Chapter 3 High‐throughput single‐molecule imaging system using nanofabricated trenches and
3.4 Discussion
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Figure 3.1│ Drawbacks of chromium barriers for use with DNA curtains
A. Schematic of DNA curtain system with chromium barriers.(top) The DNA curtain formed at a Cr nanobarrier, which is constructed in the middle of the flowcell microchannel. (bottom) Actual image of a DNA curtain, with YOYO‐1‐stained DNA molecules aligned at the Cr barrier. The
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arrowhead to the left of the image indicates the barrier position. B. Drawbacks of Cr barriers. (top) Optical image showing the degradation of barriers after cleaning. (middle) Protein aggregates remaining at the barrier after cleaning under 488 nm laser illumination. (bottom) Laser scattering and autofluorescence from the Cr barrier slide under 637 nm laser illumination. Arrowheads to the left indicate the barrier position. C. Photocleavage by YOYO‐1 under continuous laser illumination.
A substantial fraction of DNA molecules became shortened by photocleavage within 80 s, and nearly all DNA molecules were cleaved away within 160 s.
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123 Figure 3.2│ Fabrication of nanotrenches.
A. Procedure for the fabrication of nanotrenches. Step 1: Al and poly(methyl methacrylate) (PMMA) were successively coated onto a clean fused silica slide; Step 2: barrier patterns were drawn on the PMMA layer by electron‐beam lithography; Step 3: patterned areas were removed by developing solution; Step 4: Al layer at the patterned area was etched by reactive ions (Cl2 and BCl2 mixed gases, black dots); Step 5: slide glass at the patterned area was etched by reactive ions (SF4, CF4, and O2 mixed gases, red dots); Step 6: Al layer was removed by Al etchant and piranha treatment. B. Images of nanotrench patterns. (top) Optical microscope image of nanotrench barrier. (bottom) Scanning electron microscope (SEM) image of nanotrench, which has a sawtooth pattern to prevent lateral motion due to lipid diffusion. C. SEM image of a vertical cross‐section of nanotrench, which is 349 nm wide and 1.48 μm deep. EBL, electron‐beam lithography; RIE, reactive ion etching.
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125 Figure 3.3│ Optical properties of nanotrenches.
A. Total internal reflection fluorescence images of the nanotrench slide, under laser illumination at excitation wavelengths of (top) 488 nm or (bottom) 637 nm. Laser scattering and fluorescence background around nanotrenches were greatly reduced compared with Cr barriers. B. Quantitative comparison of laser scattering between Cr barriers and nanotrenches according to the excitation wavelength. Nanotrenches exhibited significantly reduced laser scattering at 488 and 637 nm. Error bars represent the standard deviations of four repeats. C. Quantitative comparison of fluorescence background between Cr barriers and nanotrenches according to the excitation wavelength. High fluorescence background at 637 nm was largely eliminated with nanotrench slides. Error bars represent the standard deviations of four repeats. D. Surface states of a Cr barrier slide before (top) and after (bottom) successive mild washing with 2% Hellmanex III overnight, 99% ethanol overnight, and 1 M sodium hydroxide for 30 min. E. Surface states of a nanotrench slide before (top) and after (bottom) harsh washing with acetone for 30 min, 2% Hellmanex III for 1 day, piranha solution (3:1 concentrated 96% H2SO4:30% H2O2) for 40 min, 99% ethanol overnight, and 1 M sodium hydroxide for 30 min. Sonication was used for each cleaning step except for piranha treatment.
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Figure 3.4│ DNA curtain with DNA molecule stained with fluorescent protein–DNA binding peptides (FP–DBPs) on nanotrench slides.
A. Schematic of DNA curtain flow‐aligned at a nanofabricated trench. B. The FP–DBP constructs consisted of a green fluorescent protein (PDB ID: 1EMA) with DNA‐binding motifs at both the N‐
and C‐termini. The fluorophore, represented by spheres, is shielded by the beta‐barrel. C. Images of a DNA curtain stained with FP–DBPs in 1X TE [7.5], without any anti‐photobleaching agents. (top) In flowing buffer, DNA molecules stained with FP–DBP was well‐aligned at the nanotrench.
(bottom) In the absence of flow, DNA molecules recoiled and diffused out of the evanescent field.
The arrowheads to the left of the images indicate the location of the nanotrench, and downward arrows to the right represent the direction of flow. D. DNA extension as the flow rate was varied.
