COMPLEXES OF NUCLEIC ACID MOLECULES AND METALS

Abstract

Provided is a conductive nucleic acid-metal complex including a polyG and PolyC consisting nucleic acids associated with a plurality of metal atoms, and methods for its preparation.

Claims

1.-60. (canceled)

61. A double-stranded nucleic acid-metal complex, comprising: a double stranded nucleic acid comprising at least one continuous region consisting guanine (G) and cytosine (C) nucleotides, and a plurality of metal atoms; wherein said at least one continuous region is associated with the plurality of said metal atoms.

62. The complex of claim 61, wherein within said continuous region one strand of the double strand nucleic acid consists essentially of G and the other strand consists essentially of C nucleotide bases, or within said continuous region each of the two strands of the double stranded nucleic acid consists a combination of G and C nucleotides.

63. The complex of claim 61, wherein the nucleic acid part of the complex comprises a combination of two or more continuous regions, wherein along at least one of said two or more continuous regions, one strand consists essentially of G and the other consists essentially of C nucleotides, and along at least one other of said two or more continuous regions, each of the strands consists a combination of G and C nucleotides.

64. The complex of claim 61, wherein the double stranded nucleic acid strand is DNA, RNA or a chimera of DNA and RNA.

65. The complex of claim 61, wherein the metal atom is a transition metal selected from Groups IIIB, IVB, VB, VIB, VIIB, VIIIB, IB and IIB of block d of the Periodic Table.

66. The complex of claim 65, wherein the metal is selected from Ag, Cu, Ni, Zn, Co, Cr and Fe.

67. The complex of claim 66, wherein the metal is Ag.

68. A double-stranded nucleic acid-metal complex, comprising: a DNA or RNA or a chimeric DNA-RNA comprising at least one continuous region consisting of guanine (G) and cytosine (C) nucleotides, and a plurality of silver metal atoms; wherein said at least one continuous region is associated with the plurality of said silver metal atoms.

69. The complex according to claim 61, wherein the complex is about one third shorter than the double stranded nucleic acid from which the complex is derived.

70. The complex according to claim 61, wherein the complex has an AFM measurable apparent height that is about a third larger than that of the double stranded nucleic acid from which the complex is derived.

71. The complex according to claim 61, being conductive.

72. A nanowire comprising a complex of claim 61.

73. A method of forming a double-stranded nucleic acid-metal complex, the method comprising contacting a double stranded nucleic acid with at least one metal particle of at least one metal, under conditions permitting said at least one metal particle to dissociate into a plurality of metal atoms; to thereby provide a metal-coated double stranded nucleic acid.

74. The method of claim 73, wherein said at least one metal particle is in a form of an aggregate or a collection or a cluster comprising a plurality of metal atoms.

75. The method of claim 74, wherein said aggregate or collection or cluster of atoms consists a single metal element.

76. The method of claim 73, further comprising a step of sintering the metal atoms.

77. The method of claim 73, wherein the conditions permitting dissociation into a plurality of metal atoms comprise contacting the double stranded nucleic acid with the at least one cluster of atoms of at least one metal in solution, or the conditions permitting dissociation into a plurality of metal atoms comprise contacting the double stranded nucleic acid with the at least one cluster of atoms of at least one metal at room temperature, or the conditions permitting dissociation into a plurality of metal atoms comprise contacting the double stranded nucleic acid with the at least one cluster of atoms of at least one metal in solution, at room temperature.

78. The method of claim 73, wherein the double stranded nucleic acid is DNA, RNA or a chimeric DNA-RNA.

79. The method of claim 73, wherein the metal is silver.

80. A device wherein at least one region thereof is associated with a complex of claim 61.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0142] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0143] FIGS. 1A-F provide AFM images and statistical length analyses of 300 molecules of 2,000 bp poly(dG)-poly(dC) before (FIGS. 1A and 1C) and after (FIGS. 1B and 1D) incubation with the silver nanoparticles. The length values (FIGS. 1C and 1D), corrected for tip convolution by subtracting the molecule's apparent width (a good approximation for the tip diameter) from the measured length, yield an average length of 60030 nm and 40020 nm for the DNA molecules before and after incubation with silver nanoparticles, respectively. The change in height was measured on a large number of cross-sections taken by AFM at various sites along 1500 bp long similarly prepared molecules that were co-deposited on the same substrate (E). Each cross-section is taken along a line scan, measuring the two types of molecules simultaneously. For the points in (FIG. 1E) the heights on each molecule were averaged. The molecules' average heights vs. their measured lengths were correlated. Clearly, two distinguishable populations of molecules are observed (FIG. 1F).

