STM and AFM Images of Nucleosome DNA Under Water

Abstract

We have imaged DNA from the calf thymus nucleosome using a scanning tunneling microscope (STM) operated in water. The fragments are deposited onto the interface between a buffer solution and an epitaxially grown gold surface using an electrochemical technique. Most of the fragments are fairly straight, and when individual polymers can be identified, their length is consistent with the expected 146 basepairs (～ 500 Å). The resolution is often adequate to show signs of the 36 Å helical pitch. Some images show a structure which appears to have abrupt kinks of the sort predicted by Crick and Klug (Nature 255, 530–533,1975). In order to check that this shape is not a consequence of binding to underlying structure on the gold substrate, we have also made images of kinked structures using an atomic force microscope (AFM) with the DNA bound to glass.     

Scanning tunneling microscopy of mercapto-hexyl-oligonucleotides attached to gold.

6-mercapto hexyl-oligonucleotides bind to a gold surface strongly enough to permit imaging by a scanning tunneling microscope (STM). STM images showed worm-like chains that were approximately 12-(A-wide for single-stranded DNA and 20-(A-wide for double-stranded DNA. The chain lengths corresponded to 3.4 +/- 0.4 A per basepair for double-stranded DNA and 2.2 +/- 0.4 A per base for single-stranded DNA. This unexpectedly short length for single-stranded DNA was confirmed using oligomers with both single- and double-stranded regions. When the attachment of the samples was weakened (by imaging in water or scraping with the STM tip) the images changed to pairs of "blobs," apparently reflecting the attachment points of the molecules to the gold surface. Given this interpretation, images of DNA containing a five-base bulge imply that the bulge bends the oligomer by 90 degrees +/- 20 degrees.     

Sequence, packing and nanometer scale structure in STM images of nucleic acids under water

Scanning tunneling microscope (STM) images of random-sequence nucleic acid polymers under water show internal structure which depends strongly on the packing density of the polymer. Images of dense aggregates have a semicrystalline order with the individual polymers adopting simple periodic structures. Loose aggregates (or isolated molecules) show structural variability with considerable local bending and curving on a nanometer scale. It is not clear to what extent this structure is induced by the operation of the microscope. In order to investigate the possibility that the structure is sequence directed, we have imaged various DNA and RNA polymers at low packing densities. We present results here for random sequence DNA, poly(dAT).poly(dAT), poly(dA).poly(dT), poly(dCG).poly(dCG) and for random sequence RNA and poly(U). In contrast to loose aggregates of the random sequence material, the homopolymers show few sharp bends. Furthermore, the homopolymers appear to yield characteristic backbone patterns, usually at resolutions in excess of that obtained with random sequence polymers. The random sequence polymers show much more evidence of image distortion due to tip-molecule interactions, suggesting that they are, on average, mechanically less stable in the STM tunnel-gap than the homopolymers. Thus, while some of the structure observed in STM images is a consequence of tip-molecule interactions, it is related to sequence-directed properties of the polymer.     

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.     

Sequence,Packing and Nanometer Scale Structure in STM Images of Nucleic Acids Under Water

Abstract

Scanning tunneling microscope (STM) images of random-sequence nucleic acid polymers under water show internal structure which depends strongly on the packing density of the polymer. Images of dense aggregates have a semicrystalline order with the individual polymers adopting simple periodic structures. Loose aggregates (or isolated molecules) show structural variability with considerable local bending and curving on a nanometer scale. It is not clear to what extent this structure is induced by the operation of the microscope. In order to investigate the possibility that the structure is sequence directed, we have imaged various DNA and RNA polymers at low packing densities. We present results here for random sequence DNA, poly(dAT) · poly(dAT), poly(dA) · poly(dT), poly(dCG) · poly(dCG) and for random sequence RNA and poly(U). In contrast to loose aggregates of the random sequence material, the homopolymers show few sharp bends. Furthermore, the homopolymers appear to yield characteristic backbone patterns, usually at resolutions in excess of that obtained with random sequence polymers. The random sequence polymers show much more evidence of image distortion due to tip-molecule interactions, suggesting that they are, on average, mechanically less stable in the STM tunnel-gap than the homopolymers. Thus, while some of the structure observed in STM images is a consequence of tip-molecule interactions, it is related to sequence-directed properties of the polymer.     

激光作用质粒DNA和小牛胸腺DNA的AFM研究

激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ产生损伤效应,导致ＤＮＡ结构变化,利用一种改进的试样制备过程和纳米显微镜--原子力显微镜（ＡＦＭ）能够获得可重现的激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ的ＡＦＭ图象,显示它们的特殊的表面结构。     

Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17.

The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.     

Potentiostatic deposition of DNA for scanning probe microscopy.

We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially.     

