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Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging.

Gorman J, Fazio T, Wang F, Wind S, Greene EC - Langmuir (2010)

Bottom Line: Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, 650 West 168th Street, Black Building Room 536, New York, New York 10032, USA.

ABSTRACT
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.

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Nanofabrication and characterization of the DNA rack elements. Panel A shows an optical image of a single barrier set collected at 100× magnification, and relevant pattern dimensions are indicated. An AFM image of a rack pattern is shown in panel B highlighting the height of the linear barriers and the pentagons, as well as the distance between these two barrier elements. An SEM image of another rack pattern with a single linear barrier and the arrayed pentagons is shown in panel C, and details of the different barrier elements are shown in panels D and E.
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fig2: Nanofabrication and characterization of the DNA rack elements. Panel A shows an optical image of a single barrier set collected at 100× magnification, and relevant pattern dimensions are indicated. An AFM image of a rack pattern is shown in panel B highlighting the height of the linear barriers and the pentagons, as well as the distance between these two barrier elements. An SEM image of another rack pattern with a single linear barrier and the arrayed pentagons is shown in panel C, and details of the different barrier elements are shown in panels D and E.

Mentions: To achieve these desired design features, chromium (Cr) or gold (Au) barrier patterns were made on a 1 in. × 3 in. fused silica slide glass by either ebeam lithography as previously described8,9 or nanoimprint lithography28,29 (see Materials and Methods). For most of the work described below, each slide contained 16 total rack patterns, arranged in a 4 × 4 array at the center of the slide. Each rack pattern was 260 μm in length with a distance of 13 μm between the linear barriers and the back of pentagons. The center-to-center distance between each of the rack patterns within the 4 × 4 array was 500 μm in the x-direction (parallel to the direction of buffer flow) and 370 μm in the y-direction (perpendicular to the direction of buffer flow). The total area encompassed by the 4 × 4 array of rack patterns was 1513 μm × 1370 μm (2.073 mm2). Figure 2 shows the characterization of a typical DNA rack pattern made by ebeam lithography. Figure 2A shows an optical image of the overall pattern design; Figure 2B shows measurements of these parameters using atomic force microscopy (AFM), and Figures 2C, D, and E show characterization of the patterns with scanning electron microscopy (SEM). As demonstrated from these images, the height of the pattern elements was typically on the order of 20 nm and the width of the channels between adjacent pentagons was 500 nm, and as indicated above, the optimal distance between the leading edge of the linear barriers and the back of the pentagon array was ∼13 μm. This distance was specifically selected for use with λ phage DNA, a commercially available linear DNA substrate that is 48502 bp with a fully extended contour length of approximately 16.5 μm (see below). The separation distance of 11−13 μm between the leading edge of the linear barrier and the front and rear edges of the pentagons corresponds to ∼65−80% mean extension of the λ DNA substrate, depending upon the precise anchoring position on the pentagon surface.


Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging.

Gorman J, Fazio T, Wang F, Wind S, Greene EC - Langmuir (2010)

Nanofabrication and characterization of the DNA rack elements. Panel A shows an optical image of a single barrier set collected at 100× magnification, and relevant pattern dimensions are indicated. An AFM image of a rack pattern is shown in panel B highlighting the height of the linear barriers and the pentagons, as well as the distance between these two barrier elements. An SEM image of another rack pattern with a single linear barrier and the arrayed pentagons is shown in panel C, and details of the different barrier elements are shown in panels D and E.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806065&req=5

fig2: Nanofabrication and characterization of the DNA rack elements. Panel A shows an optical image of a single barrier set collected at 100× magnification, and relevant pattern dimensions are indicated. An AFM image of a rack pattern is shown in panel B highlighting the height of the linear barriers and the pentagons, as well as the distance between these two barrier elements. An SEM image of another rack pattern with a single linear barrier and the arrayed pentagons is shown in panel C, and details of the different barrier elements are shown in panels D and E.
Mentions: To achieve these desired design features, chromium (Cr) or gold (Au) barrier patterns were made on a 1 in. × 3 in. fused silica slide glass by either ebeam lithography as previously described8,9 or nanoimprint lithography28,29 (see Materials and Methods). For most of the work described below, each slide contained 16 total rack patterns, arranged in a 4 × 4 array at the center of the slide. Each rack pattern was 260 μm in length with a distance of 13 μm between the linear barriers and the back of pentagons. The center-to-center distance between each of the rack patterns within the 4 × 4 array was 500 μm in the x-direction (parallel to the direction of buffer flow) and 370 μm in the y-direction (perpendicular to the direction of buffer flow). The total area encompassed by the 4 × 4 array of rack patterns was 1513 μm × 1370 μm (2.073 mm2). Figure 2 shows the characterization of a typical DNA rack pattern made by ebeam lithography. Figure 2A shows an optical image of the overall pattern design; Figure 2B shows measurements of these parameters using atomic force microscopy (AFM), and Figures 2C, D, and E show characterization of the patterns with scanning electron microscopy (SEM). As demonstrated from these images, the height of the pattern elements was typically on the order of 20 nm and the width of the channels between adjacent pentagons was 500 nm, and as indicated above, the optimal distance between the leading edge of the linear barriers and the back of the pentagon array was ∼13 μm. This distance was specifically selected for use with λ phage DNA, a commercially available linear DNA substrate that is 48502 bp with a fully extended contour length of approximately 16.5 μm (see below). The separation distance of 11−13 μm between the leading edge of the linear barrier and the front and rear edges of the pentagons corresponds to ∼65−80% mean extension of the λ DNA substrate, depending upon the precise anchoring position on the pentagon surface.

Bottom Line: Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, 650 West 168th Street, Black Building Room 536, New York, New York 10032, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus