KR102430546B1 - Single molecule array including nano trench structures for imaging - Google Patents

Abstract

It relates to a single molecule array for imaging including a nanotrench structure, and according to an aspect of the single molecule array for imaging, a method for manufacturing the same, a method for analyzing the interaction of a single molecule with an analyte protein, and a protein mapping method, it is easy to wash and It is economical as it does not affect the result even if reused several times, and it has the effect of being able to image for a long time without damaging a single molecule.

Description

Single molecule array including nano trench structures for imaging

It relates to a single-molecule array for imaging including nano-trench structures.

A biochip is a hybrid device made in the form of a conventional semiconductor chip by combining biological organic materials such as enzymes, proteins, antibodies, DNA, microorganisms, animal and plant cells and organs, nerve cells and organs, and nerve cells derived from living things and inorganic materials such as semiconductors or glass. (Hybrid device). By utilizing the unique functions of biomolecules and mimicking the functions of living things, it has the characteristics of diagnosing infectious diseases or analyzing genes and serving as new functional devices for new information processing.

Biochips can be classified into DNA chips, RNA chips, protein chips, cell chips, and neuron chips depending on the biomolecules and the degree of systematization. It can be broadly defined including a 'biosensor' capable of detecting and analyzing various biochemicals, such as a lab on a chip having an analysis function.

In order to increase the sensitivity and throughput of biochips, recent attempts have been made to integrate single-molecule technology. These advances in single-molecule spectroscopy have made it possible to accurately measure the structural dynamics and physical properties of molecules. As interest in research using single molecules has increased, techniques for fixing and manipulating single molecules have also been rapidly developed. One of those techniques is a method called DNA curtain developed by Professor Greene's research team at Columbia University in the United States in 2008 as a new method of stretching DNA by fixing only one end. In this method, avidin-bound DNA was allowed to flow over the surface of the lipid bilayer by adding avidin and biotin-labeled DNA to the lipid layer. And by creating a chromium layer on the surface and generating a flow in a certain direction, the DNA molecules were stretched out and the protein binding position on the DNA could be analyzed. However, using chrome like this is expensive and labor intensive, so it is mainly reused, but there is a disadvantage that it is not clean and can affect the results of the next experiment. In addition, YOYO-1 used for DNA staining has a disadvantage in that it can cause rapid photobleaching under continuous laser illumination and induce DNA cleavage even in the presence of a photobleaching inhibitor.

Accordingly, the present inventors have solved these problems by completing the present invention.

One aspect is to provide a single molecule array for imaging comprising, on a solid support, an etched straight or non-straight nanobarrier, a fat bilayer, and one or more single molecules bound to the fat bilayer by a linker.

Another aspect includes etching a straight or non-straight nano-barrier on a solid support; forming a fat bilayer on the solid support; attaching a single molecule to the fat bilayer by a linker; elongating the single molecule on the fat bilayer under the flow of a hydrodynamic force, an electrophoretic force, or a combination thereof; and binding a fluorescent protein to the single molecule.

Another aspect is a method comprising: etching a straight or non-straight nano-barrier on a solid support; forming a fat bilayer on the solid support; attaching a single molecule to the fat bilayer by a linker; elongating the single molecule on the fat bilayer under the flow of a hydrodynamic force, an electrophoretic force, or a combination thereof; binding a fluorescent protein to the single molecule; and binding the protein to be analyzed to the single molecule, to provide a method for analyzing the interaction between a single molecule and an analyte protein.

Another aspect is a method comprising: etching a straight or non-straight nano-barrier on a solid support; forming a fat bilayer on the solid support; attaching a single molecule to the fat bilayer by a linker; elongating the single molecule on the fat bilayer under the flow of a hydrodynamic force, an electrophoretic force, or a combination thereof; binding a fluorescent protein to the single molecule; And to provide a protein binding site mapping method comprising the step of binding the protein to be analyzed to the single molecule.

One aspect provides a single molecule array for imaging comprising, on a solid support, an etched straight or non-straight nanobarrier, a fat bilayer, and one or more single molecules bound to the fat bilayer by a linker.

