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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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Related in: MedlinePlus

Schematic of DNA rack design. Panel A shows a diagram of the total internal reflection fluorescence microscope (TIRFM) used to image single molecules of DNA. For imaging with the TIRFM, the long DNA molecules (48 kb) used in these studies must be extended parallel to the surface of the sample chamber to remain confined within the evanescent field. Panels B and C depict a cartoon illustration of the bilayer on the surface of a fused silica slide, and a single barrier set comprised of a linear barrier and a series of aligned pentagons separated by nanochannels. Also depicted is the response of tethered DNA molecules to the application of a hydrodynamic force. The magenta circles are the biotinylated ends, and the red squares are the hapten (digoxigenin, FITC, or BrdU)-labeled ends of the DNA. The top and bottom parts of panels B and C depict views from the side and above, respectively. In the absence of buffer flow, the DNA molecules are tethered to the surface but are not confined within the evanescent field, nor are they aligned at the barrier. As depicted in panel C, when flow is applied, the DNA molecules are dragged through the bilayer until they encounter the linear diffusion barrier, at which point they will align with respect to one another and the DIG-labeled ends become anchored to the antibody-coated pentagons. DNA located between the linear barriers and the pentagons passes through the nanochannels and goes to the next available linear barrier in the pattern.
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fig1: Schematic of DNA rack design. Panel A shows a diagram of the total internal reflection fluorescence microscope (TIRFM) used to image single molecules of DNA. For imaging with the TIRFM, the long DNA molecules (48 kb) used in these studies must be extended parallel to the surface of the sample chamber to remain confined within the evanescent field. Panels B and C depict a cartoon illustration of the bilayer on the surface of a fused silica slide, and a single barrier set comprised of a linear barrier and a series of aligned pentagons separated by nanochannels. Also depicted is the response of tethered DNA molecules to the application of a hydrodynamic force. The magenta circles are the biotinylated ends, and the red squares are the hapten (digoxigenin, FITC, or BrdU)-labeled ends of the DNA. The top and bottom parts of panels B and C depict views from the side and above, respectively. In the absence of buffer flow, the DNA molecules are tethered to the surface but are not confined within the evanescent field, nor are they aligned at the barrier. As depicted in panel C, when flow is applied, the DNA molecules are dragged through the bilayer until they encounter the linear diffusion barrier, at which point they will align with respect to one another and the DIG-labeled ends become anchored to the antibody-coated pentagons. DNA located between the linear barriers and the pentagons passes through the nanochannels and goes to the next available linear barrier in the pattern.

Mentions: Here we expand on our previous work and demonstrate the development of new nanofabricated barrier patterns, termed DNA “racks”, which can be used to make DNA curtains where both ends of the DNA molecules are anchored to the flow cell surface. The rack patterns utilize a combination of two distinct functional elements, and an overview of the general design is presented in Figure 1A,C. In principle, one end of the DNA is first anchored via a biotin−neutravidin interaction to a supported lipid bilayer coating the surface of the fused silica sample chamber (Figure 1B,C), as previously described.8,9 In the absence of a hydrodynamic force, the molecules are randomly distributed on the surface and lie primarily outside of the detection volume defined by the penetration depth of the evanescent field (∼150−200 nm). Application of buffer flow pushes the DNA through the sample chamber while the biotinylated DNA ends remain anchored within the mobile bilayer. The first pattern elements are linear barriers to lipid diffusion,15−17 which are oriented perpendicular to the direction of buffer flow at strategic locations in the path of the DNA (Figure 1B,C); these linear barriers are designed to halt the forward movement of the lipid-tethered DNA molecules through the sample chamber, causing them to accumulate at the leading edge of the barriers where they then extend parallel to the surface.8,9 The second elements of the pattern are a series of arrayed pentagons positioned at a defined distance behind the linear barriers. The distance between the linear barriers and the pentagons is optimized for the length of the DNA to be used for the experiments (Figure 1B,C, and see below). The channels between the adjacent pentagons are intended to minimize accumulation of lipid-tethered DNA molecules between the linear barriers and the pentagon arrays. This design feature takes advantage of our previous observation that geometric barrier patterns can be used to direct the movement of DNA by making use of barrier edges that are not perpendicular to the direction of buffer flow.(9) Any DNA molecules anchored to the bilayer between the two barrier elements are expected to slide off the angled edges of the pentagons and should be funneled through the channels. The pentagons are also designed to present a large, exposed surface that can be nonspecifically coated with antibodies directed against small molecule haptens [either digoxigenin (DIG), fluorescein isothiocyanate (FITC), or bromodeoxyuridine (BrdU) (see below)], which are covalently linked to the ends of the DNA opposite the ends bearing the biotin tag (Figure 1B). When the DNA molecules are aligned along the linear barriers and stretched perpendicular to the surface, the hapten-tagged DNA ends should bind the antibody-coated pentagons (Figure 1C). In this scenario, the DNA molecules should remain stretched parallel to the surface even when no buffer is being pushed through the sample chamber (Figure 1C).


