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1.
Chem Soc Rev ; 52(6): 1983-1994, 2023 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-36794856

RESUMO

Nanopores in thin membranes play important roles in science and industry. Single nanopores have provided a step-change in portable DNA sequencing and understanding nanoscale transport while multipore membranes facilitate food processing and purification of water and medicine. Despite the unifying use of nanopores, the fields of single nanopores and multipore membranes differ - to varying degrees - in terms of materials, fabrication, analysis, and applications. Such a partial disconnect hinders scientific progress as important challenges are best resolved together. This Viewpoint suggests how synergistic crosstalk between the two fields can provide considerable mutual benefits in fundamental understanding and the development of advanced membranes. We first describe the main differences including the atomistic definition of single pores compared to the less defined conduits in multipore membranes. We then outline steps to improve communication between the two fields such as harmonizing measurements and modelling of transport and selectivity. The resulting insight is expected to improve the rational design of porous membranes. The Viewpoint concludes with an outlook of other developments that can be best achieved by collaboration across the two fields to advance the understanding of transport in nanopores and create next-generation porous membranes tailored for sensing, filtration, and other applications.


Assuntos
Nanoporos , Membranas Artificiais , Análise de Sequência de DNA/métodos , Água
2.
J Am Chem Soc ; 141(2): 1100-1108, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30557499

RESUMO

Cells use membrane proteins as gatekeepers to transport ions and molecules, catalyze reactions, relay signals, and interact with other cells. DNA nanostructures with lipidic anchors are promising as membrane protein mimics because of their high tunability. However, the design features specifying DNA nanostructures' functions in lipid membranes are yet to be fully understood. Here, we show that altering patterns of cholesterol units on a cubic DNA scaffold dramatically changes its interaction mode with lipid membranes. This results in simple design rules that allow a single DNA nanostructure to reproduce multiple membrane protein functions: peripheral anchoring, nanopore behavior, and conformational switching to reveal membrane-binding units. Strikingly, the DNA-cholesterol cubes constitute the first open-walled DNA nanopores, as only a quarter of their wall is made of DNA. This functional diversity can increase our fundamental understanding of membrane phenomena and result in sensing, drug delivery, and cell manipulation tools.


Assuntos
Materiais Biomiméticos/metabolismo , Colesterol/metabolismo , DNA/metabolismo , Nanoporos , Lipossomas Unilamelares/metabolismo , Materiais Biomiméticos/química , Colesterol/química , DNA/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Lipossomas Unilamelares/química
3.
Langmuir ; 34(49): 15084-15092, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30350681

RESUMO

Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA-membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The Kd values range from 8.5 ± 4.9 to 466 ± 134 µM whereby negatively charged headgroups led to weak binding due to the electrostatic repulsion with respect to the negatively charged DNA. Atomistic MD simulations explain the findings and elucidate the dynamic nature of anchored DNA such as the mushroom-like conformation of single-stranded DNA hovering over the bilayer surface in contrast to a straight-up conformation of double-stranded DNA. The biophysical insight into the binding strength to membranes as well as the molecular accessibility of DNA for hybridization to molecular cargo is expected to facilitate the creation of biomimetic DNA versions of natural membrane nanopores and cytoskeletons for research and nanobiotechnology.


Assuntos
DNA de Cadeia Simples/metabolismo , Bicamadas Lipídicas/metabolismo , Lipossomas Unilamelares/metabolismo , Colesterol/análogos & derivados , DNA de Cadeia Simples/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/metabolismo , Eletricidade Estática , Lipossomas Unilamelares/química
4.
J Am Chem Soc ; 136(28): 9902-5, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-24992159

RESUMO

Biological channels embedded in cell membranes regulate ionic transport by responding to external stimuli such as pH, voltage, and molecular binding. Mimicking the gating properties of these biological structures would be instrumental in the preparation of smart membranes used in biosensing, drug delivery, and ionic circuit construction. Here we present a new concept for building synthetic nanopores that can simultaneously respond to pH and transmembrane potential changes. DNA oligomers containing protonatable A and C bases are attached at the narrow opening of an asymmetric nanopore. Lowering the pH to 5.5 causes the positively charged DNA molecules to bind to other strands with negative backbones, thereby creating an electrostatic mesh that closes the pore to unprecedentedly high resistances of several tens of gigaohms. At neutral pH values, voltage switching causes the isolated DNA strands to undergo nanomechanical movement, as seen by a reversible current modulation. We provide evidence that the pH-dependent reversible closing mechanism is robust and applicable for nanopores with opening diameters of up to 14 nm. The concept of creating an electrostatic mesh may also be applied to different organic polymers.


Assuntos
Canais de Cálcio/química , DNA/química , Polímeros/química , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Potenciais da Membrana , Membranas Artificiais , Eletricidade Estática
5.
Nano Lett ; 13(8): 3890-6, 2013 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-23819625

RESUMO

The voltage-driven passage of biological polymers through nanoscale pores is an analytically, technologically, and biologically relevant process. Despite various studies on homopolymer translocation there are still several open questions on the fundamental aspects of pore transport. One of the most important unresolved issues revolves around the passage of biopolymers which vary in charge and volume along their sequence. Here we exploit an experimentally tunable system to disentangle and quantify electrostatic and steric factors. This new, fundamental framework facilitates the understanding of how complex biopolymers are transported through confined space and indicates how their translocation can be slowed down to enable future sensing methods.


Assuntos
Biopolímeros/química , DNA/química , Nanoporos , Peptídeos/química , Modelos Moleculares , Estrutura Molecular , Eletricidade Estática
6.
Nano Lett ; 12(4): 1983-9, 2012 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-22376238

RESUMO

We present a generic and flexible method to nanopattern biomolecules on surfaces. Carbon-containing nanofeatures are written at variable diameter and spacing by a focused electron beam on a poly(ethylene glycol) (PEG)-coated glass substrate. Proteins physisorb to the nanofeatures with remarkably high contrast factors of more than 1000 compared to the surrounding PEG surfaces. The biological activity of model proteins can be retained as shown by decorating avidin spots with biotinylated DNA, thereby underscoring the universality of the nano-biofunctionalized platform for the binding of other biotinylated ligands. In addition, biomolecule densities can be tuned over several orders of magnitude within the same array, as demonstrated by painting a microscale image with nanoscale pixels. We expect that these unique advantages open up entirely new ways to design biophysical experiments, for instance, on cells that respond to the nanoscale densities of activating molecules.


Assuntos
Avidina/química , Carbono/química , DNA/química , Imunoglobulina G/química , Nanoestruturas/química , Pinturas , Biotina/química , Vidro/química , Ligantes , Polietilenoglicóis/química , Propriedades de Superfície
7.
Nat Commun ; 14(1): 1314, 2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36898984

RESUMO

Building synthetic protocells and prototissues hinges on the formation of biomimetic skeletal frameworks. Recreating the complexity of cytoskeletal and exoskeletal fibers, with their widely varying dimensions, cellular locations and functions, represents a major material hurdle and intellectual challenge which is compounded by the additional demand of using simple building blocks to ease fabrication and control. Here we harness simplicity to create complexity by assembling structural frameworks from subunits that can support membrane-based protocells and prototissues. We show that five oligonucleotides can anneal into nanotubes or fibers whose tunable thicknesses and lengths spans four orders of magnitude. We demonstrate that the assemblies' location inside protocells is controllable to enhance their mechanical, functional and osmolar stability. Furthermore, the macrostructures can coat the outside of protocells to mimic exoskeletons and support the formation of millimeter-scale prototissues. Our strategy could be exploited in the bottom-up design of synthetic cells and tissues, to the generation of smart material devices in medicine.


Assuntos
Células Artificiais , Nanotubos , Células Artificiais/química , DNA/química
8.
Small ; 8(1): 89-97, 2012 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-22083943

RESUMO

The bottom-up approach of DNA nano-biotechnology can create biomaterials with defined properties relevant for a wide range of applications. This report describes nanoscale DNA tetrahedra that are beneficial to the field of biosensing and the targeted immobilization of biochemical receptors on substrate surfaces. The DNA nanostructures act as immobilization agents that are able to present individual molecules at a defined nanoscale distance to the solvent thereby improving biomolecular recognition of analytes. The tetrahedral display devices are self-assembled from four oligonucleotides. Three of the four tetrahedron vertices are equipped with disulfide groups to enable oriented binding to gold surfaces. The fourth vertex at the top of the bound tetrahedron presents the biomolecular receptor to the solvent. In assays testing the molecular accessibility via DNA hybridization and protein capturing, tetrahedron-tethered receptors outperformed conventional immobilization approaches with regard to specificity and amount of captured polypeptide by a factor of up to seven. The bottom-up strategy of creating DNA tetrahedrons is also compatible with the top-down route of nanopatterning of inorganic substrates, as demonstrated by the specific coating of micro- to nanoscale gold squares amid surrounding blank or poly(ethylene glycol)-passivated glass surfaces. DNA tetrahedra can create biofunctionalized surfaces of rationally designed properties that are of relevance in analytical chemistry, cell biology, and single-molecule biophysics.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Nanoestruturas/química , Polietilenoglicóis/química
9.
Nat Protoc ; 16(1): 86-130, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33349702

