Assembly of DNA Curtains Using Hydrogen Silsesquioxane As a

Sep 1, 2012 - ABSTRACT: We have established a single-molecule imaging experimental platform called “DNA curtains” in which DNA molecules tethered ...
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Assembly of DNA Curtains Using Hydrogen Silsesquioxane As a Barrier to Lipid Diffusion T. A. Fazio,†,⊥,∥ Ja Yil Lee,‡,∥ S. J. Wind,*,† and E. C. Greene*,‡,§ †

Department of Applied Physics and Applied Mathematics, Center for Electron Transport in Molecular Nanostructures, NanoMedicine Center for Mechanical Biology, Columbia University, 1020 Schapiro CEPSR, 530 West 120th Street, New York, New York 10027, United States ‡ Department of Biochemistry and Molecular Biophysics, Department of Biological Sciences, and the §Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, New York 10032, United States ABSTRACT: We have established a single-molecule imaging experimental platform called “DNA curtains” in which DNA molecules tethered to a lipid bilayer are organized into patterns at nanofabricated metallic barriers on the surface of a microfluidic sample chamber. This technology has wide applications for real-time single-molecule imaging of protein−nucleic acid interactions. Here, we demonstrate that DNA curtains can also be made from hydrogen silsesquioxane (HSQ). HSQ offers important advantages over metallic barriers because it can be lithographically patterned directly onto fused silica slides without any requirement for further processing steps, thereby offering the potential for rapid prototype development and/or scale up for manufacturing.

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molecule imaging. HSQ is a spin-on glass originally developed as a low-k dielectric insulator for interconnect levels in semiconductor chips. In 1997, Namatsu et al. discovered that HSQ can act as a negative tone electron beam resist that can be patterned with a resolution of 4 h at room temperature. The lengthy development time is required because the O2 plasma Received: August 6, 2012 Accepted: September 1, 2012 Published: September 1, 2012 7613

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Figure 1. Schematic of microscope, DNA curtains, and device fabrication: (A) simplified overview of the microscope system highlighting the TIRF illumination geometry, (B) schematic diagram of a single-tethered DNA curtain (side view). The left panel illustrates how biotinylated DNA is anchored by one end to a lipid bilayer. Also shown is a barrier on the surface of the slide, which disrupts the bilayer. The right panel illustrates how DNA is pushed into the barrier and extended parallel to the slide surface through the application of buffer flow. (C) Overview of the HSQ barrier fabrication procedure and (D) SEM image highlighting the detail of a typical barrier pattern.

under constant N2 flow and cooled slowly to room temperature to let the porous HSQ condense into a glassy surface. The optical density (OD) of chromium (Cr) at 1064 nm was calculated from optical constants of chromium. The absorption coefficient (α) is defined as I/I0 = exp(−αT), where I is light intensity transmitted through a material, I0 is a reference light intensity, and T is the thickness of the material. The absorption coefficient (α) is given as 4πκ(λ)/λ, where λ is a wavelength of light and κ(λ) is an imaginary part of the refractive index at the

treatment used to make HSQ hydrophilic also increases the density of the HSQ layer.15 After development, the samples were sonicated in developer for 15 min, rinsed with deionized H2O, and dried with ultrapure N2. Alternatively, the HSQ was coated with a thermally evaporated layer of aluminum (Al). This allowed a reduction of the development time to just 3 min in MF CD-26 (2.4% tetramethyl ammonium hydroxide). After development, slides were annealed on a hot plate at 540 °C 7614

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Figure 2. Linear HSQ barriers: (A) schematic of lipid-tethered DNA molecules aligned along a linear barrier that disrupts the continuity of the lipid bilayer coating the surface of a microfluidic sample chamber. (B and C) TIRFM images of a YOYO1-stained DNA curtain made using the HSQ barriers, in the presence and absence of buffer flow, respectively.