As used herein, the term “array” may mean that one or more single molecules are attached to a support in a regular arrangement.

The solid support is, for example, silica, fused silica, quartz, borosilicate glass, functional glass, polydimethylsiloxane, Langmure-Blodgett film, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , silicone, modified silicone, or a polymer (eg, (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, or polycarbonate). Also, the solid support may be, for example, circular, square, square, or triangular. In one embodiment, the solid support is made of fused silica, and may be a slide used in a fluorescence microscope.

In one embodiment, the solid support may be, for example, to form a flow cell (flow cell). For example, it may be a microfluidic flow cell comprising an array of the present invention. The solid support may further comprise two or more openings, eg, one or more inlets and one or more outlets. The inlet and outlet are continuous, and the solid support and a cover adhesively attached at the boundary thereof, for example, a glass cover such as a glass coverslip, may be attached to a flow cell to create a chamber between the solid support and the cover. According to the present invention, a buffer can be injected into the inlet to elongate single molecules on the lipid bilayer and discharged to the outlet. The flow cell further includes an intermediate region, bifurcated nanochannels, at least one pair of parallel channels, at least one aperture, or a combination thereof.

The etched straight or non-straight nano-barrier may prevent lipid diffusion. The etching may mean forming an intaglio on a solid support, and in one embodiment, may be etched by dry etching including electron beam lithography or photolithography. The dry etching may be performed by reactive ion etching (RIE) or inductively coupled plasma (ICP) etching. In addition, in the present invention, the etching may be uniformly etched because the etching is not performed by applying another physical pressure. In addition, since it is not a barrier made of a metal such as chromium (Cr), it does not corrode even if washed under strong conditions, and it can be washed cleanly, so even if it is reused several times, the result may not be affected. The straight or non-linear nano-barriers may have various lengths and depths and widths. For example, the depth may be 100 nm to 5 μm, and the width may be 50 nm to 1 μm. In one embodiment, the barrier pattern may form a jagged barrier with a repeating triangular waveform. The barrier is non-linear and forms a nanowell by connecting the vertices of the geometric barrier, and the nanowell may include nanopores for entry and exit of nucleic acids.

In one embodiment, the single molecule may be a nucleic acid molecule, for example, single-stranded DNA, double-stranded DNA or RNA. A nucleic acid molecule may consist of about 10, 20, 30, 40, 50, 100, 150, 200, 500, 1000, 2000, 5000, 10000, 50000, 100000, 200000 or more nucleotides. The number of nucleic acid molecules that can be attached to the solid support may be determined by the size of the solid support and the design of the array. For example, about 50, 100, 250, 500, 1000, 2000, 5000 or more nucleic acid molecules may be attached.

In one embodiment, the linker may include one or more proteins. The linker is attached to the lipid bilayer, and the linker may include biotin-streptavidin. Streptavidin may be bound to a lipid head, and biotin may be bound to a single molecule and attached to a lipid bilayer by a biotin-streptavidin bond. In one embodiment, the one or more single molecules may be bound to the fat bilayer by separate linkers. For example, it may have one linker per each single molecule. In one embodiment, one end of a single molecule may be bound by a linker, and the single molecule may be straight or non-linear and aligned along a geometric barrier through hydrodynamic force, electrophoretic force, or a combination thereof. can Also, in one embodiment, the single molecule may be positioned in a desired direction depending on the direction in which a hydrodynamic force, an electrophoretic force, or a combination thereof is applied, and the hydrodynamic force may be due to injection of a buffer solution. In one embodiment, one end of the single molecule is aligned and attached along the barrier, and the non-adherent end of the single molecule is extended on the bilayer according to a flow direction such as a hydrodynamic force.