RESUMO

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores' chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore-membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2-3 weeks for DNA origami pores.


Assuntos
DNA/química , Bicamadas Lipídicas/química , Nanoporos , Nanotecnologia/métodos , Lipossomas Unilamelares/química , Nanoporos/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura
10.
Bioconjug Chem ; 20(3): 466-75, 2009 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-19196022

RESUMO

We describe microarrays of receptors on gold/glass substrates for the selective capturing of viral particles at high density. Microscale gold squares were surface-modified with alkanethiol derivatives which enabled the immobilization of the His(6)-tagged virus-binding domain from the very-low density lipoprotein (VLDL) receptor. The free glass areas surrounding the gold squares were passivated with a dense film of poly(ethylene glycol) (PEG). As assessed by atomic force microscopy, human rhinovirus particles were captured onto the VLDL-receptor patches with a high surface coverage but were effectively repelled by the PEG layer, resulting in a 330 000-fold higher density of the particles on the gold as compared to the glass surfaces. The metal chelate-based coupling strategy was found to be superior to two alternative routes, which used the covalent coupling of viral particles or viral receptors to the substrate surface. The high-density receptor arrays were employed for sensing and characterizing viral particles with so far unprecedented selectivity.


Assuntos
Vidro/química , Ouro/química , Receptores de LDL/metabolismo , Rhinovirus/isolamento & purificação , Vírion/isolamento & purificação , Lipoproteínas VLDL/química , Microscopia de Força Atômica , Polietilenoglicóis/química , Receptores de LDL/química , Rhinovirus/metabolismo , Propriedades de Superfície , Vírion/metabolismo
12.
Nat Commun ; 9(1): 1521, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29670084

RESUMO

Synthetically replicating key biological processes requires the ability to puncture lipid bilayer membranes and to remodel their shape. Recently developed artificial DNA nanopores are one possible synthetic route due to their ease of fabrication. However, an unresolved fundamental question is how DNA nanopores bind to and dynamically interact with lipid bilayers. Here we use single-molecule fluorescence microscopy to establish that DNA nanopores carrying cholesterol anchors insert via a two-step mechanism into membranes. Nanopores are furthermore shown to locally cluster and remodel membranes into nanoscale protrusions. Most strikingly, the DNA pores can function as cytoskeletal components by stabilizing autonomously formed lipid nanotubes. The combination of membrane puncturing and remodeling activity can be attributed to the DNA pores' tunable transition between two orientations to either span or co-align with the lipid bilayer. This insight is expected to catalyze the development of future functional nanodevices relevant in synthetic biology and nanobiotechnology.


Assuntos
DNA/genética , Bicamadas Lipídicas/química , Nanoestruturas/química , Membrana Celular/metabolismo , Colesterol/química , DNA/química , Lipídeos/química , Lipídeos de Membrana/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanoporos , Nanotubos , Polímeros/química , Biologia Sintética
13.
ACS Nano ; 9(11): 11209-17, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26506011

RESUMO

Recently developed synthetic membrane pores composed of folded DNA enrich the current range of natural and engineered protein pores and of nonbiogenic channels. Here we report all-atom molecular dynamics simulations of a DNA nanotube (DNT) pore scaffold to gain fundamental insight into its atomic structure, dynamics, and interactions with ions and water. Our multiple simulations of models of DNTs that are composed of a six-duplex bundle lead to a coherent description. The central tube lumen adopts a cylindrical shape while the mouth regions at the two DNT openings undergo gating-like motions which provide a possible molecular explanation of a lower conductance state observed in our previous experimental study on a membrane-spanning version of the DNT (ACS Nano 2015, 9, 1117-26). Similarly, the central nanotube lumen is filled with water and ions characterized by bulk diffusion coefficients while the gating regions exhibit temporal fluctuations in their aqueous volume. We furthermore observe that the porous nature of the walls allows lateral leakage of ions and water. This study will benefit rational design of DNA nanopores of enhanced stability of relevance for sensing applications, of nanodevices with tunable gating properties that mimic gated ion channels, or of nanopores featuring defined permeation behavior.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Movimento (Física) , Nanoporos , Difusão , Íons , Nanotubos/química , Conformação de Ácido Nucleico , Tamanho da Partícula , Porosidade , Água/química
14.
ACS Nano ; 9(2): 1117-26, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25338165