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

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

Schematic of DNA rack design. Panel A shows a diagram of the total internal reflection fluorescence microscope (TIRFM) used to image single molecules of DNA. For imaging with the TIRFM, the long DNA molecules (48 kb) used in these studies must be extended parallel to the surface of the sample chamber to remain confined within the evanescent field. Panels B and C depict a cartoon illustration of the bilayer on the surface of a fused silica slide, and a single barrier set comprised of a linear barrier and a series of aligned pentagons separated by nanochannels. Also depicted is the response of tethered DNA molecules to the application of a hydrodynamic force. The magenta circles are the biotinylated ends, and the red squares are the hapten (digoxigenin, FITC, or BrdU)-labeled ends of the DNA. The top and bottom parts of panels B and C depict views from the side and above, respectively. In the absence of buffer flow, the DNA molecules are tethered to the surface but are not confined within the evanescent field, nor are they aligned at the barrier. As depicted in panel C, when flow is applied, the DNA molecules are dragged through the bilayer until they encounter the linear diffusion barrier, at which point they will align with respect to one another and the DIG-labeled ends become anchored to the antibody-coated pentagons. DNA located between the linear barriers and the pentagons passes through the nanochannels and goes to the next available linear barrier in the pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Schematic of DNA rack design. Panel A shows a diagram of the total internal reflection fluorescence microscope (TIRFM) used to image single molecules of DNA. For imaging with the TIRFM, the long DNA molecules (48 kb) used in these studies must be extended parallel to the surface of the sample chamber to remain confined within the evanescent field. Panels B and C depict a cartoon illustration of the bilayer on the surface of a fused silica slide, and a single barrier set comprised of a linear barrier and a series of aligned pentagons separated by nanochannels. Also depicted is the response of tethered DNA molecules to the application of a hydrodynamic force. The magenta circles are the biotinylated ends, and the red squares are the hapten (digoxigenin, FITC, or BrdU)-labeled ends of the DNA. The top and bottom parts of panels B and C depict views from the side and above, respectively. In the absence of buffer flow, the DNA molecules are tethered to the surface but are not confined within the evanescent field, nor are they aligned at the barrier. As depicted in panel C, when flow is applied, the DNA molecules are dragged through the bilayer until they encounter the linear diffusion barrier, at which point they will align with respect to one another and the DIG-labeled ends become anchored to the antibody-coated pentagons. DNA located between the linear barriers and the pentagons passes through the nanochannels and goes to the next available linear barrier in the pattern.
Mentions: Here we expand on our previous work and demonstrate the development of new nanofabricated barrier patterns, termed DNA “racks”, which can be used to make DNA curtains where both ends of the DNA molecules are anchored to the flow cell surface. The rack patterns utilize a combination of two distinct functional elements, and an overview of the general design is presented in Figure 1A,C. In principle, one end of the DNA is first anchored via a biotin−neutravidin interaction to a supported lipid bilayer coating the surface of the fused silica sample chamber (Figure 1B,C), as previously described.8,9 In the absence of a hydrodynamic force, the molecules are randomly distributed on the surface and lie primarily outside of the detection volume defined by the penetration depth of the evanescent field (∼150−200 nm). Application of buffer flow pushes the DNA through the sample chamber while the biotinylated DNA ends remain anchored within the mobile bilayer. The first pattern elements are linear barriers to lipid diffusion,15−17 which are oriented perpendicular to the direction of buffer flow at strategic locations in the path of the DNA (Figure 1B,C); these linear barriers are designed to halt the forward movement of the lipid-tethered DNA molecules through the sample chamber, causing them to accumulate at the leading edge of the barriers where they then extend parallel to the surface.8,9 The second elements of the pattern are a series of arrayed pentagons positioned at a defined distance behind the linear barriers. The distance between the linear barriers and the pentagons is optimized for the length of the DNA to be used for the experiments (Figure 1B,C, and see below). The channels between the adjacent pentagons are intended to minimize accumulation of lipid-tethered DNA molecules between the linear barriers and the pentagon arrays. This design feature takes advantage of our previous observation that geometric barrier patterns can be used to direct the movement of DNA by making use of barrier edges that are not perpendicular to the direction of buffer flow.(9) Any DNA molecules anchored to the bilayer between the two barrier elements are expected to slide off the angled edges of the pentagons and should be funneled through the channels. The pentagons are also designed to present a large, exposed surface that can be nonspecifically coated with antibodies directed against small molecule haptens [either digoxigenin (DIG), fluorescein isothiocyanate (FITC), or bromodeoxyuridine (BrdU) (see below)], which are covalently linked to the ends of the DNA opposite the ends bearing the biotin tag (Figure 1B). When the DNA molecules are aligned along the linear barriers and stretched perpendicular to the surface, the hapten-tagged DNA ends should bind the antibody-coated pentagons (Figure 1C). In this scenario, the DNA molecules should remain stretched parallel to the surface even when no buffer is being pushed through the sample chamber (Figure 1C).

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