[0144] FIGS. 2A-C depicts morphology evolution of the E-DNA formation. Poly(dG)-poly(dC) DNA (FIG. 2A) was incubated with AgNPs for: 3 (FIG. 2B) and 40 (FIG. 2C) hours. The same color scale is used in all the images. One can note the difference in height of segments along the molecules, corresponding to the height color bar. The E-DNA formation progresses mostly from the molecules termini towards the center, but not in a uniform rate and with some variation from molecule to molecule. Occasionally, it starts from a certain point on the molecule and progresses along the molecule. After 16 hours many of the molecules seem smooth, more rigid and uniform.

[0145] FIGS. 3A-C provide images: TEM (FIG. 3A) and SEM (FIGS. 3B-C) images of E-DNA. Elongated DNA-based molecules are observed as well as some roughly spherical features which are attributed to silver aggregates.

[0146] FIGS. 4A-B is a scheme of E-DNA formation (FIG. 4A) and AFM imaging of an intermediate stage of E-DNA formation (FIG. 4B). FIG. 4Astep (1): The AgNPs bind to DNA and donate its atoms to the nucleic acid. As a result, silver atoms and few atoms clusters are positioned within or on the DNA molecules. Step (2): The NPs dissociate, leaving some of their atoms bound to the DNA. Step (3): A number of binding-dissociation cycles yield E-DNA. FIG. 4BThe DNA was incubated with AgNPs for 20 h and imaged by AFM. AgNPs bound to the DNA molecules are indicated by the arrows.

DETAILED DESCRIPTION OF THE INVENTION

[0147] The complexes of the invention are referred to also as E-DNA (Electrical DNA) and are exemplified by the following non-limiting examples.

[0148] Incubation of poly(dG)-poly(dC) DNA, with silver nanoparticles (NPs) yields uniform linear DNA-based molecules, which are thicker and shorter than the parent dsDNA, as shown by atomic force microscopy (AFM). The resulting DNA-based molecules are visible in transmission and scanning electron microscopy (TEM and SEM), in contrast to the parent dsDNA, which is invisible by both techniques. The morphological changes induced in the DNA can be completely reversed by incubation with dithiothreitol that strongly binds to silver atoms and ions. The morphology of neither poly(dA)-poly(dT) nor of random sequenced plasmid DNA are affected by the presence of the NPs. It is suggested that adsorption of silver atoms by the GC-sequences, which is termed here metallization, takes place during the incubation of the poly(dG)-poly(dC) dsDNA with the NPs. The selectively metalized hybrid DNA-based molecules conduct current and may be used as nanowires in nanoelectronic devices and DNA-based programmable circuits.

[0149] Atomic-force microscopy (AFM) imaging analysis demonstrates that incubation of oligonucleotide-coated 15 nm (in diameter) silver NPs (AgNPs) with poly(dG)-poly(dC) DNA yields uniform DNA-based molecules which length is shorter by one-third and which height is larger by about one-third than that of the parent DNA. The resulting DNA-based molecules are more rigid and more resistant to mechanical deformation than the canonical dsDNA. They can be visualized by TEM and SEM unlike the parent molecules, probably because of the metal atoms adsorption. The presence of silver atoms in the molecules is further indicated by X-ray photoelectron spectroscopy (XPS).

[0150] The dsDNA morphology can be recovered by complexation of the metal using dithiothreitol (DTT). The morphology evolution of formation and decomposition of the molecules was followed by AFM snapshot imaging. Moreover, their circular dichroism (CD) spectrum was changing upon transition and recovered upon treatment with DTT. The molecules were not digested by DNAse I, unlike the parent and recovered molecules. The transition of the dsDNA to E-DNA is sequence (GC) specific. Neither poly(dA)-poly(dT), nor random sequenced plasmid (Puc19) DNA undergo the transition during incubation with silver nanoparticles.

