The potential and challenges of nanopore sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.     

Nanocolonies: Detection, cloning, and analysis of individual molecules

Nanocolonies (other names molecular colonies or polonies) are formed upon template nanomolecule (DNA or RNA) amplification in immobilized medium with efficient pore size in the nanometer range. This work deals with the principle, invention, development, and diverse nanocolony applications based on their unique abilities to compartmentalize amplification and expression of individual DNA and RNA molecules, including studying reactions between single molecules, digital molecular diagnostics, in vitro gene cloning and expression, as well as identification of the molecular cis-elements including DNA sequencing, analysis of single-nucleotide polymorphism, and alternative splicing investigation.     

Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel

RNA and DNA strands produce ionic current signatures when driven through an alpha-hemolysin channel by an applied voltage. Here we combine this nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale. Measurable properties include duplex stem length, base pair mismatches, and loop length. This nanopore instrument can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.     

Nucleic acid aptamers and enzymes as sensors

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.     

Curcumin-embedded DBPC liposomes coated with chitosan layer as a fluorescence nanosensor for the selective detection of ribonucleic acid

One of the limitations of fluorescence probe molecules during biomedical estimation is their lack of ability to selectively determine the targeted species. To overcome this there have been various approaches that involve attaching a functional group or aptamers to the fluorescence probe. However, encapsulating probe molecules in a matrix using nanotechnology can be a viable and easier method. Curcumin (Cur) as a fluorescence marker cannot distinguish DNA and RNA. This research reports a novel selective approach involving the use of nanocapsules composed of liposomal curcumin coated with chitosan for the selective detection of RNA molecules using a fluorescence method. The increase in RNA concentration enhanced the electrostatic interaction between the negatively charge surface of RNA and the positively charged nanocapsule, which was further verified by zeta potential measurement. This method had a low limit of detection (36 ng/ml) and higher linear dynamic ranges compared with other studies found in the literature. Moreover, the method was not affected by DNA and was selective for the detection of RNA molecules for which the site of interaction was confined only to uracil. The selectivity for RNA molecules towards other analogues species was also examined and recovery range found was between 99 and 100.33%.     

Passive and facilitated transport in nuclear pore complexes is largely uncoupled

Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.     

Intrinsic aqueduct orifices facilitate K+ channel gating

The ion-conducting pore of potassium channels, which can open and close to regulate ion passage, was at long thought to be a one-dimensional pore structure with a water-filled central cavity. Here, we find four orifices in the KcsA potassium channel, which are perpendicular to the pore and stretch out from the cavity. Equilibrium molecular dynamics simulations show that water molecules can flow between the cavity and orifices. Targeted molecular dynamics simulations show that during the opening process, water molecules can move into the cavity through the orifices to facilitate channel gating, whereas blocking the aqueduct orifices makes the channel difficult to open.     

Wide nanoscopic pore of maxi-anion channel suits its function as an ATP-conductive pathway

The newly proposed function of the maxi-anion channel as a conductive pathway for ATP release requires that its pore is sufficiently large to permit passage of a bulky ATP(4-) anion. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the first time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cutoff radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of approximately 1.3 nm. These three estimates are considerably larger than the radius of ATP(4-) (0.57-0.65 nm) and MgATP(2-) (approximately 0.60 nm). We therefore conclude that the nanoscopic maxi-anion channel pore provides sufficient room to accommodate ATP and is well suited to its function as a conductive pathway for ATP release in cell-to-cell communication.     

Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.     

ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis.

The "molecular Coulter counter" concept has been used to study transport of ATP molecules through the nanometer-scale aqueous pore of the voltage-dependent mitochondrial ion channel, VDAC. We examine the ATP-induced current fluctuations and the change in average current through a single fully open channel reconstituted into a planar lipid bilayer. At high salt concentration (1 M NaCl), the addition of ATP reduces both solution conductivity and channel conductance, but the effect on the channel is several times stronger and shows saturation behavior even at 50 mM ATP concentration. These results and simple steric considerations indicate pronounced attraction of ATP molecules to VDAC's aqueous pore and permit us to evaluate the effect of a single ATP molecule on channel conductance. ATP addition also generates an excess noise in the ionic current through the channel. Analysis of this excess noise shows that its spectrum is flat in the accessible frequency interval up to several kilohertz. ATP exchange between the pore and the bulk is fast enough not to display any dispersion at these frequencies. By relating the low-frequency spectral density of the noise to the equilibrium diffusion of ATP molecules in the aqueous pore, we calculate a diffusion coefficient D = (1.6-3.3)10(-11) m2/s. This is one order of magnitude smaller than the ATP diffusion coefficient in the bulk, but it agrees with recent results on ATP flux measurements in multichannel membranes using the luciferin/luciferase method.     

