Characterization of individual polynucleotide molecules using a membrane channel
26 Nov 1996-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 93, Iss: 24, pp 13770-13773
TL;DR: It is shown that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane, which could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.
Abstract: We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.
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University of Cambridge1, Istituto Italiano di Tecnologia2, Lancaster University3, University of Manchester4, Catalan Institution for Research and Advanced Studies5, Technical University of Denmark6, Nokia7, Queen Mary University of London8, fondazione bruno kessler9, University of Trento10, Technische Universität München11, Polytechnic University of Milan12, Centre national de la recherche scientifique13, University of Trieste14, University of Ioannina15, University of Geneva16, Trinity College, Dublin17, Texas Instruments18, University of Paris19, Spanish National Research Council20, Leiden University21, Delft University of Technology22, University of Patras23, École Normale Supérieure24, Radboud University Nijmegen25, Nest Labs26, Airbus UK27, Seoul National University28, Yonsei University29, University of Oxford30, Chalmers University of Technology31, University of Groningen32, STMicroelectronics33, Chemnitz University of Technology34, Max Planck Society35, Aalto University36
TL;DR: An overview of the key aspects of graphene and related materials, ranging from fundamental research challenges to a variety of applications in a large number of sectors, highlighting the steps necessary to take GRMs from a state of raw potential to a point where they might revolutionize multiple industries are provided.
Abstract: We present the science and technology roadmap for graphene, related two-dimensional crystals, and hybrid systems, targeting an evolution in technology, that might lead to impacts and benefits reaching into most areas of society. This roadmap was developed within the framework of the European Graphene Flagship and outlines the main targets and research areas as best understood at the start of this ambitious project. We provide an overview of the key aspects of graphene and related materials (GRMs), ranging from fundamental research challenges to a variety of applications in a large number of sectors, highlighting the steps necessary to take GRMs from a state of raw potential to a point where they might revolutionize multiple industries. We also define an extensive list of acronyms in an effort to standardize the nomenclature in this emerging field.
2,560 citations
Harvard University1, University of California, Santa Cruz2, University of British Columbia3, University of Oxford4, University of Washington5, University of California, San Diego6, Oak Ridge National Laboratory7, Arizona State University8, Brown University9, Case Western Reserve University10, Boston University11, University of North Carolina at Chapel Hill12, North Carolina State University13, National Institutes of Health14
TL;DR: 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, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility.
Abstract: 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 ∼$1,000 in ∼24 h.
2,512 citations
Cites background from "Characterization of individual poly..."
...doc\100309\10:32 The components of an ideal commercial sequencing system using electrical measurements would then be (1) a disposable detector chip containing an array of nanopores having the required integrated microfluidics and electronic probes; and (2) a bench-top instrument, or portable system, that would control the fluidics and electronic elements of the chip and process the raw sequence data....
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TL;DR: A general strategy for the synthesis of highly ordered, rigid arrays of nanoporous carbon having uniform but tunable diameters is described, which gives rise to promising electrocatalytic activity for oxygen reduction and could prove to be practically relevant for fuel-cell technologies.
Abstract: Nanostructured carbon materials are potentially of great technological interest for the development of electronic1,2, catalytic3,4 and hydrogen-storage systems5,6. Here we describe a general strategy for the synthesis of highly ordered, rigid arrays of nanoporous carbon having uniform but tunable diameters (typically 6 nanometres inside and 9 nanometres outside). These structures are formed by using ordered mesoporous silicas as templates, the removal of which leaves a partially ordered graphitic framework. The resulting material supports a high dispersion of platinum nanoparticles, exceeding that of other common microporous carbon materials (such as carbon black, charcoal and activated carbon fibres). The platinum cluster diameter can be controlled to below 3 nanometres, and the high dispersion of these metal clusters gives rise to promising electrocatalytic activity for oxygen reduction, which could prove to be practically relevant for fuel-cell technologies. These nanomaterials can also be prepared in the form of free-standing films by using ordered silica films as the templates.
2,352 citations
TL;DR: The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.
Abstract: The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring nanopores, but advances in technology now allow artificial solid-state nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.
1,861 citations
TL;DR: It is shown that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%.
Abstract: A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications. A protein nanopore with a permanent adaptor molecule can continuously identify unlabelled DNA bases with ∼99.8% accuracy. This level of performance could provide the foundation for the development of nanopore-based DNA sequencing technologies that are faster and less expensive than existing approaches.