[0151] Incubation of poly(dG)-poly(dC) DNA with 15 nm (in diameter) spherical AgNPs coated with (dA).sub.10, an oligonucleotide composed of 10 deoxyadenosines, led to noticeable changes in morphology of the dsDNA, measured by AFM. It is clearly seen that DNA-based molecules obtained by the incubation are smooth, uniform, less wavy and more rigid as compared to the parent DNA (compare FIGS. 1A and 1B). The contour length of a 2,000 base pair long poly(dG)-poly(dC) DNA (FIG. 1A) was 60030 nm (FIG. 1C). The contour length was reduced by approximately one-third during incubation of the DNA with AgNPs (compare the length histograms in FIGS. 1C and 1D). This shrinkage of the molecules was accompanied by an increase of their height from 0.70.1 nm to 1.10.1 nm (see FIG. 1E). A comprehensive height and height versus length analyses of a large number of cross-sections taken on a large number of similarly prepared 1500 bp long molecules, co-deposited on the same mica substrate, is shown in FIGS. 1E and 1F, respectively. Each cross-section is from the same scan line and taken simultaneously on the two types of molecules. The analysis reveals the height increase, as well as a clear height-length correlation within each one of the two types of molecules. Co-deposition enables a true comparison between both molecule populations, since their morphological features are not scan-dependent and attained under the same measuring conditions.

[0152] The process of E-DNA formation is gradual and takes about 2-3 days to completely convert poly(dG)-poly(dC) to E-DNA. FIG. 2 shows snapshots of the morphology evolution of the E-DNA formation, as depicted by AFM. Poly(dG)-poly(dC) is shown in FIG. 2A, and molecules that were incubated with AgNPs for 3 and 40 hours are shown in FIGS. 2B and 2C, respectively. The morphology change, observed in many molecules even after tens of minutes, progresses slowly on a time scale of hours (see FIG. 1). After 16 hours most of these molecules seem to adopt a nearly final morphological configuration. The transformation process is generally completed within 40 hours. A sharp difference in the height of different segments along the molecules, corresponding to dsDNA and E-DNA is clearly observed. In many cases, E-DNA formation starts at the DNA termini and progresses towards the center. Sometimes, however, it starts from a certain point on the molecule, possibly at the position of a structural defect, and progresses along the dsDNA.

[0153] The metallization process is selective to poly(dG)-poly(dC). The structure of neither poly(dA)-poly(dT), nor plasmid DNA (Puc19) is affected by incubation with the AgNPs. The process is therefore strictly selective and only GC-rich sequences undergo transition to E-DNA.

[0154] CD spectroscopy is a valuable method for studying the DNA conformation. Incubation with AgNPs led to a strong reduction of the signal amplitude in the 250-280 nm range of the spectrum. These data also support the suggestion that the poly(dG)-poly(dC) conformation has been changed during incubation. The new structure cannot, however, be interpreted from these spectra. E-DNA seems to be very stable: incubation with AgNPs at ambient temperature for two or even six months did not lead to any noticeable changes in the molecules morphology. The E-DNA structure seems to reach a thermodynamic equilibrium in the solution after a few days.

[0155] Incubation of E-DNA with DTT for various durations results in gradual but non-linear restoration of the dsDNA morphology. After 16 hours the dsDNA morphology is fully restored. It is known that DTT as well as other SH-containing compounds strongly bind silver atoms. The effect of DTT can thus be explained by assuming that the silver atoms that were bound to the E-DNA are scavenged by the dithiol. The molecule lacking Ag-atoms is then likely transformed back to the canonical dsDNA.

[0156] E-DNA is also resistant to DNAse in contrast to the parent DNA that is almost completely cleaved by the enzyme. The poly(dG)-poly(dC) that results from incubation of the E-DNA with DTT for 16 hours is completely cleaved by DNAse I, similar to the parent molecule as demonstrated by gel electrophoresis.

[0157] To verify the presence of silver atoms in E-DNA, elemental analysis was performed using XPS of molecules deposited on a cystamine modified flame annealed gold substrates. Clear peaks corresponding to silver are seen in the E-DNA sample in contrast to poly(dG)-poly(dC) and bare samples. A control sample, i.e. AgNPs incubated in the absence of DNA and centrifuged to remove the nanoparticles as described in the Experimental section, gave a 60% weaker signal than the E-DNA sample. These results suggest that the silver signal originates from E-DNA molecules and not from AgNPs that might be brought with the E-DNA solution.