短杆菌肽A-DMPC通道内离子输运的分子动力学模拟

用最近提出的构建膜体系初始构象的有效方法 ,构建了在DMPC脂膜环境下短杆菌肽A通道模型 (GA -DMPC)。通过对Na 、Ca2 、Cl-三种不同离子在GA -DMPC通道内不同位置的分子动力学模拟 ,研究离子在通道内输运过程中与通道及通道内水分子的相互作用 ,从分子动力学的角度阐明离子在通道内的输运机制。主要计算结果表明 :(1)离子在通道内的输运使GA的构象发生变化 ,GA的柔性是离子在通道内通透的重要因素 ;(2)Cl- 离子可扩大通道半径 ,Na 离子和Ca2 离子则减小通道半径。Cl-离子不能在GA通道内通透 ;(3)离子的出现使通道内水分子的偶极方向发生变化。上述结果均与实验相符。     

Vaccinia virus RNA helicase: nucleic acid specificity in duplex unwinding.

Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate-dependent unwinding of duplex RNAs containing a single-stranded 3' RNA tail. In this study, we examine the structural features of the nucleic acid substrate that are important for helicase activity. Strand displacement was affected by the length of the 3' tail. Whereas NPH-II efficiently unwound double-stranded RNA substrates with 19- or 11-nucleotide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding by an order of magnitude. Processivity of the helicase was inferred from its ability to unwind a tailed RNA substrate containing a 96-bp duplex region. NPH-II exhibited profound asymmetry in displacing hybrid duplexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unwound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less effective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as helicase substrates. On the basis of current models for RNA-DNA hybrid structures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynucleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary structure impedes protein translocation through DNA duplexes.     

Introns, protein syntheses and aging

In the fungus Podospora, a correlation has recently been established between the presence of circular DNA molecules arising from the mitochondrial genome (SEN-DNAs) and the senescence syndrome. Here, I propose a hypothesis which accounts for the initial event which leads to the first SEN-DNA. A molecule in the most frequent situation where the SEN-DNA is an intron which might code for a maturase. This hypothesis is based upon several observations made either in Podospora or in the yeast S. cerevisiae. It assumes that mitochondrially synthesized maturases are unspecific nucleases able to work at the level of RNA and DNA molecules. Their specificity for RNA splicing instead of DNA is given by cytoplasmic proteins. Therefore, if the balance between cytoplasmic and mitochondrial protein syntheses is disturbed in favour of the mitochondrial compartment, the maturase would be accumulated and allowed to splice introns from DNA instead of RNA molecules. This hypothesis can account for aging of higher eucaryotic cells by postulating analogous processes in their nuclear compartment.     

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.     

R环的形成及对基因组稳定性的影响

R-环是由一个RNA:DNA杂交体和一条单链状态的DNA分子共同组成的三链核酸结构。其中, RNA:DNA杂交体的形成起因于基因转录所合成的RNA分子不能与模板分开, 或RNA分子重新与一段双链DNA分子中的一条链杂交。在基因转录过程中, 当转录泡遇到富含G碱基的非模板链区或位于某些与人类疾病有关的三核苷酸卫星DNA时, 转录泡后方累积的负超螺旋可促进R环形成。同时, 新生RNA分子未被及时加工、成熟或未被快速转运到细胞质等因素也会催生R环。研究表明, 细胞拥有多种管理R环的方法, 可以有效地管理R环的形成和处理已经形成的R环, 以尽量避免R环对DNA复制、基因突变和同源重组产生不利影响。文章重点分析了R-环的形成机制及R环对DNA复制、基因突变和同源重组的影响, 并针对R-环诱导的DNA复制在某些三核苷酸重复扩增有关的神经肌肉退行性疾病发生过程中的作用进行了分析和讨论。     

RNA as a catalyst: Natural and designed ribozymes

RNA can catalyse chemical reactions through its ability to fold into complex three-dimensional structures and to specifically bind small molecules and divalent metal ions. The 2′-hydroxyl groups of the ribose moieties contribute to this exceptional reactivity of RNA, compared to DNA. RNA is not only able to catalyse phosphate ester transfer reactions in ribonucleic acids, but can also show aminoacyl esterase activity, and is probably able to promote peptide bond formation. Bearing its potential for functioning both as a genome and as a gene product, RNA is suitable for in vitro evolution experiments enabling the selection of molecules with new properties. The growing repertoire of RNA catalysed reactions will establish RNA as a primordial molecule in the evolution of life.     

On the possible permeation of water across the glucose transporter

The possibility that the glucose transporter may serve as water channel is explored with the help of theoretical and experimental arguments. A model for a pore is drawn based on a hypothetical water channel structure, subject to the constraints that: molecules will bind to the channel wall in successive rings, forming a hollow sleeve; an integer number of molecules will exist in each ring; the pore radius will not be large enough to allow water molecules along its center, but will be large enough to allow glucose molecules across. The only configurations that meet these conditions exhibit either 5 or 6 water molecules abreast in each ring, with pore radii of 4.1 and 4.5 Å, respectively. The kinetic characteristics of such pores are estimated and found to conform to available evidence.