1,783 citations
Cites background from "Characterization of individual poly..."
...Each base would be registered, in sequence, by a characteristic decrease in current amplitud...
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References
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Book•
01 Jan 1984
TL;DR: The Ionic Channel of Excitable Membranes (ICOMB) as discussed by the authors is an extended version of ICOMB with new chapters on fast chemical synapses, modulation through G protein coupled receptors and second messenger systems, molecules cloning, site directed mutagenesis, and cell biology.
Abstract: This new, fully revised and expanded edition of "Ionic Channels of Excitable Membranes" includes new chapters on fast chemical synapses, modulation through G protein coupled receptors and second messenger systems, molecules cloning, site directed mutagenesis, and cell biology. It begins with the classical biophysical work of Hodgkin and Huxley and then weaves a description of the known ionic channels together with their biological functions. The book continues by developing the physical and molecular principles needed for explaining permeation, gating, pharmacological modification, and molecular diversity, and ends with a discussion of channel evolution. "Ionic Channels of Excitable Membranes" is written to be accessible and interesting to biological and physical scientists of all kinds.
7,096 citations
TL;DR: A more sensitive method of conductance measurement is reported, which, in appropriate conditions, reveals discrete changes in conductance that show many of the features that have been postulated for single ionic channels.
Abstract: THE ionic channel associated with the acetylcholine (ACh) receptor at the neuromuscular junction of skeletal muscle fibres is probably the best described channel in biological membranes. Nevertheless, the properties of individual channels are still unknown, as previous studies were concerned with average population properties. Macroscopic conductance fluctuations occurring in the presence of ACh were analysed to provide estimates for single channel conductance and mean open times1–3. The values obtained, however, depended on assumptions about the shape of the elementary conductance contribution—for example, that the elementary contribution is a square pulse-like event2. Clearly, it would be of great interest to refine techniques of conductance measurement in order to resolve discrete changes in conductance which are expected to occur when single channels open or close. This has not been possible so far because of excessive extraneous background noise. We report on a more sensitive method of conductance measurement, which, in appropriate conditions, reveals discrete changes in conductance that show many of the features that have been postulated for single ionic channels.
2,377 citations
TL;DR: There is a significant difference between the capacity of bilayers made from mono-layers and that of hydrocarbon-containing bilayer made by phase transition; the average values are 0.9 and 0.45 muF cm(-2), respectively, which approximates that of biological membranes.
Abstract: Bimolecular membranes are formed from two lipid monolayers at an air-water interface by the apposition of their hydrocarbon chains when an aperture in a Teflon partition separating two aqueous phases is lowered through the interface. Formation of the membrane is monitored by an increase of the electrical capacity, as measured with a voltage clamp. Electrical resistance of the unmodified membrane is analogous to that of conventional planar bilayers (black lipid membranes) prepared in the presence of a hydrocarbon solvent, i.e., 106-108 ohm cm2; the resistance can be lowered to values of 103 ohm cm2 by gramicidin, an antibiotic that modifies the conductance only when the membranes are of biomolecular thickness. In contrast to the resistance, there is a significant difference between the capacity of bilayers made from mono-layers and that of hydrocarbon-containing bilayers made by phase transition; the average values are 0.9 and 0.45 μF cm-2, respectively. The value of 0.9 μF cm-2 approximates that of biological membranes. Assuming a dielectric constant of 2.1 for the hydrocarbon region, the dielectric thickness, as calculated from a capacity of 0.9 μF cm-2, is 22 A. This value is 6-10 A smaller than the actual thickness of the hydrocarbon region of bilayers and cell membranes, as determined by x-ray diffraction. The difference may be due to a limited penetration of water into the hydrocarbon region near the ester groups that would lower the electrical resistance of this region and reduce the dielectric thickness. Asymmetric membranes have been formed by adjoining two lipid monolayers of different chemical composition.
1,668 citations
01 Jan 1980
TL;DR: That system is specific in publications sharing across various users and nations, and e-book Biophysical Chemistry Part Iii The Behavior Of Biological Macromolecules Their Biophysical chemistry Pt 3 Download PDF can be also downloaded from here.
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TL;DR: Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former and well-studied phenomena include the stimulation of arachidonic acid metabolism, triggering of granule exocytosis, and contractile dysfunction.
698 citations