Probing DNA base pairing energy profiles using a nanopore

We experimentally show that the voltage driven unzipping of long DNA duplexes by an α-hemolysin pore is sensitive to the shape of the base pairing energy landscape. Two sequences of equal global stability were investigated. The sequence with an homogeneous base pairing profile translocates faster than the one with alternative weak and strong regions. We could qualitatively account for theses observations by theoretically describing the voltage driven translocation as a biased random walk of the unzipping fork in the sequence dependent energy landscape. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nanopore-based Fourth-generation DNA Sequencing Technology

Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein.Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale.In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.     

Nanopore detection of antibody prion interactions

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. A prion peptide, PrP(143-169), can readily translocate through the pore, but on the addition of monoclonal antibody M2188, which binds the peptide, the number of translocations is reduced because the complex is too large to translocate. At a peptide-to-immunoglobulin G (IgG) ratio of 2:1, only bumping events were observed. The event profile of a control peptide that does not bind the antibody was unchanged. Similarly, the presence of the antibody prevents translocation of the full-length prion protein. Because a nanopore can detect a single molecule, these experiments represent an important first step towards the development of a sensitive prion detector.     

Biocatalytic synthesis of oligosaccharides

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.     

Primary sequence dependence of conformation in oligomannose oligosaccharides

The oligomannose series of oligosaccharides from bovine thyroglobulin (BTG) and the variant surface glycoprotein (VSG) ofTrypanosoma brucei have been isolated and sequenced by¹H NMR. The structure of Man₉GlcNAc₂, the parent molecule of the series, is shown below. Structural isomerism occurs within this series through the removal of residues D1, D2, D3, and C. Using spin-spin coupling and chemical shift data the rotamer distributions about the dihedral angle ω for the Manα1-6Man\ and Manα1-6Manα linkages were determined for each member of the series. It is shown that the dihedral angle ω of the Manα1-6Man\ linkage exhibits low flexibility with a preference for the ω = 180° conformation when residue D2 is present and high flexibility when this residue is absent. Flexibility of ω for the Manα1-6Manα is largely independent of primary sequence and is intermediate between the two Manoα1-6Man\ extremes, again with a preference for the ω = 180° conformation. There are, however, data which indicate that removal of residue D3 may confer additional flexibility upon the dihedral angle ω of the Manα1-6Manα linkage. Molecular graphics modelling, together with chemical and enzymatic modification studies, suggest that the origin of the observed primary sequence dependence of the Manα1-6Man\ linkage arises from steric factors. On the basis of these observations taken together with previous work, it is postulated that recognition of individual oligomannose conformations may play a role in the control of N-linked oligosaccharide biosynthesis. Offprint requests to: T W Rademacher     

Synthesis of natural beta-D-(1-->3)-glucopyranosyl oligosaccharides

beta-D-(1-->3)-Glucan core structure derivatives corresponding to schizophyllan, epiglucan and lentinan were synthesized efficiently. 4,6-O-Benzylidenated glucopyranosyl acceptors were found to be helpful in the attempted beta-D-(1-->3) bond formation. The epiglucan pentasaccharide showed a weak anti-tumor activity in preliminary mice tests.     

Developing a quantitative approach for determining the in vitro prebiotic potential of dietary oligosaccharides

Prebiotics are nondigestible carbohydrates that beneficially affect the host by selectively stimulating the growth and/or activity of one, or a limited number of, bacteria present in the colon. The selected genera should have the capacity to improve host health (e.g. Bifidobacterium, Lactobacillus). To help identify preferred types, for inclusion into the diet, a quantitative equation [measure of the prebiotic effect (MPE)] is suggested. This will help evaluate, in vitro, the fermentation of dietary carbohydrates and compare their prebiotic effect. Although the approach is not meant to define health values, it is formulated to better inform the choice of prebiotic. It therefore, compares measurements of bacterial changes through the determination of maximum growth rates of predominant groups present in faeces, rate of substrate assimilation and the production of lactic, acetic, propionic and butyric acids. The equation will allow further in vitro comparisons of MPE, leading towards further studies (e.g. in humans) to determine the success of dietary intervention.     

Synthesis of biantennary beta-D-(1-->6) glucosamine oligosaccharides

Biantennary beta-D-(1-->6) glucosamine hexa-, octa-, and dodecaoligosaccharide derivatives were synthesized convergently using isopropyl thioglycosides as donors in NIS/TMSOTf-catalyzed glycosylation.     

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le^a and A, Le^b). Neutral and acidic components were separated from each other by anion-ecchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore, the molar response factors after HPAEC-PAD have been determined for 8 components.     

Preparation and structural determination of large oligosaccharides derived from acharan sulfate

The structures of a series of large oligosaccharides derived from acharan sulfate were characterized. Acharan sulfate is an unusual glycosaminoglycan isolated from the giant African snail, Achatina fulica. Oligosaccharides from decasaccharide to hexadecasaccharide were enzymatically prepared using heparin lyase II and purified. Capillary electrophoresis and gel electrophoresis confirmed the purity of these oligosaccharides. Their structures, determined by ESI-MS and NMR, were consistent with the major repeating sequence in acharan sulfate, -->4)-alpha-d-GlcN(p)Ac-(1-->4)-alpha-l-IdoA(p)2S-(1-->, terminated by 4-linked alpha-d-GlcN(p)Ac residue at the reducing end and by 4,5-unsaturated pyranosyluronic acid 2-sulfate at the non-reducing end.     

Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of alpha-hemolysin channel and the channel formation of alpha-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29+/-4% is observed between 0 and 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.     