wavelength.17 The κ of Cr at 1064 nm is 4.28.18 Therefore, the absorption coefficient of Cr is 0.0505/nm at 1064 nm. For 25nm thick Cr, I/I0 is about 0.283 at 1064 nm and the OD, defined as −log10(I/I0), is ∼0.29. The OD of HSQ barrier at 1064 nm was measured using a spectrophotometer (Agilent). HSQ was spin-coated on a fused silica slide as described above, and the transmitted intensity through the HSQ layer (I) was measured by placing the HSQ slide in the front of the aperture of a spectrophotometer, while the reference (I0) was measured with a bare fused silica slide. The OD of HSQ layer at 1064 nm was calculated from log10(I/I0), yielding an OD of 0.55. Lipids and DNA Curtains. Lipid bilayers were assembled as described.2−5 Lipid vesicles comprised of DOPC (1,2dioleoyl-sn-glycerophosphocholine), 0.5% biotinylated-DPPE (1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(cap biotinyl)), and 8% mPEG 550-DOPE (1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]) were diluted in buffer containing 10 mM Tris-HCl (pH 7.8) and 100 mM NaCl and incubated within the sample chamber for 30 min. Streptavidin (660 nM) was then injected into the sample chamber and incubated for 10 min. The surface was further passivated with buffer A (40 mM Tris-HCl [pH 7.8], 1 mM DTT, 1 mM MgCl2, and 0.2 mg/mL bovine serum albumin (BSA)). Biotinylated λ-DNA (∼10 pM; 48.5 kb) prestained with YOYO1 (∼1 dye per 600 base pairs) was injected into the sample chamber in buffer B (buffer A plus YOYO1 and an oxygen scavenging system comprised of 1% (w/v) glucose, 60 mM β-mercaptoethanol, glucose oxidase (100 units/mL), and catalase (1,560 units/mL)). The DNA was then injected into the flowcell, where it is bound to the lipid bilayer via streptavidin−biotin interactions. Microscopy. All microscopy was done using a prism-type total internal reflection fluorescence microscope (Nikon

TE2000) equipped with a 488 nm laser excitation source (200 mW Sapphire; Coherent Inc.). Images were collected using a 60× water immersion objective lens and a backilluminated EMCCD (Photometrics, Cascade 512B). Optical tweezers were combined with the TIRFM through the same objective lens, and a high-power infrared (IR) laser (1 W, 1064 nm; Crystalaser) was used for the optical trapping (Figure 1B). The beam was then reflected by a control mirror, which was mounted on a motorized optical mount (8807; New Focus) and controlled remotely with iPicomotor modules (New Focus). The IR beam entered the microscope through the back-port and reflected toward the objective lens by a dichroic mirror (z780dcspxr; Chroma Technology). All images were captured using NIS-Elements software (Nikon) and processed and analyzed in ImageJ (http://rsbweb.nih.gov/ij/).

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RESULTS AND DISCUSSION Supported lipid bilayers can be readily assembled on fused silica surfaces, and the continuity of these bilayers can be disrupted by either manually etching the surface or through the deposition of nanofabricated metallic patterns.19−21 The ability of a barrier to disrupt the continuity of a supported lipid bilayer is the basis for the DNA curtain technology.1−5 To make the curtains, the DNA molecules are first anchored to a biotinstreptavidin interaction (Figure 1B). Application of buffer flow is then used to push the DNA into the diffusion barriers, which disrupts the bilayer and halts the movement of the DNA (Figure 1B). The DNA is then extended parallel to the sample chamber surface. Previous curtains utilized chromium (Cr) barriers.5 HSQ is a negative electron beam resist, which can be used to generate patterns with fewer processing steps (Figure 1C).16,22,23 Linear barrier patterns comprised of HSQ were 7615

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Figure 3. Sawtooth HSQ barriers: (A) schematic of a DNA curtain assembled using a sawtooth HSQ barrier, in which the individual DNA strands are pushed into the apex of each tooth of the pattern. (B) SEM image highlighting the detail of a typical sawtooth barrier pattern, and (C) TIRFM image of DNA aligned along the sawtooth barrier. Variation in YOYO1 signal intensity arises from overlapping DNA molecules. In part B, the distance between the pattern elements is 2 μm, which corresponds to the 2 μm separation distance between adjacent DNA molecules in part C.

problem with the Cr barriers is that they are not transparent in the infrared spectrum, and a 25-nm layer of Cr is expected to have an optical density of 2.9 at a wavelength of 1064 nm, suggesting that that if the IR beam were to cross the Cr barrier it might cause local heating of the sample. To determine the effect of the IR beam on the DNA curtains, the focal point of the optical trap was placed at the left edge of a DNA curtain aligned at a Cr barrier and then swept across the leading edge of the DNA curtain (Figure 4A). When the IR laser crossed the Cr barrier, it caused the constituent molecules to be immediately released from the surface. This effect was only evident when the IR beam crossed the Cr barriers, and even transient exposure of the beam to the barrier edges caused disruption of the curtains. The DNA itself could withstand extended exposures to the IR beam with no evidence of breakage so long as the IR beam was not placed at the Cr barriers. There are two potential explanations for this phenomenon. Exposure to the IR laser may heat the Cr barriers, which in turn could lead to localized disruption of the bilayer causing release of the anchored DNA molecules. Alternatively, bilayer and/or the DNA may be damaged by high-intensity irradiation, causing the molecules to break away from the surface. Regardless of which is the correct mechanism, both would arise from the high absorbance of Cr barriers at the infrared wavelength necessary for optical trapping, which immediately suggests one way to eliminate the effects of laser-induced disruption would be to use barriers made from transparent materials. An attractive feature of HSQ is that it is nearly transparent in the visible and IR spectra. Therefore, we next asked whether DNA curtains made from HSQ barriers could tolerate exposure to a focused IR laser. When the IR laser was focused on the HSQ barriers, it had no effect on the DNA curtain.