[0158] The presence of metal atoms in the E-DNA was further supported by TEM and SEM analyses. FIG. 3 shows TEM (FIG. 3A) and SEM (FIGS. 3B and 3C) images of E-DNA. The TEM and SEM in FIGS. 3A and 3B were measured on the same grid. Elongated DNA-based molecules, which are 3-4 nm in diameter and about 200-400 nm long, were observed. No clear structures were observed in samples on which the same concentration of the parent DNA was deposited either by TEM or SEM. As a further control, E-DNA (approximately 1,200 bp long) was mixed with circular plasmid (puc 19) DNA in equal molar concentrations and imaged by both Cryo-TEM and SEM. Only linear molecules corresponding to the E-DNA length were observed by both techniques as shown in FIG. 3C. The poly(dG)-poly(dC) and the circular puc19 plasmids were not observed presumably due to their very low contrast. The visibility of the E-DNA is attributed to the presence of metal atoms which are likely to increase the contrast since they scatter electrons better than the light organic elements of the non-modified DNA.

[0159] Taking together the results presented here, it is concluded that metallization of poly(dG)-poly(dC) molecules during incubation with the AgNPs takes place. Because in most molecules the transformation to E-DNA starts from the termini and sometimes from some central point, the termini and possibly sequence or other defects along the molecules are empirically more likely to bind AgNPs. The attachment probably triggers the metallization process speculated below, in which the silver is transferred to the molecule and the change of the molecular structure initiates. The directionality of the transition indicates that the gradual change along the molecule occurs base-pair by base-pair. Possibly metal atoms or few atoms clusters translocate to specific positions within or between the base-pairs, initiating the formation of a new equilibrium hybrid structure. The process progresses until the whole molecule is transformed to E-DNA. A tentative scheme, presented in FIG. 4A, illustrates the suggested process of E-DNA formation. The first step includes binding of the particle to the DNA molecule, either on its side or in the termini. AgNPs attached to the DNA are seen in many AFM images (see FIG. 4B), supporting the proposed step. It is unlikely that in the complex silver atoms are directly transferred from the NP to the DNA. The metallization process seems to involve oxidation of silver atoms on the surface of the NP by one of the bases, followed by their binding to the DNA molecule. Guanine, having the lowest ionization potential among the four nucleic bases is the most probable candidate for oxidation of silver atoms in the NP. In addition, the affinity of silver ions to G- and C-bases is higher than to A- and T-bases leading to specific binding of the metal ions to GC-rich DNA. Higher affinity of G and C bases to silver ions as compared to A and T ones together with the highest oxidation potential of guanines among the nucleic bases may account for the sequence-specific metallization of the DNA demonstrated here. After oxidation and binding to DNA a silver atom can get its electron back from the reduced guanine radical. A number of successive cycles of Ag atoms oxidation and transferring from the NP to the DNA results in positioning of the atoms in specific positions along the DNA or in the formation of few atoms silver clusters next to the site of the particle binding on the Poly(dG)-Poly(dC) molecule.

[0160] The metal atoms positioned along the DNA molecules improve the charge transport properties and make E-DNA an attractive candidate for nanoelectronics.

Experimental Details

[0161] Unless otherwise stated, reagents were obtained from Sigma-Aldrich (USA) and were used without further purification. Klenow fragment exonuclease minus of DNA polymerase I from E. coli lacking the 3-, 5-exonuclease activity (Klenow exo.sup.) was purchased from Epicenter Biotechnologies (USA) and puc 19 was from Thermo Fisher Scientific (USA).

Oligonucleotide Purification and DNA Synthesis

[0162] All the DNA samples, A.sub.10, C.sub.12 and G.sub.12 comprising 10 adenines, 12 cytosines and 12 guanines, correspondingly, were purchased from Alpha DNA (Montreal, Canada). Each oligonucleotide (1 mg) was dissolved in 200 L of double distillate water (DDW) and subsequently passed through a pre-packed Sephadex G-25 DNA-Grade column (Amersham, Biosciences) equilibrated with 2 mM Tris-acetate, pH 7.5. The oligonucleotide eluted in the void volume, was collected in 0.4-0.5 mL and purified by ion-exchange HPLC to homogeneity.

Enzymatic Synthesis of DNA

[0163] A standard reaction mixture contained: 60 mM K-Pi (pH 6.5), 5 mM MgCl.sub.2, 5 mM DTT, 1.5 mM dGTP, 1.5 mM dCTP, 0.2 M Klenow exo.sup., and HPLC purified template-primers, (dG).sub.12-(dC).sub.12. The enzymatic reaction was conducted for 1-2 h at 37 C. and was halted by the addition of EDTA to a final concentration of 10 mM.