Computer simulation of polypeptide translocation through a nanopore

A simplified model of polypeptide chains was designed and studied by means of computer simulations. Chains were represented by a sequence of united atoms located at the positions of the -carbons. A further assumption was the lattice approximation for the chains. We used a (310) lattice, which was found useful for studying properties of proteins. The force field used consisted of a long-range contact potential between amino-acid residues and a local preference for forming -helical states. The chain consisted of two kinds of residues: hydrophilic (P) and hydrophobic (H) ones forming model helical septets –HHPPHPP– in a sequence. The chains were placed near an impenetrable surface with a square hole in it. The size of the hole was comparable or smaller than the size of a chain. The properties of these model chains were determined using the Monte-Carlo simulation method. During the simulations, translocation of the chain through the hole in the wall was observed. The influence of the chain length, the temperature differences on both sides of the wall and the force field on the chain properties were investigated. It was shown that the translocation time scales as N^2.2 and it was found that the presence of the local helical potential significantly slows down the process of translocation.Figure: The snapshots of typical chains conformation obtained during the simulation for chain consisted of N = 60. The values of the local potential _loc = -8.     

Translocation of a single-stranded DNA through a conformationally changing nanopore

We investigate the translocation of a single-stranded DNA through a pore which fluctuates between two conformations, using coupled master equations. The probability density function of the first passage times of the translocation process is calculated, displaying a triple-, double-, or monopeaked behavior, depending on the interconversion rates between the conformations, the applied electric field, and the initial conditions. The cumulative probability function of the first passage times, in a field-free environment, is shown to have two regimes, characterized by fast and slow timescales. An analytical expression for the mean first passage time of the translocation process is derived, and provides, in addition to the interconversion rates, an extensive characterization of the translocation process. Relationships to experimental observations are discussed.     

Simulation and optimization of batch autohydrolysis of wheat straw to monosaccharides and oligosaccharides

Twenty-four non-isothermal wheat straw autohydrolysis experiments were performed in a batch reactor in order to support the development of a new kinetic model. An optimum of 76% w/w total xylose was obtained due to 5% w/w xylose degradation at 180 °C for 70 min. An optimum of 31% w/w total glucose was obtained due to 22% w/w glucose degradation at 240 °C for 82 min. The autohydrolysis of cellulose and hemicelluloses was simulated using a new kinetic model, in which a new phenomenological first-order reaction was introduced to take into account the increasing concentration of acids that are produced during the complex cascade of reactions. The new model simulated experimental results more accurately than the severity factor (R0) model.     

Enzymatic synthesis of oligosaccharides from branched cyclodextrins

6'-alpha-Maltosyl-maltotriose and 6'-alpha-D-glucosyl-maltotriose were prepared from Novamyl degradation of 6-O-alpha-maltosyl-alpha-cyclodextrin and 6-O-alpha-D-glucosyl-alpha-cyclodextrin, respectively. NMR spectroscopy was used to elucidate their structural identities, in a combination of COSY experiments. Further, a mechanism for the degradation was proposed based on the Novamyl active site geometry.     

Observing structure,function and assembly of single proteins by AFM

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.     

Fermentation effects of oligosaccharides of Radix Ophiopogonis on alloxan-induced diabetes in mice

In this study, oligosaccharides extracted from Ophiopogon japonicus vinegar (OOV) by alcoholic and acetic acid fermentation with water extracts from Radix Ophiopogon and oligosaccharides extracted from Radix Ophiopogonis (OOJ) were investigated. Characterization of the extracts indicated that OOV are proteoglycans, whereas OOJ are not. Moreover, compared with OOJ, monosaccharide compositions of OOV only include fructose and galactose and not glucose. MALDI-TOF-mass spectrometric results showed that the molecular weight of OOV was smaller after fermentation. Changes in the characteristics of OOV would inevitably lead to changes in its hypoglycemic properties. The OOV inhibition activity against α-glucosidase was stronger than that of OOJ. The inhibition activity became stronger with higher dosages of OOV. The hypoglycemic effect of OOV on alloxan-induced diabetic mice was stronger than that of OOJ. More important, the ability of OOV to reduce damage on islets in diabetic mice was stronger than that of OOJ. Overall, alcoholic and acetic acid fermentation improved the hypoglycemic activity of OOJ.     

Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Determination of RNA orientation during translocation through a biological nanopore

We investigate single-molecule electrophoretic translocation of A(50), C(50), A(25)C(50), and C(50)A(25) RNA molecules through the alpha-hemolysin transmembrane protein pore. We observe pronounced bilevel current blockages during translocation of A(25)C(50) and C(50)A(25) molecules. The two current levels observed during these bilevel blockages are very similar to the characteristic current levels observed during A(50) and C(50) translocation. From the temporal ordering of the two levels within the bilevel current blockages, we infer whether individual A(25)C(50) and C(50)A(25) molecules pass through the pore in a 3'-->5' or 5'-->3' orientation. Correlation between the level of current obstruction and the inferred A(25)C(50) or C(50)A(25) orientation indicates that 3'-->5' translocation of a poly C segment causes a significantly deeper current obstruction than 5'-->3' translocation. Our analysis also suggests that the 3' ends of C(50) and A(25)C(50) RNA molecules are more likely to initiate translocation than the 5' ends. Orientation dependent differences in a smaller current blockage that immediately precedes many translocation events suggest that this blockage also contains information about RNA orientation during translocation. These findings emphasize that the directionality of polynucleotide molecules is an important factor in translocation and demonstrate how structure within ionic current signals can give new insights into the translocation process.