In one embodiment, a fluorescent protein may be bound to the single molecule. In the present invention, it may be to bind a fluorescent protein to visualize individual single molecules. The fluorescent protein is a green fluorescent protein (GFP), a modified green fluorescent protein (mGFP), an enhanced green fluorescent protein (EGFP), a red fluorescent protein (red fluorescent protein) ; RFP), modified red fluorescent protein (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (Yellow Fluorescent Protein) ; YFP), Enhanced Yellow Fluorescent Protein (EYFP), Cyan Fluorescent Protein (CFP), Cyan Green Fluorescent Protein (CGFP), Enhanced Cyan Fluorescent Protein (Cyan) Fluorescent Protein (ECFP), Azami Green (AzG), Heteractis crispa red fluorescent protein (HcRed), or Discosoma red fluorescent protein (DsRed), but is not limited thereto. In addition, a motif capable of binding to a single molecule may be bound to the fluorescent protein. In one embodiment, a motif capable of binding to a nucleic acid molecule may be bound to a fluorescent protein, and the motif may be a peptide. The amino acid sequence constituting the peptide includes a sequence capable of binding to a nucleic acid molecule and may consist of 5 to 20 amino acids, and as long as it is a sequence capable of binding to a nucleic acid molecule, it may vary without being limited to a specific sequence. According to one embodiment of the present invention, EGFP has an amino acid sequence consisting of N-KWKWKKA-C or N-AKKWKWK-C at the N-terminus or C-terminus of EGFP, or may have a combination thereof, It may also include an amino acid sequence. In addition, the fluorescent protein may not cleave a single molecule, and may be further labeled with a quantum dot.

Another aspect includes etching a straight or non-straight nano-barrier on a solid support; forming a fat bilayer on the solid support; attaching a single molecule to the fat bilayer by a linker; elongating the single molecule on the fat bilayer with buffer flow; and binding a fluorescent protein to the single molecule.

Another aspect is a method comprising: etching a straight or non-straight nano-barrier on a solid support; forming a fat bilayer on the solid support; attaching a single molecule to the fat bilayer by a linker; elongating the single molecule on the fat bilayer with buffer flow; binding a fluorescent protein to the single molecule; and binding the protein to be analyzed to the single molecule.

etching a straight or non-straight nano-barrier on a solid support; forming a fat bilayer on the solid support; attaching a single molecule to the fat bilayer by a linker; elongating the single molecule on the fat bilayer with buffer flow; binding a fluorescent protein to the single molecule; And it provides a protein binding site mapping method comprising the step of binding the protein to be analyzed to the single molecule.

The method for mapping the protein binding site may mean indicating by analyzing at which position in the single molecule the protein binding to a single molecule binds and at which position it interacts in the present invention.

The terms and methods described for the above invention are equally applied between the respective inventions.

According to the single molecule array for imaging, the manufacturing method thereof, the single molecule-analyzed protein interaction analysis method, and the protein mapping method according to an aspect, it is easy to wash and does not affect the result even if it is reused several times. Imaging is possible for a long time without damage.

1A shows a schematic diagram when a chromium barrier is used and a TIRFM image when it is used; Figure 1B is an image showing the cleavage of the barrier after washing (top) when using a chromium barrier, a TIRFM image at 488 nm showing aggregation of proteins when tested using it after washing (middle), and after washing Images showing laser scattering when tested using this (bottom), and the arrows on the left of each image indicate the location of the barrier; and FIG. 1C is an image showing photocleavage of DNA molecules by YOYO-1 under continuous laser illumination when YOYO-1 is used.
Figure 2A is a schematic diagram of the manufacturing process of the nano-trench barrier; Figure 2B is a figure showing the resulting nano-trench pattern (top) and an optical microscopy image (bottom) of the nano-trench barrier; and FIG. 2C is an SEM image showing a vertical cross-section of the nanotrench.
3A is a TIRFM image at 488 nm (top) and a TIRFM image at 637 nm when using a nano-trench barrier; 3B is a graph quantitatively comparing laser scattering when a chromium barrier or a nano-trench barrier is used, and the error bars represent the standard deviation after 4 repetitions; Figure 3C is a graph quantitatively comparing the fluorescence background when a chromium barrier or a nano-trench barrier is used, and the error bars represent the standard deviation after 4 repetitions; 3D is an image showing the surface condition before and after cleaning of the chromium barrier; and FIG. 3E is an image showing the surface state before and after cleaning of the nano-trench barrier.
Figure 4A is a schematic diagram of a DNA curtain using a nano-trench barrier; Figure 4B is a diagram showing the structure of the FP-DBP consisting of a DNA binding motif and a fluorescent protein; 4C is a TIRFM image showing a DNA curtain stained with FP-DBP with and without buffer flow, respectively; 4D is a TIRFM image showing DNA elongation as a function of flow rate and a graph showing DNA relative elongation versus flow rate with and without free FP-DBP; 4E is a TIRFM image of DNA molecules stained with FP-DBP under continuous illumination; Figure 4F is a graph showing the photobleaching curve of the DNA curtain when stained with FP-DBP or YOYO-1; and FIG. 4G is an image showing the chymograph of FP-DBP according to pH change.
Figure 5A is a diagram schematically showing that EcoRI ^E111Q bound to quantum dots is bound to a DNA molecule; 5B is a TIRFM image of DNA molecules stained with FP-DBP (top), a fluorescent speckle image showing a quantum dot-tagged EcoRI ^E111Q (middle), and an image of DNA and EcoRI ^E111Q superimposed (bottom); Figure 5C is an image showing the chymograph of a single DNA molecule to which EcoRI ^E111Q is bound; and FIG. 5D is a histogram showing the binding site of EcoRI ^E111Q bound to λ-DNA.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Nano Trench Fabrication