DNA molecules were stained with 0.1 nM FP–DBP, and the DNA extension was measured at different flow rates in the presence (magenta filled circle) or absence (blue blank square) of free FP–DBP. Plots of relative extension of DNA (<x>/L) versus flow rates were fitted by the worm‐like chain model, with persistence length as the only parameter (red dashed line: with free FP–DBP and blue dashed line: without free FP–DBP). The observed DNA persistence length was 187 nm in the presence of FP–DBP, and 53 nm in its absence. The solid black line shows the curve predicted for naked DNA, whose persistence length is 50 nm. E. Images of DNA molecules stained with FP–DBPs at the curtain at 0 s (top) and at 80 s (bottom) of continuous illumination (488 nm laser, 6.7 W/mm2). DNA density at the curtain was not reduced, indicating that DNA molecules were not cleaved by photoactivated FP–DBPs. F. Photobleaching curves of DNA curtains stained with FP–DBP (filled square) or with YOYO‐1 (blank circle) at 6.7 W/mm2 of 488 nm laser. The photobleaching curves were fit by a single exponential decay function, giving characteristic photobleaching times for FP–DBP (τ_FP‐DBP) and YOYO‐1 (τ_YOYO‐1) of 42.4 and 4.3 s, respectively. Error bars represent standard deviations (n = 10). G. (top) Kymograph showing FP–
DBP binding to and dissociation from DNA, depending on pH change. FP–DBPs were bound to DNA at pH 7.5. Upon the injection of a pH 11.0 buffer without FP–DBP, the fluorescence of FP–
DBP momentarily increased and then abruptly decreased. When the pH 7.5 buffer containing FP–
DBP was reinjected, the fluorescence returned. The arrowhead to the left of the graph indicates the nanotrench position. (bottom) Kymograph of background fluorescence under the same conditions, measured in the absence of DNA. Injection of pH 11.0 buffer increased background fluorescence, mirroring its effect on DNA fluorescence intensity. Arrows above the kymograph indicate the time points of buffer exchange. TE: Tris‐EDTA (10 mM Tris‐HCl [7.5] and 1 mM EDTA)
128 Figure 3.5│ Kymograph for a single DNA molecule
Kymograph for a single DNA molecule showing the removal of FP-DBP from DNA at high salt concentration. When 300 mM NaCl and 10 mM MgCl2 were injected into the flowcell, fluorescent proteins aggregated momentarily, giving a transient increase in fluorescence intensity. Then FP-DBPs dissociated from DNA, and DNA fluorescence disappeared. Following reinjection of FP-DBP in 1X TE [7.5], DNA fluorescence returned. The arrowhead to the left of the kymograph indicates the location of the nano-trench.-
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Figure 3.6│ Mapping the sites of fluorescently labeled protein binding to DNA, using DNA curtain formed with nanotrenches
A. Schematic of a DNA curtain underflow and binding of EcoRIE111Q conjugated with quantum dots (Qdots). B. Total internal reflection fluorescence microscope images of a DNA curtain with DNA‐bound EcoRIE111Q. (top) DNA molecules stained with fluorescent protein–DNA binding peptide (FP–DBP; green). (middle) Puncta are EcoRIE111Q tagged with Qdots (magenta). (bottom) Merged image of both DNA and EcoRIE111Q. C. Kymograph for a single DNA molecule, with EcoRIE111Q molecules bound. With the flow off, DNA recoiled from the evanescent field, and hence proteins bound to DNA disappeared along with DNA. D. Histogram for sites of EcoRIE111Q binding to λ‐DNA. The histogram was constructed collecting the center coordinates determined in two‐dimensional Gaussian fits of the fluorescent puncta. The histogram was then fitted with multiple Gaussian functions, giving peaks centered at 4.4, 9.8, 17.9, 23.9, and 29.5 kbp. The known EcoRI binding sites on λ‐DNA occur at 3.53, 9.334, 16.755, 22.398, and 27.276 kbp. Error bars in the histogram represent 70% confidence intervals obtained by bootstrapping. The total number of molecules analyzed was 583.
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Figure 3.7│ 𝐄𝐜𝐨𝐑𝐈𝐄𝟏𝟏𝟏𝐐 binding on lambda DNA that was pre-stained with FP-DBP
A. Representative image of single DNA in a DNA curtain and kymograph of EcoRIE111Q binding on λ-DNA pre-stained with 0.1 nM FP-DBP. EcoRIE111Q did not locate EcoRI cognate sites. B. Average binding number of EcoRIE111Q molecules on single λ-DNA. Error bars were obtained from standard deviation (number of DNA molecules was greater than 70). C. Binding distribution of EcoRIE111Q on λ-DNA that was pre-stained with FP-DBP. Green dotted lines represented EcoRI binding sites on λ- DNA. There were no distinct peaks around the EcoRI binding sites. The number of molecules (N) was 407 and the error bars were obtained from bootstrapping with 70% confidence interval.
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