Results

Chapter 3 High‐throughput single‐molecule imaging system using nanofabricated trenches and

3.3 Results

3.3.1 Optical performance of nanotrenches for DNA curtain

Nanotrenches were fabricated on fused silica slides using EBL and RIE (Figure 3.2A). The details of fabrication are described in Section 2. EBL allowed us to draw patterns at a high spatial resolution up to 50 nm, while RIE‐etched substrates without any broadening of the pattern width.

Unlike manual scratches, nanotrenches that had been fabricated using EBL and RIE were uniformly carved (Figure 3.2B). Each nanotrench had a sawtooth pattern, preventing lateral diffusion of DNA molecules within the lipid bilayer. The scanning electron microscope image for the vertical cross‐section (Figure 3.2C) showed a very sharp cliff, 1.38 μm deep, so that the nanotrenches provided an effective barrier to diffusion of lipid molecules. The width was about 350 nm, compatible

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We tested whether the optical properties of the nanotrench slide were suitable for use with DNA curtains. It has been reported that manually scratched slides displayed laser scattering, and that scatter was reduced by the use of Cr nanobarriers⁹³. Far less laser scattering was apparent at the nanotrenches compared with Cr nanobarriers, under the same illumination conditions (Figure 3.3A and Figure 3.1B). Quantitatively, nanotrenches significantly reduced scattering at excitation wavelengths of 488 and 637 nm, while there was no significant difference between nanotrenches and Cr barriers at 532 or 561 nm (Figure 3.3B). Furthermore, under 637 nm excitation, autofluorescence was dramatically suppressed for nanotrench slides compared with Cr barriers (Figure 3.3C). Our results demonstrated that the optical properties of nanotrenches were better suited to fluorescence imaging of DNA curtains than Cr or manually scratched barriers.

We next examined surface cleanliness after washing the patterned slides, because the removal of surface impurities is crucial for their reuse. Because Cr barriers are easily degraded by sonication, heating, and/or harsh reagents such as strong solvents or acids, they must be mildly cleaned. Although mild washing conditions sometimes succeed in removing surface residues, we found that their removal was incomplete. (Figure 3.3D). By contrast, nanotrenches etched on the slide were quite robust to any chemical reagents tolerated by the fused silica. To clean nanotrench slides, we treated them with acetone and strongly oxidizing piranha solution, in addition to the Hellmanex/ethanol/sodium hydroxide series used for Cr barrier slides. Moreover, except for piranha treatment, these cleaning steps were performed with sonication. This harsh cleaning process resulted in the complete removal of residues, leaving a pristine slide surface (Figure 3.3E).

3.3.2 FP–DBP for the DNA curtain formed at nanotrenches

We next tested the performance of a DNA curtain formed using the nanotrench slide (Figure 3.4A). We applied FP–DBP to fluorescently stain the DNA molecules (Figure 3.4B). In the presence of 0.1 nM FP–DBP, without any anti‐photobleaching agents, λ‐DNA molecules were well‐ stained. In the flowing buffer, the end‐bound DNA molecules were stretched out but recoiled from the evanescent field when the flow was stopped (Figure 3.4C). To determine whether the mechanical property of DNA was altered by binding of FP–DBPs, we measured the DNA persistence length versus flow rate, at 0.1 nM FP–DBP (Figure 3.4D). The data were fitted by a worm‐like chain model given as:

𝐹 =^𝑘𝐵𝑇

𝑃 ( ¹

4(1−^<𝑥>

𝐿 )²−¹

4+^<𝑥>

𝐿 ),

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where F is the hydrodynamic force due to flow, <x> is DNA extension, L is the contour length of λ‐DNA, 𝑘_𝐵 is Boltzmann constant, T is the absolute temperature, and P is the DNA persistence length¹⁰². The value of 𝑘_𝐵𝑇 is 4.1 pN nm at room temperature. In the presence of 0.1 nM FP–DBP, P, the only fitting parameter, was estimated at 187nm, which is about 3.7 times the value for naked DNA (∼50nm). In contrast, previous literature reported no significant difference in the persistence length of DNA stained with FP–DBP from that of naked DNA and concluded that FP–DBP binding does not alter the flexibility of DNA¹⁰⁰. Those previous persistence length measurements were made on stained DNA after washing out free FP–DBPs. We thus measured DNA extension versus flow rate after washing free FP–DBPs with 1X TE [7.5] and obtained a persistence length of 53 nm, consistent with the earlier reports that included washout (Figure 3.4D). In the presence of free FP–DBPs, the increase of flow rate extends DNA to greater length so that more FP–DBPs can bind. In addition, the aromatic ring of tryptophan residues in the DNA‐binding motifs can intercalate between DNA bases.

Consequently, the binding of FP–DBPs causes DNA to become more rigid, increasing its persistence length.

