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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.


The lack of co-localization of Cy3 and Cy5-labeled DNA confirms that individual DNA strands bind onto avidin-coated 50 nm islands.Fluorescence microscopic images of an 10 × 10 island array written at 0.87 pC and scanned (A) in the Cy3 channel at 532 nm and (B) in the Cy5 channel at 646 nm. (C) Overlay of both fluorescence images.
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f6: The lack of co-localization of Cy3 and Cy5-labeled DNA confirms that individual DNA strands bind onto avidin-coated 50 nm islands.Fluorescence microscopic images of an 10 × 10 island array written at 0.87 pC and scanned (A) in the Cy3 channel at 532 nm and (B) in the Cy5 channel at 646 nm. (C) Overlay of both fluorescence images.

Mentions: As additional proof for the 1:1 stoichiometry of DNA strands per small nanoislands (Fig. 5), experiments on the anti-colocalization were conducted. In this assay, island arrays were incubated with a mixture of otherwise identical strands that are labeled with either Cy3 or Cy5 fluorophores. Provided that solely a single strand of DNA binds per islands, the islands should either have a Cy3 or Cy5 but not both fluorophore signals. The anti-colocalization of the two fluorescence signals was successfully established with fluorescence scanning in the Cy3 and Cy5 channel and in an overlay (Fig. 6). The results demonstrate that out of 58 DNA-covered islands, 45 were Cy3, 13 and Cy5 and only 1 had both fluorophores. A 1:1 stoichiometry of DNA molecule per islands was also established with DNA origami nanoplates of 50 × 50 nm onto small nanoislands (Supplementary Information, Figs S-10 to S-12). As expected, larger 100 nm islands with multiple DNA strands generated frequent co-localization of the Cy3 and Cy5 signal (Supplementary Information, Fig. S-9).


Arrays of Individual DNA Molecules on Nanopatterned Substrates
The lack of co-localization of Cy3 and Cy5-labeled DNA confirms that individual DNA strands bind onto avidin-coated 50 nm islands.Fluorescence microscopic images of an 10 × 10 island array written at 0.87 pC and scanned (A) in the Cy3 channel at 532 nm and (B) in the Cy5 channel at 646 nm. (C) Overlay of both fluorescence images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5304548&req=5

f6: The lack of co-localization of Cy3 and Cy5-labeled DNA confirms that individual DNA strands bind onto avidin-coated 50 nm islands.Fluorescence microscopic images of an 10 × 10 island array written at 0.87 pC and scanned (A) in the Cy3 channel at 532 nm and (B) in the Cy5 channel at 646 nm. (C) Overlay of both fluorescence images.
Mentions: As additional proof for the 1:1 stoichiometry of DNA strands per small nanoislands (Fig. 5), experiments on the anti-colocalization were conducted. In this assay, island arrays were incubated with a mixture of otherwise identical strands that are labeled with either Cy3 or Cy5 fluorophores. Provided that solely a single strand of DNA binds per islands, the islands should either have a Cy3 or Cy5 but not both fluorophore signals. The anti-colocalization of the two fluorescence signals was successfully established with fluorescence scanning in the Cy3 and Cy5 channel and in an overlay (Fig. 6). The results demonstrate that out of 58 DNA-covered islands, 45 were Cy3, 13 and Cy5 and only 1 had both fluorophores. A 1:1 stoichiometry of DNA molecule per islands was also established with DNA origami nanoplates of 50 × 50 nm onto small nanoislands (Supplementary Information, Figs S-10 to S-12). As expected, larger 100 nm islands with multiple DNA strands generated frequent co-localization of the Cy3 and Cy5 signal (Supplementary Information, Fig. S-9).

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.