Analysis of recombinant protein expression using localized surface plasmon resonance (LSPR)

The localized surface plasmon resonance (LSPR)-based optical biosensor using nano-structures of noble metals has been considered as a useful tool for label-free detection of DNA hybridization and protein-protein interactions. We fabricated LSPR-based optical biosensors using gold nano-islands (nominal thickness; 75 A) on glass substrates that were easily made using the conventional fabrication methods. The formation of gold nano-islands on glass substrates was realized by heat treatment of thin gold film deposited with a low deposition rate (approximately 0.05 A/s). The morphologies of sensor surfaces composed of gold nano-islands were observed using an atomic force microscope (AFM) with a non-contact mode. To investigate the sensing capacity of the gold nano-island sensor for the binding of proteins by affinity interactions, the streptavidin and biotin interaction was used as a model system. In addition, detection of recombinant glutathione-S-transferase (GST)-tagged human interleukin-6 (hIL6) expressed in Escherichia coli was carried out by LSPR. It is expected that the LSPR sensors composed of gold nano-islands can be an alternative to traditional methods such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for fast analysis of protein expression.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Architectures based on the use of gold nanoparticles and ruthenium complexes as a new route to improve genosensor sensitivity

The preparation of DNA-sensing architectures based on gold nanoparticles (Au-NPs) in conjunction with an "in situ" prepared ruthenium complex as a new route to improve the analytical properties of genosensors is described. In the development of these architectures several strategies to obtain Au-NPs modified gold electrodes (Au-NP/Au) have been essayed, in particular covalent binding and electrochemical deposition from a solution containing Au-NPs previously synthesized. UV-vis absorption measurements in conjunction with transmission electron microscope (TEM) images reveal that the synthesized Au-NPs are stable for at least 4 weeks and have a narrow size distribution. Atomic force microscopy (AFM) was employed to characterize the morphology and to estimate the Au-NPs surface coverage of the modified gold electrodes obtained following the different modification strategies. In order to assess the utility of these architectures as DNA-sensing devices, a thiolated capture probe sequence from Helicobacter pylori was immobilized onto the as-prepared surface. This sequence was chosen as a case of study within the framework of developing approaches of wide applicability. The hybridization event is detected using a water-soluble pentaamin ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] complex (Ru(NH(3))(5)L) prepared "in situ". This complex, due to its intercalative character, is able to bind to double stranded DNA more efficiently than to single stranded DNA. In addition, the metal provides with a redox center that can be used as an electrochemical indicator. On the basis of this strategy, complementary target sequences of H. pylori have been detected over the range of 40-800pmol with a detection limit of 25+/-2pmol.     

Fungus mediated synthesis of gold nanoparticles and their conjugation with genomic DNA isolated from Escherichia coli and Staphylococcus aureus

Biological systems employing microorganisms have been used as an alternative to conventional chemical techniques for synthesizing gold nanoparticles. In the present study, gold nanoparticles have been synthesized from the supernatant broth (SB) and live cell filtrate (LCF) of the industrially important fungus Penicillium rugulosum. Additionally, potato dextrose broth (PDB) medium which is used for the growth of the fungus has also been able to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification as well as after purification to find the difference in morphology pattern of the nanoparticles. Different characterization techniques like X-ray diffraction (XRD), infra-red (FTIR), X-ray photoelectron (XPS) and UV–vis spectroscopy have been used for analysis of the particles. SB of the fungus has yielded nanoparticles with better morphology and hence further optimization studies were conducted for controlling the size and shape of the above by altering pH and concentration of gold salt. A pH range of 4–6 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. The optimized nanoparticles have been used to conjugate with isolated genomic DNA of bacteria Escherichia coli and Staphylococcus aureus. Visual observation of agarose gel electrophoresis images confirmed the binding of gold nanoparticles (4 μL and 6 μL) with isolated DNA (2 μL) fragments of both the organisms. The slight red shift of the surface plasmon (SP) band and minor aggregations noticed in Bio-TEM images for the DNA conjugated gold nanoparticles indicates that the genomic DNA could stabilize the particles against aggregation owing to negatively charged phosphate backbone.     

The orientation of DNA fragments in the agarose gels

A microscopic method of measuring the orientation of nucleic acids in the agarose gels is described. A nucleic acid undergoing electrophoresis is stained with the dye ethidium bromide and is viewed under high magnification with a polarization microscope. A high-numerical-aperture microscope objective is used to illuminate and to collect the fluorescence signal, and therefore the orientation of the minute quantities of nucleic-acid can be measured: in a typical experiment we can detect the orientation of one-tenth of a picogram (10(13)g) of DNA. Polarization properties of the fluorescent light emitted by the separate bands corresponding to different molecular weights of the DNA are examined. A linear dichroism equation relates the measured fluorescence to the mean orientation of the absorption dipole of the ethidium bromide (and therefore DNA) and to the extent to which it is disorganized. As an example, we measured the orientation of phi X174 DNA RF/HaeIII fragments undergoing electrophoresis in a field of 10 V/cm. Ethidium bromide bound to the fragments with an angle of the absorption dipole largely perpendicular to the direction of the electrophoretic current. The dichroism declined as the molecular weight of the fragments decreased which is interpreted as an increase in the degree of disorder for shorter DNA.     