HPLC Purifications

[0164] The separation of synthesized DNA molecules from nucleotides and other reaction components was on a TSK-gel G-DNA-PW HPLC column (7.8300 mm) from TosoHaas (Japan) by isocratic elution with 20 mM Tris-acetate (pH 7.5) for 30 min at a flow rate of 0.5 mL/min. The purification was conducted on a Finnigan Surveyor LC (Thermo Electron Corporation, USA) HPLC system with a photodiode array detector. Peaks were identified from their retention times obtained from the absorbance at 260 nm for DNA. Eluted products were concentrated by Amicon Ultra-30K-50K MWCO filter devices (Millipore, USA). The length of the synthesized molecules was determined by 1.5% Agarose gel electrophoresis.

Synthesis of AgNPs

[0165] Spherical silver NPs with a diameter of 152 nm were prepared by AgNO.sub.3 reduction in the presence of citric acid and borohydride (NaBH.sub.4) as follows: 180 mL of DDW/filtered water were added into a 0.5 L glass beaker standing in an ice-water bath. 0.45 mL of 0.1M AgNO.sub.3, 0.90 mL of 50 mM sodium citrate and 0.75 mL of 0.6 M NaBH.sub.4 were consequently added into the beaker under vigorous stirring. The yellow solution was stored at 4 C. for 12-16 h. 0.72 mL of 2.5 M LiCl were then added under constant stirring at ambient temperature. The solution was transferred into 15 mL capacity DuPont Pyrex tubes and centrifuged at 14,000 rpm for 1.5 h at 20 C. in a Sorval SS-34 rotor. A fluffy pellet was collected.

[0166] Coating of AgNPs with (dA).sub.10, an oligonucleotide composed of 10 deoxyadenosines, was conducted at ambient temperature as follows: 20 M (dA).sub.10 was added to 4 mL of AgNPs (OD90 at 400 nm). The mechanism of the oligonucleotide binding includes interaction of the nucleic bases with the surface of nanoparticles. The oligonucleotide-coated nanoparticles are relatively stable in aqueous solutions and do not aggregate even at relatively high salt concentrations (up to 150 mM) in contrast to citrate-protected nanoparticles. (dC).sub.10 and (dG).sub.10 oligonucleotides produce similar effect on the nanoparticles stability. In contrast to the above oligonucleotides, incubation of citrate-protected NPs with (dT).sub.10 does not yield stable nanoparticles. The coating procedure includes stepwise increase of the NaCl concentration during the incubation with the oligonucleotides. First the particles were treated with 20 M (dA).sub.10 for 1 h in 25 mM NaCl at room temperature (RT); then the salt concentration was increased to 50 mM and the incubation was continued for additional 16 hours. Finally NaCl concentration was adjusted to 100 mM and the solution was incubated for 2 more hours and subsequently loaded onto a Sepharose 6B-CL column (1.635 cm). Elution was done with 10 mM Na-Pi (pH 7.4). The yellow eluate was collected into Eppendorf tubes and centrifuged at 13,000 rpm for 40 mM at RT on bench-top centrifuge 5424 (Eppendorf, Germany). The fluffy pellet was suspended by pipetting and stored in dark at ambient temperature. The resulting AgNPs were screened for their size and uniformity by TEM, revealing an average diameter of 152 nm. The visible spectra showed a characteristic absorption peak at 400 nm. The concentration of the NPs was calculated using an extinction coefficient () of 210.sup.9 Mol.sup.1 cm.sup.1 at 400 nm.

Preparation of E-DNA

[0167] 200 M (expressed in base pairs), poly(dG)-poly(dC) ranging in length from 1000 to 2000 bp was incubated with (dA).sub.10-coated AgNPs (OD at 400 nm30) in 5 mM Na-Pi (pH=7.5) containing 100 mM NaCl for 2-4 days at RT. AgNPs coated with (dC).sub.10 and (dG).sub.10 can be used for preparation of the E-form as well. The NPs were separated from the DNA by centrifugation for 5 min at 50,000 rpm on an ultracentrifuge (Beckman Coulter Optima TLX, RotorTLA-120.1) at 18 C. The supernatant was collected and stored at RT.