Nano trenches were fabricated in fused silica slides as shown in Fig. 2a (Fig. 2A). Slides with two holes were washed with Pirana solution (3:1 mixture of 30% H ₂ O ₂ and concentrated 96% H ₂ SO ₄ ) and rinsed with deionized water. A 250 nm thick aluminum layer (Al) was deposited by DC sputtering (SRN-120, Sorona) using 10 mTorr of argon gas, followed by a 310 nm thick 950 K PMMA layer (4%, AR-P 671.04, Allresist GmbH) at 4000 rpm for 1 min (Fig. 2a, step 1). The PMMA layer was cured on a hotplate at 180 °C for 3 minutes. The sawtooth pattern was drawn by EBL (NB3, Nanobeam Ltd.) with a current of 0.724 nA at 80 kV (Fig. 2a, step 2). The PMMA area exposed to the e-beam was removed in developer (MIBK (methyl isobutyl ketone) and isopropanol in a 1:3 ratio) for 2 min and rinsed with isopropanol (Fig. 2a, step 3). The PMMA and Al layers were etched by ICP-RIE (FabStar, Top Technology Ltd.) using a mixture of Cl ₂ and BCl ₂ gases (Fig. 2a, step 4). Nano-trenches were then formed by further ICP-RIE etching with a gas mixture of SF ₄ , CF ₄ and O ₂ ( FIG. 2a , step 5 ). The remaining Al layer was removed by immersion in Al etchant AZ-MIF300 for 10 minutes (Fig. 2a, step 6). Then, the residual Al was completely removed by immersion in the pirana solution for 10 minutes to finally prepare a nano-trench structure.

As a result, unlike manual scratching, the nanotrenches fabricated using EBL and RIE were uniformly engraved (Fig. 2B). Each nano-trench has a sawtooth pattern to prevent lateral diffusion of DNA molecules within the lipid bilayer. Also, scanning electron microscopy (SEM) images of vertical sections (Fig. 2C) show a very sharp cliff with a width of about 350 nm and a depth of 1.38 μm, suggesting that the nano-trench can provide an effective barrier to the diffusion of lipid molecules. indicates that there is

Figure 2A is a schematic diagram of the manufacturing process of the nano-trench barrier; Figure 2B is a figure showing the resulting nano-trench pattern (top) and an optical microscopy image (bottom) of the nano-trench barrier; and FIG. 2C is an SEM image showing a vertical cross-section of the nanotrench.

Example 2. DNA Preparation

For DNA curtain experiments, 4 nM lambda phage (λ) DNA (NEB) was mixed with 100-fold excess of oligomers in 200 μl of T4 DNA ligase reaction solution. The oligomers are biotinylated L-COS (5'-phosphate-GGG CGG CGA CCT-biotin-3') and R-COS (5'-phosphate-AGG TCG CCG CCC-3'), both of which are Biooneer (Korea). was synthesized by The mixture was heated to 65 °C on a heat block and then slowly cooled to 23 °C. After addition of 1 μl of T4 DNA ligase (2 U/μl, NEB), ligation was performed at 23° C. overnight. After heat-inactivation at 65° C., ligase and excess oligomers were removed with an S-400 micro-spin column (Illusta MicroSpin™ S-400, GE Healthcare) to finally prepare DNA for DNA curtain experiment.

