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Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging
[Image: see text] Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2009
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806065/ https://www.ncbi.nlm.nih.gov/pubmed/19736980 http://dx.doi.org/10.1021/la902443e |
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author | Gorman, Jason Fazio, Teresa Wang, Feng Wind, Shalom Greene, Eric C. |
author_facet | Gorman, Jason Fazio, Teresa Wang, Feng Wind, Shalom Greene, Eric C. |
author_sort | Gorman, Jason |
collection | PubMed |
description | [Image: see text] Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and maintained in an extended configuration by anchoring both ends of the DNA to the surface of a microfluidic sample chamber that is otherwise coated with an inert lipid bilayer. This “double-tethered” DNA substrate configuration is established through the use of nanofabricated rack patterns comprised of two distinct functional elements: linear barriers to lipid diffusion that align DNA molecules anchored by one end to the bilayer and antibody-coated pentagons that provide immobile anchor points for the opposite ends of the DNA. These devices enable the alignment and anchoring of thousands of individual DNA molecules, which can then be visualized using total internal reflection fluorescence microscopy under conditions that do not require continuous application of buffer flow to stretch the DNA. This unique strategy offers the potential for studying protein−DNA interactions on large DNA substrates without compromising measurements through application of hydrodynamic force. We provide a proof-of-principle demonstration that double-tethered DNA curtains made with nanofabricated rack patterns can be used in a one-dimensional diffusion assay that monitors the motion of quantum dot-tagged proteins along DNA. |
format | Text |
id | pubmed-2806065 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-28060652010-01-13 Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging Gorman, Jason Fazio, Teresa Wang, Feng Wind, Shalom Greene, Eric C. Langmuir [Image: see text] Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and maintained in an extended configuration by anchoring both ends of the DNA to the surface of a microfluidic sample chamber that is otherwise coated with an inert lipid bilayer. This “double-tethered” DNA substrate configuration is established through the use of nanofabricated rack patterns comprised of two distinct functional elements: linear barriers to lipid diffusion that align DNA molecules anchored by one end to the bilayer and antibody-coated pentagons that provide immobile anchor points for the opposite ends of the DNA. These devices enable the alignment and anchoring of thousands of individual DNA molecules, which can then be visualized using total internal reflection fluorescence microscopy under conditions that do not require continuous application of buffer flow to stretch the DNA. This unique strategy offers the potential for studying protein−DNA interactions on large DNA substrates without compromising measurements through application of hydrodynamic force. We provide a proof-of-principle demonstration that double-tethered DNA curtains made with nanofabricated rack patterns can be used in a one-dimensional diffusion assay that monitors the motion of quantum dot-tagged proteins along DNA. American Chemical Society 2009-09-09 2010-01-19 /pmc/articles/PMC2806065/ /pubmed/19736980 http://dx.doi.org/10.1021/la902443e Text en Copyright © 2009 American Chemical Society http://pubs.acs.org This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org. |
spellingShingle | Gorman, Jason Fazio, Teresa Wang, Feng Wind, Shalom Greene, Eric C. Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging |
title | Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging |
title_full | Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging |
title_fullStr | Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging |
title_full_unstemmed | Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging |
title_short | Nanofabricated Racks of Aligned and Anchored DNA Substrates for Single-Molecule Imaging |
title_sort | nanofabricated racks of aligned and anchored dna substrates for single-molecule imaging |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806065/ https://www.ncbi.nlm.nih.gov/pubmed/19736980 http://dx.doi.org/10.1021/la902443e |
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