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Arrays of Individual DNA Molecules on Nanopatterned Substrates

View Article: PubMed Central - PubMed

ABSTRACT

Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells.

No MeSH data available.


Cy3-labeled DNA strands bind electrostatically with 1:1 stoichiometry onto 50 nm avidin-coated carbon nanoislands while multiple DNA strands adsorb onto 100 nm islands.DNA was dissolved in TAE buffer supplemented with 14 mM MgCl2. (A) Fluorescence microscopic image of an unpatterned substrate with non-specifically bound labeled DNA strands. (B,C) Fluorescence microscopic images of arrays of 10 × 10 islands with (B) 50 nm diameter and (C) 100 nm side length written with an electron dose of 0.87 pC. Image size: 39 × 39 μm. The insets for A to C show the distribution of fluorescence on the islands obtained from the microscopy images. The x-axis scale is the same as in Figure 2. (D) Total fluorescence signal of individual non-specific bound DNA, and DNA-derivatized 50 nm and 100 nm islands.
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f3: Cy3-labeled DNA strands bind electrostatically with 1:1 stoichiometry onto 50 nm avidin-coated carbon nanoislands while multiple DNA strands adsorb onto 100 nm islands.DNA was dissolved in TAE buffer supplemented with 14 mM MgCl2. (A) Fluorescence microscopic image of an unpatterned substrate with non-specifically bound labeled DNA strands. (B,C) Fluorescence microscopic images of arrays of 10 × 10 islands with (B) 50 nm diameter and (C) 100 nm side length written with an electron dose of 0.87 pC. Image size: 39 × 39 μm. The insets for A to C show the distribution of fluorescence on the islands obtained from the microscopy images. The x-axis scale is the same as in Figure 2. (D) Total fluorescence signal of individual non-specific bound DNA, and DNA-derivatized 50 nm and 100 nm islands.

Mentions: The electrostatically adhesive 50 nm islands were exploited to bind individual DNA strands of negative charge (Fig. 1, step 3). We used single stranded DNA of 7249 nucleotides length which can form nanoballs with a hydrodynamic diameter of 50–60 nm in buffers with ionic strength of 0.05 to 0.1 M3948. This hydrodynamic diameter of the nanoball matches the size of the smaller nanoislands and is expected to lead to a 1:1 stoichiometry when the DNA binds to the island. Upon binding to a substrate surface, the nanoball can adopt a flattened shape which is further compressed when imaged by AFM to yield an inverted cup with a height of 14.9 ± 4.3 nm and a diameter of 119 ± 22 nm (n = 20)38 (Supplementary Information, Fig. S-4). To facilitate fluorescence detection of the bound DNA strand, labeling with the Cy3 fluorophore was achieved by the covalent attachment at nitrogen 7 of the guanine base (Supplementary Information, Fig. S-5). DNA strands were labeled with multiple fluorophores to avoid issues that arise from bleaching of single fluorophores. The successful fluorescence tagging was confirmed by gel electrophoresis (Supplementary Information, Fig. S-5). The average number of Cy3 dyes per DNA strands was obtained by isolating individual molecules via non-specific binding onto non-arrayed substrates and subjecting them to fluorescence scanning (Fig. 3A). The average brightness of single strands was 4290 ± 2990 counts (n = 101) which corresponds to approximately 100 fluorophores per strand calculated from the known average brightness of individual Cy3 fluorophores, if other optical effects such as quenching is excluded. The fluorescence distribution of the Cy-3 labeled DNA (Fig. 3A) was in line with the stochastic number of labeled guanine bases within the strand.


Arrays of Individual DNA Molecules on Nanopatterned Substrates
Cy3-labeled DNA strands bind electrostatically with 1:1 stoichiometry onto 50 nm avidin-coated carbon nanoislands while multiple DNA strands adsorb onto 100 nm islands.DNA was dissolved in TAE buffer supplemented with 14 mM MgCl2. (A) Fluorescence microscopic image of an unpatterned substrate with non-specifically bound labeled DNA strands. (B,C) Fluorescence microscopic images of arrays of 10 × 10 islands with (B) 50 nm diameter and (C) 100 nm side length written with an electron dose of 0.87 pC. Image size: 39 × 39 μm. The insets for A to C show the distribution of fluorescence on the islands obtained from the microscopy images. The x-axis scale is the same as in Figure 2. (D) Total fluorescence signal of individual non-specific bound DNA, and DNA-derivatized 50 nm and 100 nm islands.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Cy3-labeled DNA strands bind electrostatically with 1:1 stoichiometry onto 50 nm avidin-coated carbon nanoislands while multiple DNA strands adsorb onto 100 nm islands.DNA was dissolved in TAE buffer supplemented with 14 mM MgCl2. (A) Fluorescence microscopic image of an unpatterned substrate with non-specifically bound labeled DNA strands. (B,C) Fluorescence microscopic images of arrays of 10 × 10 islands with (B) 50 nm diameter and (C) 100 nm side length written with an electron dose of 0.87 pC. Image size: 39 × 39 μm. The insets for A to C show the distribution of fluorescence on the islands obtained from the microscopy images. The x-axis scale is the same as in Figure 2. (D) Total fluorescence signal of individual non-specific bound DNA, and DNA-derivatized 50 nm and 100 nm islands.
Mentions: The electrostatically adhesive 50 nm islands were exploited to bind individual DNA strands of negative charge (Fig. 1, step 3). We used single stranded DNA of 7249 nucleotides length which can form nanoballs with a hydrodynamic diameter of 50–60 nm in buffers with ionic strength of 0.05 to 0.1 M3948. This hydrodynamic diameter of the nanoball matches the size of the smaller nanoislands and is expected to lead to a 1:1 stoichiometry when the DNA binds to the island. Upon binding to a substrate surface, the nanoball can adopt a flattened shape which is further compressed when imaged by AFM to yield an inverted cup with a height of 14.9 ± 4.3 nm and a diameter of 119 ± 22 nm (n = 20)38 (Supplementary Information, Fig. S-4). To facilitate fluorescence detection of the bound DNA strand, labeling with the Cy3 fluorophore was achieved by the covalent attachment at nitrogen 7 of the guanine base (Supplementary Information, Fig. S-5). DNA strands were labeled with multiple fluorophores to avoid issues that arise from bleaching of single fluorophores. The successful fluorescence tagging was confirmed by gel electrophoresis (Supplementary Information, Fig. S-5). The average number of Cy3 dyes per DNA strands was obtained by isolating individual molecules via non-specific binding onto non-arrayed substrates and subjecting them to fluorescence scanning (Fig. 3A). The average brightness of single strands was 4290 ± 2990 counts (n = 101) which corresponds to approximately 100 fluorophores per strand calculated from the known average brightness of individual Cy3 fluorophores, if other optical effects such as quenching is excluded. The fluorescence distribution of the Cy-3 labeled DNA (Fig. 3A) was in line with the stochastic number of labeled guanine bases within the strand.

View Article: PubMed Central - PubMed

ABSTRACT

Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells.

No MeSH data available.