Independently-addressable micron-sized biosensor elements

With the continuing development of micro-total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules onto a surface in a small well-defined area. A novel method was developed to electrochemically attach DNA probes to micron-sized regions of a gold surface using biotin-LC-hydrazide (BH). Previously, we have found that the radical produced during the oxidation of BH will attach to a wide variety of electroactive surfaces. An array of micron-sized gold band electrodes (75 microm wide) was fabricated onto glass microscope slides and BH was deposited onto each electrode through the application of an oxidizing potential. Subsequent attachment of avidin to the biotinylated surface created the 'molecular sandwich' architecture necessary for further immobilization of biotinylated biomolecules to the surface. In this work, we utilized biotinylated DNA probes of varying sequence to illustrate the specificity of the attachment scheme. The immobilization of avidin, DNA probe, and hybridization of DNA target is visualized with fluorescence tags and the spatially selective attachment and hybridization of unique DNA sequences is demonstrated.     

Mapping of intracellular halogenous molecules by low and high resolution SIMS microscopy.

The subcellular distribution of halogenous molecules has been studied by SIMS microscopy in cultured cells of a human breast carcinoma (MCF-7 cell line). Two instruments of microanalysis were used. A low lateral resolution ion microscope (SMI 300 CAMECA) and a prototype scanning ion microscope equipped with a cesium gun that gives high lateral resolution images. This apparatus has been developed by G Slodzian, in Onera Laboratories (Office National d'Etudes et de Recherches Aérospatiales). Molecules studied by low lateral resolution ion microscope were halogenous steroids: fluorometholone, triamcinolone, bromocriptine and bromoandrosterone. Analytical images show that the first two compounds are mainly localized in the nuclear structure of MCF-7 cells whereas the last two molecules are localized in cytoplasm of these cells. Images were obtained with a resolution of 1 micron. With the scanning ion microscope, it is now possible to obtain images at the ultrastructural level. Four analytical images can be simultaneously obtained by a single scan of the imaged area, corresponding to a depth of erosion of the section of ten nm. The intranuclear distributions of three pyrimidine analogs, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine and 5-fluorouracil have been studied in phase S and M of MCF-7 cells and these images have been compared to the distribution of sulfur, nitrogen and phosphorus. All these images have been obtained with a lateral resolution better than 100 nm.     

Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation

An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ～1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen–phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.     

Multiphase method for automatic alignment of transmission electron microscope images using markers

In order to successfully perform the 3D reconstruction in electron tomography, transmission electron microscope images must be accurately aligned or registered. So far, the problem is solved by either manually showing the corresponding fiducial markers from the set of images or automatically using simple correlation between the images on several rotations and scales. The present solutions, however, share the problem of being inefficient and/or inaccurate. We therefore propose a method in which the registration is automated using conventional colloidal gold particles as reference markers between images. We approach the problem from the computer vision viewpoint; hence, the alignment problem is divided into several subproblems: (1) finding initial matches from successive images, (2) estimating the epipolar geometry between consecutive images, (3) finding and localizing the gold particles with subpixel accuracy in each image, (4) predicting the probable matching gold particles using the epipolar constraint and its uncertainty, (5) matching and tracking the gold beads through the tilt series, and (6) optimizing the transformation parameters for the whole image set. The results show not only the reliability of the suggested method but also a high level of accuracy in alignment, since practically all the visible gold markers can be used.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.     

A gold nanoparticle-based chronocoulometric DNA sensor for amplified detection of DNA

We report a protocol for the amplified detection of target DNA by using a chronocoulometric DNA sensor (CDS). Electrochemistry is known to be rapid, sensitive and cost-effective; it thus offers a promising approach for DNA detection. Our CDS protocol is based on a 'sandwich' detection strategy, involving a capture probe DNA immobilized on a gold electrode and a reporter probe DNA loaded on gold nanoparticles (AuNPs). Each probe flanks one of two fragments of the target sequence. A single DNA hybridization event brings AuNPs, along with hundreds of reporter probes, in the proximity of the electrode. We then employ chronocoulometry to interrogate [Ru(NH3)6]3+ electrostatically bound to the captured DNA strands. This AuNP-amplified DNA sensor can selectively detect as low as femtomolar (zeptomoles) concentrations of DNA targets and conveniently analyze a breast cancer-associated BRCA-1 mutant DNA. The time range for the entire protocol is approximately 3 d, whereas the DNA sensing takes less than 2 h to complete.