RESUMO

Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.


Assuntos
DNA/química , Condutividade Elétrica , Bicamadas Lipídicas/química , Conformação Molecular , Nanoporos , Membrana Celular/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Polietilenoglicóis/química
15.
Curr Opin Biotechnol ; 22(4): 485-91, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21664809

RESUMO

Multimeric protein assemblies are essential components in viruses, bacteria, eukaryotic cells, and organisms where they act as cytoskeletal scaffold, storage containers, or for directional transport. The bottom-up structures can be exploited in nanobiotechnology by harnessing their built-in properties and combining them with new functional modules. This review summarizes the design principles of natural protein assemblies, highlights recent progress in their structural elucidation, and shows how rational engineering can create new biomaterials for applications in vaccine development, biocatalysis, materials science, and synthetic biology.


Assuntos
Proteínas/química , Animais , Materiais Biocompatíveis/química , Humanos , Engenharia de Proteínas , Multimerização Proteica , Biologia Sintética
16.
Langmuir ; 23(20): 10244-53, 2007 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-17715951

RESUMO

We describe the formation and characterization of surface-passivating poly(ethylene glycol) (PEG) films on indium tin oxide (ITO) glass substrates. PEG chains with a molecular weight of 2000 and 5000 D were covalently attached to the substrates in a systematic approach using different coupling schemes. The coupling strategies included the direct grafting with PEG-silane, PEG-methacrylate, and PEG-bis(amine), as well as the two-step functionalization with aldehyde-bearing silane films and subsequent coupling with PEG-bis(amine). Elemental analysis by X-ray photoelectron spectroscopy (XPS) confirmed the successful surface modification, and XPS and ellipsometry provided values for film thicknesses. XPS and ellipsometry thickness values were almost identical for PEG-silane films but differed by up to 400% for the other PEG layers, suggesting a homogeneous layer for PEG-silane but an inhomogeneous distribution for other PEG coatings on the molecularly rough ITO substrates. Atomic force microscopy (AFM) and water contact angle goniometry confirmed the different degrees of surface homogeneity of the polymer films, with PEG-silane reducing the AFM rms surface roughness by 50% and the water contact angle hysteresis by 75% compared to uncoated ITO. The ability of the PEG layers to passivate the substrate against the nonspecific adsorption of biopolymers was tested using fluorescence-labeled immunoglobulin G and DNA oligonucleotides in combination with fluorescence microscopy. The results indicate a positive relationship between film density and homogeneity on one hand and the ability to passivate against biopolymer adhesion on the other hand. The most homogeneous layers prepared with PEG-silane reduced the nonspecific adsorption of fluorescence-labeled DNA by a factor of 300 compared to uncoated ITO. In addition, the study finds that the ratio of film thicknesses derived by ellipsometry and XPS is a useful parameter to quantify the structural integrity of PEG layers on molecularly rough ITO surfaces. The findings may be applied to characterize PEG or other polymeric films on similarly coarse substrates.


Assuntos
Polietilenoglicóis/química , Compostos de Estanho/química , Sequência de Bases , Primers do DNA , Microscopia de Força Atômica , Microscopia de Fluorescência , Análise Espectral/métodos
17.
J Am Chem Soc ; 129(31): 9640-9, 2007 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-17636906

RESUMO

We describe nanoscale protein pores modified with a single hyperbranched dendrimer molecule inside the channel lumen. Sulfhydryl-reactive polyamido amine (PAMAM) dendrimers of generations 2, 3 and 5 were synthesized, chemically characterized, and reacted with engineered cysteine residues in the transmembrane pore alpha-hemolysin. Successful coupling was monitored using an electrophoretic mobility shift assay. The results indicate that G2 and G3 but not G5 dendrimers permeated through the 2.9 nm cis entrance to couple inside the pore. The defined molecular weight cutoff for the passage of hyperbranched PAMAM polymers is in contrast to the less restricted accessibility of flexible linear poly(ethylene glycol) polymers of comparable hydrodynamic volume. Their higher compactness makes sulfhydryl-reactive PAMAM dendrimers promising research reagents to probe the structure of porous membrane proteins with wide internal diameters. The conductance properties of PAMAM-modified proteins pores were characterized with single-channel current recordings. A G3 dendrimer molecule in the channel lumen reduced the ionic current by 45%, indicating that the hyperbranched and positively charged polymer blocked the passage of ions through the pore. In line with expectations, a smaller and less dense G2 dendrimer led to a less pronounced current reduction of 25%. Comparisons to recordings of PEG-modified pores revealed striking dissimilarities, suggesting that differences in the structural dynamics of flexible linear polymers vs compact dendrimers can be observed at the single-molecule level. Current recordings also revealed that dendrimers functioned as ion-selectivity filters and molecular sieves for the controlled passage of molecules. The alteration of pore properties with charged and hyperbranched dendrimers is a new approach and might be extended to inorganic nanopores with applications in sensing and separation technology.


Assuntos
Nanoestruturas/química , Poliaminas/química , Proteínas/química , Cromatografia Líquida de Alta Pressão , Dendrímeros , Dissulfetos/química , Íons/química , Modelos Moleculares , Estrutura Molecular , Poliaminas/síntese química , Polietilenoglicóis/química , Porosidade , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Enxofre/química
18.
Langmuir ; 23(17): 8916-24, 2007 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-17636991

RESUMO

Indium tin oxide (ITO) substrates were modified with a layer of poly(amidoamine) (PAMAM) dendrimers to change their surface properties and, in particular, the substrates' work function. The functionalization procedure involved the electrostatic adsorption of positively charged PAMAM dendrimers of generation five onto negatively polarized ITO surfaces. Three different PAMAM dendrimers were used: PAMAM-NH2 and PAMAM-OH with terminal amine and hydroxyl groups, respectively, as well as Q-PAMAM-NH2, which had been prepared from PAMAM-NH2 by quaternization of the dendrimer's terminal and internal amine groups with methyl iodide. The resulting organic films were analyzed by contact angle goniometry, X-ray photoelectron spectroscopy, ellipsometry, and Kelvin probe force microscopy to confirm the presence of a dense layer. A Langmuir isotherm was derived from surface densities of fluorescence-labeled PAMAM-NH2 dendrimers from which we deduced an equilibrium binding constant, K(eq), of (1.3 +/- 0.3) x 10(5) M(-1). Kelvin probe measurements of the contact potential difference revealed a high reduction of the work function from 4.9 eV for bare ITO to 4.3 eV for ITO with a dense film of PAMAM-NH2 of generation five. PAMAM-OH and Q-PAMAM-NH2 resulted in slightly smaller work function changes. This study illustrates that the work function of ITO can be tuned by adlayers composed of PAMAM dendrimers.


Assuntos
Dendrímeros/química , Nylons/química , Compostos de Estanho/química , Aminas/química , Elétrons , Microscopia de Força Atômica , Estrutura Molecular , Fotoquímica , Espectrofotometria , Propriedades de Superfície , Água/química , Raios X
19.
Langmuir ; 22(1): 277-85, 2006 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-16378432

RESUMO

Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Materiais Revestidos Biocompatíveis , Vidro , Teste de Materiais , Microscopia de Força Atômica , Polietilenoglicóis , Análise Espectral , Raios X
20.
Biophys J ; 83(6): 3202-10, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12496089

RESUMO

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.


Assuntos
Técnicas Biossensoriais/instrumentação , Sondas de DNA/química , Proteínas Hemolisinas/química , Técnicas de Sonda Molecular/instrumentação , Oligonucleotídeos/química , Técnicas Biossensoriais/métodos , Membrana Celular/química , Proteínas de Ligação a DNA/química , Eletroquímica/instrumentação , Eletroquímica/métodos , Desenho de Equipamento , Bicamadas Lipídicas/química , Potenciais da Membrana/fisiologia , Membranas Artificiais , Microquímica/métodos , Sondas Moleculares , Nanotecnologia/métodos , Porinas/química , Sensibilidade e Especificidade
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