Atomic Force Microscopy

[0168] AFM imaging was performed on molecules adsorbed on muscovite mica surfaces. 100 L aliquots of 0.2 M (in base pairs) DNA solution in 1 mM MgCl.sub.2, were deposited on freshly cleaved 0.50.5 cm mica plates for 5 min. The surface was then washed with ultra-pure distilled water and dried by a nitrogen flow. AFM imaging was performed with two AFM systems: a Solver PRO AFM system (NT-MDT, Russia), in a semi-contact (tapping) mode, using 130 m Si-gold-coated cantilevers (NT-MDT, Russia) with a resonance frequency of 100-120 kHz, and an Aist-NT SmartSPM AFM system, in AC (tapping) mode, using 240 m Medium-Soft Silicon cantilevers (Olympus) with a resonance frequency of 60-80 kHz. The images were flattened (each line of the image was fitted to a second-order polynomial, and the polynomial was then subtracted from the image line) by the Nova image processing software (NT-MDT, Russia). The images were analyzed and visualized using a Nanotec Electronica S.L (Madrid) WSxM imaging software.

CD Spectroscopy

[0169] The spectra were recorded with an Aviv Model 202 series (Aviv Instrument Inc., USA) CD Spectrometer. Each spectrum was recorded from 220 to 350 nm and was an average of 4 measurements. Recording specifications were: wavelength step 1 nm, settling time 0.333 sec, average time 1.0 sec, bandwidth 1.0 nm, path length 1 cm.

X-Ray Photoelectron Spectroscopy (XPS)

[0170] Prior to the DNA deposition flame-annealed gold substrates were treated with cystamine as follows: the substrate was immersed into 0.5 mL of 10 mM cystamine solution and left for 24 hours. The substrate was then rinsed with distilled water and dried with a flow of nitrogen gas. This treatment introduces positive charges (amino groups) to the surface and promotes binding of a negatively charged DNA. A drop of a sample solution containing DNA was poured on the surface and incubated for 40 min. The surface was then washed with ultra-pure distilled water and dried by a nitrogen flow. The X-ray Photoelectron Spectroscopy (XPS) measurements were performed on a Kratos Axis Ultra X-ray photoelectron spectrometer (Karatos Analytical Ltd., Manchester, UK). High resolution XPS spectra were acquired with monochromatic Al K X-ray radiation source (1,486.6 eV) with 90 takeoff angle (normal to the analyzer). The pressure in the chamber was 1.8.Math.10.sup.9 Torr. The high-resolution XPS spectra were collected for Ag 3 d, N 1 s and C 1 s levels with pass energy 20 eV and step 0.1 eV. The samples were prepared on gold substrates, so for high resolution XPS analyses the gold peaks were not measured. Data analyses were performed using Casa XPS (Casa Software Ltd.) and Vision data processing program (Kratos Analytical Ltd.).

TEM Measurements

[0171] Imaging was performed with a FEI Tecnai 12 G.sup.2 Spirit TWIN TEM operated at an acceleration voltage of 120 kV, and images were recorded on a FEI Eagle 4K4K CCD camera in low dose mode and with a 3-5 m defocus.

[0172] For EM, 3 L of sample was applied to a glow discharged ultrathin carbon on carbon lacey support film on 400 mesh copper grid (Ted Pella, Ltd). The excess liquid was blotted with a filter paper, and the grid was allowed to dry in air.

[0173] For Cryo-TEM, a drop (3 L) of the solution was applied to a glow discharged TEM grid (300-mesh Cu grid) coated with a holey carbon film (Lacey substrate, Ted Pella, Ltd.). The excess liquid was blotted, and the specimen was vitrified by a rapid plunging into liquid ethane pre-cooled with liquid nitrogen using Vitrobot Mark IV (FEI).

[0174] The vitrified samples were examined at 177 C. using a FEI Tecnai 12 G.sup.2 Spirit TWIN TEM equipped with a Gatan 626 cold stage, and the images were recorded (4K4K FEI Eagle CCD camera) at 120 kV in low-dose mode.

SEM Measurements

[0175] Scanning electron microscope (SEM) images of the same TEM grids to which the samples were applied and that were first observed by TEM were acquired using a FEI Magellan 400 L XHR SEM (without any further treatment).

[0176] A drop of water solution of GM was placed on a freshly cleaved HOPG and was subsequently removed from the surface with a flow of nitrogen in 5 min. 10 L of the E-DNA solution was applied on a GM-treated HOPG surface. A drop of fresh DDW (100 L) was gently placed above the drop of the sample solution and the liquid was removed from the surface with a flow of nitrogen. This sample was visualized by Zeiss Merlin with GEMINI 11 Electron Optics SEM.