prepared, and the quality of the patterns was assessed by atomic force microscopy (AFM) and scanning electron microscopy (SEM), revealing a height of 80−100 nm and a corresponding width of 100−200 nm for HSQ patterns (Figure 1D and not shown). Flowcells were then made from slides bearing HSQ patterns, lipid bilayers containing 0.5% biotinylated-DPPE were deposited, and biotinylated λ-DNA was tethered to the bilayer through a streptavidin linkage (Figure 2A).1−5 When flow was applied, the tethered DNA molecules aligned along the leading edges of the HSQ barriers (Figure 2B,C). When flow was paused, the DNA molecules diffused up out of the evanescent field. This is a standard control used to verify that the DNA molecules are not nonspecifically adsorbed to the surface.1−3,5 These results confirm that slides bearing patterns made from HSQ support bilayer deposition, in which the HSQ barriers disrupt the bilayer, and that HSQ can be used to align DNA curtains. We have previously shown that the geometry of the Cr barriers can be used to control the lateral separation distance between adjacent DNA molecules within a curtain (Figure 3A),2,5 which is often crucial in biochemical experiments where it may be necessary to ensure that adjacent DNA molecules do not overlap with one another.10 To determine whether HSQ could also be used to control DNA separation, we made HSQ barriers in a sawtooth pattern where the peak-to-peak distance between each “tooth” was 2 μm (Figure 3B). When the DNA was aligned along these sawtooth patterns, the distance between the adjacent molecules was highly uniform and occurred at 2 μm intervals, confirming that the position of each molecule within the curtain was dictated by the geometry of the barrier pattern (Figure 3C). The combination of TIRFM with laser tweezers offers the opportunity to measure and manipulate molecules in ways not possible with previous DNA curtain designs.5 One potential 7616

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded in part by the Initiatives in Science and Engineering (ISE; awarded to E.C.G. and S.J.W.) program through Columbia University, and by an NIH Grant and an NSF PECASE Award to E.C.G. T.A.F. was supported by an NSF Graduate Research fellowship. This work was partially supported by the Nanoscale Science and Engineering Initiative of the National Science Foundation under NSF Award No. CHE-0641523 and by the New York State Office of Science, Technology, and Academic Research (NYSTAR). This work was also funded in part by the National Institutes of Health (NIH) Common Fund Nanomedicine Program (Grant PN2 EY016586). E.C.G is an Early Career Scientist with the Howard Hughes Medical Institute.

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Figure 4. IR-induced disruption of DNA curtains. (A) TIRFM images showing the effects of IR-laser exposure on DNA curtains aligned at chromium (Cr) barriers. The upper panel shows the DNA curtain before exposure to the IR laser, and the lower panel shows the curtain after exposure to the IR laser. The focal spot of the laser is indicated as an orange circle, and the movement of the focal spot along the barrier edge is highlighted with dashed arrows. As the beam moves along the barrier edge, the DNA molecules are sheared from the curtain and flushed away by buffer flow. (B) An identical IR-laser experiment is shown using nanofabricated HSQ barriers. In this case, the DNA molecules are unaffected by exposure to the IR beam. In both examples, the IR beam intensity after the objective was 50 mW and was moved at an average of 10 μm/s along the barrier edges.

In summary, we have shown that HSQ can be used to generate DNA curtains for single-molecule imaging applications. One primary advantage of HSQ is that this negative resist eliminates the need for metal evaporation, thereby simplifying barrier manufacturing and reducing process time. By establishing HSQ barriers, we expand the catalog of materials that can be used to make DNA curtains for single-molecule imaging applications; combined with its fine resolution and biocompatibility, this “rinse-and-go” method offers a broadening of substrate fabrication resources for high-throughput singlemolecule experiments. Another advantage of HSQ is that it is transparent across the ultraviolet to infrared spectrum, whereas metallic barriers can absorb, scatter, and reflect light. Thus, HSQ barriers offer the potential for use in experiments that will combine fluorescence visualization with laser trap-based force measurements and/or physical manipulation of the DNA molecules.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (E.C.G). Present Address ⊥

Columbia Technology Ventures, 630 W. 168th Street, New York, NY, 10032. Author Contributions ∥

Equal contribution. 7617

dx.doi.org/10.1021/ac302149g | Anal. Chem. 2012, 84, 7613−7617