pore. A better understanding of the cellular and molecular bases of the physical gene-transfer process may provide strategies to overcome those obstacles that highly limit the efficiency and use of gene-delivery methods.     

Microscopic Kinetics of DNA Translocation through synthetic nanopores

We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 x 2.6 nm(2) cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.     

Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids

We here characterize local conformations of site-specifically placed pairs of guanine (G) residues in RNA and DNA, using 6-methyl isoxanthopterin (6-MI) as a conformational probe. 6-MI is a base analog of G and spectroscopic signals obtained from pairs of adjacent 6-MI residues reflect base-base interactions that are sensitive to the sequence context, local DNA conformation and solvent environment of the probe bases. CD signals show strong exciton coupling between stacked 6-MI bases in double-stranded (ds) DNA; this coupling is reduced in single-stranded (ss) DNA sequences. Solvent interactions reduce the fluorescence of the dimer probe more efficiently in ssDNA than dsDNA, while self-quenching between 6-MI bases is enhanced in dsDNA. 6-MI dimer probes closely resemble adjacent GG residues, in that these probes have minimal effects on the stability of dsDNA and on interactions with solvent additive betaine. They also serve as effective template bases, although further polymerase-dependent extension of DNA primers past 6-MI template bases is significantly inhibited. These probes are also used to monitor DNA 'breathing' at model replication forks. The 6-MI dimer probe can serve in many contexts as a useful tool to investigate GG conformations at specific sites within the nucleic acid frameworks of functioning macromolecular machines in solution.     

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac⁴C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac⁶az⁸c⁷A). It was found that PDP containing ac⁴C and ac⁶az⁸c⁷A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.     

DNA translocation governed by interactions with solid-state nanopores

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.     

DNA Strands Attached Inside Single Conical Nanopores: Ionic Pore Characteristics and Insight into DNA Biophysics

Single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume. Tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution. Here, we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single-stranded DNA molecules. We find that the DNA occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte. The results are explained by the ionic strength dependence of the persistence length of DNA. At low KCl concentrations (10 mM) the molecules assume an extended and rigid conformation, thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted DNA at high salt concentrations. Attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species.     

Detection of DNA recognition events using multi-well field effect devices

We proposed the multi-well field effect device for detection of charged biomolecules and demonstrated the detection principle for DNA recognition events using quasi-static capacitance-voltage (QSCV) measurement. The multi-well field effect device is based on the electrostatic interaction between molecular charges induced by DNA recognition and surface electrons in silicon through the Si(3)N(4)/SiO(2) thin double-layer. Since DNA molecules and DNA binders such as Hoechst 33258 have intrinsic charges in aqueous solutions, respectively, the charge density changes due to DNA recognition events at the Si(3)N(4) surface were directly translated into electrical signal such as a flat band voltage change in the QSCV measurement. The average flat band shifts were 20.7 mV for hybridization and -13.5 mV for binding of Hoechst 33258. From the results of flat band voltage shifts due to hybridization and binding of Hoechst 33258, the immobilization density of oligonucleotide probes at the Si(3)N(4) surface was estimated to be 10(8) cm(-2). The platform based on the multi-well field effect device is suitable for a simple and arrayed detection system for DNA recognition events.     

SNP detection for cytochrome P450 alleles by target-assembled tandem oligonucleotide systems based on exciplexes

We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (approximately 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A-->C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.     

Microscopic Mechanics of Hairpin DNA Translocation through Synthetic Nanopores

Nanoscale pores have proved useful as a means to assay DNA and are actively being developed as the basis of genome sequencing methods. Hairpin DNA (hpDNA), having both double-helical and overhanging coil portions, can be trapped in a nanopore, giving ample time to execute a sequence measurement. In this article, we provide a detailed account of hpDNA interaction with a synthetic nanopore obtained through extensive all-atom molecular dynamics simulations. For synthetic pores with minimum diameters from 1.3 to 2.2 nm, we find that hpDNA can translocate by three modes: unzipping of the double helix and—in two distinct orientations—stretching/distortion of the double helix. Furthermore, each of these modes can be selected by an appropriate choice of the pore size and voltage applied transverse to the membrane. We demonstrate that the presence of hpDNA can dramatically alter the distribution of ions within the pore, substantially affecting the ionic current through it. In experiments and simulations, the ionic current relative to that in the absence of DNA can drop below 10% and rise beyond 200%. Simulations associate the former with the double helix occupying the constriction and the latter with accumulation of DNA that has passed through the constriction.     

SNP Detection for Cytochrome P450 Alleles by Target-assembled Tandem Oligonucleotide Systems Based on Exciplexes

Abstract

We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (～ 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A→C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.     

电场驱动微悬臂生物传感器及对DNA 的快速、高灵敏度检测

微悬臂列阵传感器在生物检测方面具有快速、痕量和非标记的特性. 我们以镀金并在其上固定了 DNA 探针的微悬臂为正极,在靶杂交液槽内引入另一电极作为负极,构成电场驱动微悬臂 DNA 生物传感器. 对该传感器系统施加静电场,驱动 DNA 分子朝正极迁移,使溶液中的 DNA 分子富集在微悬臂上,促进 DNA 分子的杂交. 结果表明： a. DNA 在微悬臂上的杂交时间仅需 3 min,加快了微悬臂生物传感器对 DNA 分子的检测速度; b. 提高了微悬臂生物传感器的灵敏度,可以检测到皮克级的 DNA 分子.     

Molecular dynamics simulations of DNA within a nanopore: arginine-phosphate tethering and a binding/sliding mechanism for translocation

Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore α-hemolysin (αHL). In the present study, we have developed simplified models of the wild-type αHL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for αHL and its mutants.     

DNA electromagnetic properties and interactions -An investigation on intrinsic bioelectromagnetism within DNA

The question whether intrinsic bioelectromagnetism exists within DNA or not is an important and so far unexplored area of biology. We carried out a study of isolated genetic material, utilizing both prokaryotic and eukaryotic DNA, to measure any possible intrinsic electromagnetic effects or fields emanated within the molecules. Studies were carried out with extremely sensitive ultra-low-noise trans-impedance amplifiers and a high-precision data acquisition system to record any possible faintest electromagnetic signals from the concentrated, as well as diluted DNA, in vitro. Some experiments were performed to investigate any possible electromagnetic effects of high-frequency (HF) RF fields on the DNA under test. However, after extensive testing and careful measurements, we failed to detect any possible intrinsic or induced electromagnetic activity from the DNA as compared to simple water or empty chambers. We reached a conclusion that there does not seem to be any measurable intrinsic electromagnetic activity or fields present in the DNA material, whether in concentrated or diluted form, and if there were, any such activity or fields would be extremely minuscule to be detected with scientific precision by current human measurement methods.     

Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips

We have developed a rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips. The method was validated by the accurate discrimination of blinded DNA samples for the complex quadra-allelic SNP of mannose binding protein. The microchip directed the transport, concentration, and attachment of amplified patient DNA to selected electrodes (test sites) creating an array of DNA samples. Through control of the electric field, the microchip enabled accurate genetic identification of these samples using fluorescently labeled DNA reporter probes. The accuracy of this approach was established by internal controls of dual labeled reporters and by using mismatched sequences in addition to the wild-type and variant reporter sequences to validate the SNP-genotype. The ability to customize this assay for multiple genes has advantages over other existing approaches.