Example 3. Purification of FP-DBP (Fluorescent Protein-DNA Binding Peptide)

FP-DBP for fluorescent labeling of DNA was purified. FP-DBP used in this Example is a peptide capable of binding to the DNA backbone to EGFP (enhanced green fluorescence protein), and as shown in FIG. 4B, the N-terminus of EGFP is N-KWKWKKA-C, It has an amino acid sequence of N-AKKWKWK-C at the C-terminus. An FP-DBP construct with an N-terminal 3xFLAG-tag and C-terminal 10xHis was constructed from the pET15b plasmid and then transformed into E. coli strain BL21 (DE3). Cells were grown in 1 L LB broth supplemented with carbenicillin at 37°C. When the OD ₆₀₀ reached about 0.8, expression of FP-DBP was induced with 1 mM isopropyl-β-D-thiogalactoside and further expressed at 16°C overnight. Cells were collected, pelleted and resuspended in lysis buffer (50 mM sodium phosphate [8.0], 300 mM NaCl and 10 mM imidazole). All subsequent purification processes were performed at 4 °C. Cells were lysed by sonication and then the clarified lysate was loaded onto a 10 ml column of Talon resin (GE Healthcare) under gravity flow. The column was washed with 80 ml wash buffer (50 mM sodium phosphate [8.0], 300 mM NaCl and 20 mM imidazole) followed by elution buffer (50 mM sodium phosphate [8.0], 300 mM NaCl and 250 mM imidazole). eluted. Fractions containing FP-DBP were pooled and diluted with 1xTE (10 mM Tris-HCl [8.0] and 1 mM EDTA) to give 100 mM NaCl, and buffer A (20 mM Tris-HCl [8.0], 100 mM NaCl, 1 mM) EDTA and 1 mM DTT) and loaded onto a 1 ml heparin column (HiTrap Heparin HP, GE Healthcare). The column was then washed with 10 ml of 95% Buffer A/5% Buffer B, where Buffer B comprised 20 mM Tris-HCl [8.0], 1 M NaCl, 1 mM EDTA and 1 mM DTT. Proteins were eluted with a gradient of 90% Buffer A/10% Buffer B to 100% Buffer B and fractions containing FP-DBP were pooled. Glycerol was added to a final concentration of 50% and the final purified protein stock was snap-frozen in liquid N ₂ and stored at −80° C. until use.

Example 4. EcoRI ^E111Q purification of

In order to confirm the protein-DNA interaction, EcoRI ^E111Q , a catalytically inactive E111Q mutant of EcoRI, was purified.

N-terminal 3xFLAG-tagged EcoRI ^E111Q with a C-terminal intein was expressed in E. coli strain BL21 (DE3). After growth to an OD of _600-0.6 in 2 L of LB medium supplemented with carbenicillin, protein expression was induced with 1 mM IPTG, and the protein was expressed overnight at 16°C. After cell lysis, the clarified lysate was passed through a 5 ml column of chitin resin (S6651L, NEB) under gravity flow. The column was washed with 20 ml of wash buffer (20 mM Tris-HCl [8.5], 500 mM NaCl and 1 mM EDTA). While binding to the column, the intein was cleaved with 50 mM DTT overnight in wash buffer. Finally, EcoRI ^E111Q was eluted in chitin elution buffer (40 mM Tris-HCl [7.5], 300 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 50% glycerol and 0.15% Triton X-100). The final purified protein stock was flash frozen in liquid N ₂ and stored at -80 °C until use.

Experimental Example 1. Analysis of Optical Characteristics and Reusability of Nano Trench

The optical properties of the nano-trench slides were tested to be suitable for use in DNA curtains.