We also compared the photochemical properties of FP–DBP to those of YOYO‐1 at the same concentration (0.1 nM). YOYO‐1 led to severe breakage of many DNA molecules within the first 80 s (Figure 3.1C). In contrast, DNA density at the curtain was not reduced under continuous laser illumination (488 nm laser, 6.7 W/mm2) when DNA was stained with FP–DBP, indicating that FP–

DBP did not cleave DNA even when photoactivated (Figure 3.4E). We did observe that FP–DBP became photobleached, indicated by a gradual decrease in its fluorescence. We measured the relative photobleaching of FP–DBP and YOYO‐1 at 6.7 W/mm² of 488 nm laser. For this comparison, because of DNA photocleavage by YOYO‐1, only undamaged DNA molecules were included in the comparison. Under the same illumination conditions, FP–DBP bleached much more slowly than YOYO‐1 (Figure 3.4F). The measured lifetime of FP–DBP (τ_FP‐DBP) was 42.4 s, 10 times greater than that of YOYO‐1(τ_YOYO‐1: 4.3 s; Figure 3.4B). Generally, fluorescent proteins have longer fluorescence lifetime when the fluorophore moiety is buried in the protein interior. Our results demonstrated that FP–DBP had better photochemical properties for fluorescence imaging of DNA molecules in the DNA curtain than YOYO‐1, enabling us to observe DNA for a longer period of time.

YOYO‐1 intercalates DNA and disrupts protein–DNA interactions. Furthermore, it is difficult to remove YOYO‐1 from DNA even at high salt concentrations. To test whether FP–DBPs could be removed from DNA in our system, we washed DNA‐bound FP–DBP with a high pH buffer (1X TE [11.0]) that did not contain FP–DBP. As the solution flowed in, fluorescence momentarily increased, then gradually decreased (Figure 3.4G). The fluorescence increase resulted from the fact that the intrinsic fluorescence of enhanced green fluorescent protein increases as the solution becomes basic¹⁰³. This was shown by a comparable transient pH‐dependent increase in background fluorescence

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occurring in the absence of DNA (Figure 3.4G). The gradual disappearance of fluorescence at high pH was due to the dissociation of FP–DBP from DNA⁹⁸. Afterward, reinjection of FP–DBPs at pH [7.5] led to the recovery of fluorescence upon reassociation of FP–DBPs with DNA, suggesting reversibility of binding to DNA by pH exchange (Figure 3.4G). Likewise, FP–DBPs dissociated from DNA in the presence of high salt buffer (300 mM NaCl and 10 mM MgCl₂) and rebound to DNA when the buffer was exchanged to 1X TE [7.5] buffer containing FP–DBP (Figure 3.5)¹⁰¹. Interestingly, the high salt buffer caused fluorescence to increase due to DNA shrinking, which presumably resulted from the oligomerization of fluorescent proteins at high salt ¹⁰⁴. Taken together, our data demonstrated that FP–DBPs could be removed from DNA by high pH or salt.

3.3.3 Mapping of protein binding using nanotrenches

DNA curtain is a universal system for investigating diverse protein– DNA interactions, for example, mapping protein binding sites²⁶. We thus examined whether DNA curtains formed at nanotrenches could be applied to probe protein–DNA interactions. To this end, we purified an EcoRI^E111Q that is catalytically inactive but retains very high‐affinity binding to its cognate sequence (Kd ∼ fM)¹⁰⁵. After labeling with Qdots, the protein was injected onto DNA curtains, where FP–

DBPs were washed out (Figure 3.6A). EcoRI^E111Q molecules were well bound to λ‐DNA on the curtains, displaying vertically aligned fluorescent puncta (Figure 3.6B). A kymograph for EcoRI^E111Q binding to an individual DNA molecule demonstrated several distinct lines of fluorescent puncta, which corresponded to the EcoRI binding sites on λ‐DNA (Figure 3.6C). Binding of the protein to DNA was distinguished from its nonspecific binding to surfaces by the disappearance of fluorescent puncta when the flow was turned off, because of the recoil of DNA, along with bound EcoRI^E111Q molecules, out of the evanescent field (Figure 3.6C). To further analyze the binding locations of EcoRI^E111Q on λ‐DNA, individual fluorescent puncta were fitted with two‐dimensional Gaussian functions, and the center coordinates were used to construct a histogram for EcoRI^E111Q binding positions (Figure 3.6D). The histogram exhibited five distinct peaks, the centers of which coincided with the predicted EcoRI binding sites on λ‐DNA. Our results are consistent with the previous study using the chromium barrier, demonstrating that DNA curtains formed by nanotrenches can be used to map protein binding sites⁴⁴. We also tested EcoRI binding in the presence of FP– DBP on DNA to examine the influence of FP–DBP on protein binding (Figure 3.7). When λ‐DNA was prestained with FP–DBP, EcoRI^E111Q bound to λ‐DNA but the binding number of EcoRI^E111Q on single DNA was reduced by half (Figure 3.7AB). In the binding distribution, there were no distinct peaks around EcoRI binding sites, indicating that EcoRI^E111Qbinding was not specific to its cognate sites (Figure 3.7C). Our results suggested that FP–DBPs disturb the protein binding when they are bound to DNA.

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