Specifically, a prismatic total internal reflection fluorescence microscope (TIRFM) was fabricated with an inverted fluorescence microscope (Nikon Eclipse Ti-2) and a solid-state laser (200 mW; OBIS, Coherent Laser) was used. The laser beam was incident at an angle above the critical angle through a custom dove prism to induce total internal reflection due to the presence of an evanescent field at the boundary between the fused silica slide and the buffer. Fluorescence signals were collected with a 60x water immersion objective (CFI Plan Apo VC 60XWI, Nikon) using an appropriate long-pass filter to block laser excitation. Fluorescence was imaged using an EM-CCD camera (iXon 897, Andor Technology). Data were collected using NIS-Element software (Nikon).

As a result, under the same illuminance condition, much less laser scattering was observed in the nano-trench compared to the Cr nano-barrier (compare FIGS. 3A and 1B). Quantitatively, the nanotrench significantly reduced scattering at excitation wavelengths of 488 nm and 637 nm, and there was no significant difference between the nanotrench and the Cr barrier at 532 nm or 561 nm (Fig. 3B). Also, under 637 nm excitation, autofluorescence was highly suppressed for the nano-trench slides compared to the Cr barrier (Fig. 3C). These results indicate that the optical properties of nanotrenches are more suitable for fluorescence imaging of DNA curtains than Cr or passive scratch barriers.

Surface cleanliness was tested after washing the patterned slides because removal of surface impurities is important for reuse. Cr barriers should be cleaned lightly as they are easily degraded by sonication, heating and/or harsh reagents such as strong solvents or acids. Therefore, after overnight in 2% Hellmanex III, then in 99% ethanol overnight, and then in 1M sodium hydroxide for 30 minutes to wash successively. Rinse with deionized water between each step. As a result, this washing indicated that the removal of impurities was incomplete (Fig. 3D).

In contrast, the nano trenches etched onto the slides were fairly robust against any chemical reagents acceptable to fused silica. Therefore, nano-trench cleaning was further treated with acetone and strong oxidized pirana solution compared to the cleaning of the Cr barrier. Specifically, to wash the nano-trench slides, they were placed in acetone for 30 minutes, placed in 2% Hellmanex III for one day, and then pirana solution (concentrated 96% H ₂ SO ₄ in a 3: 1 ratio: 30% H ₂ O ₂ ) ) for 40 minutes, placed in 99% ethanol overnight, and then placed in 1M sodium hydroxide for 30 minutes, followed by continuous washing. Rinse with deionized water between each step. Also, with the exception of pirana treatment, these washing steps were performed by sonication. As a result, the residue was completely removed by this strong washing process, resulting in a clean slide surface (Fig. 3E). These results indicate that nanotrenches are more suitable for reuse compared to Cr barriers.

1A shows a schematic diagram when a chromium barrier is used and a TIRFM image when it is used; Figure 1B is an image showing the cleavage of the barrier after washing (top) when using a chromium barrier, a TIRFM image at 488 nm showing aggregation of proteins when tested using it after washing (middle), and after washing It is an image showing laser scattering in the case of an experiment using this (bottom), and the arrow on the left of each image indicates the position of the barrier.

3A is a TIRFM image at 488 nm (top) and a TIRFM image at 637 nm when using a nano-trench barrier; 3B is a graph quantitatively comparing laser scattering when a chromium barrier or a nano-trench barrier is used, and the error bars represent the standard deviation after 4 repetitions; Figure 3C is a graph quantitatively comparing the fluorescence background when a chromium barrier or a nano-trench barrier is used, and the error bars represent the standard deviation after 4 repetitions; 3D is an image showing the surface condition before and after cleaning of the chromium barrier; and FIG. 3E is an image showing the surface state before and after cleaning of the nano-trench barrier.

Experimental Example 2. DNA elongation analysis of DNA molecules stained with FP-DBP on a slide on which a nanotrench structure is formed

The performance of the DNA curtain formed using nano-trench slides was tested (Fig. 4A). FP-DBP was applied to fluorescently stain DNA molecules (Fig. 4B).

Specifically, to prepare a DNA curtain, a flow cell was made by attaching a nano-trench slide to a coverslip using double-sided tape, which is also a spacer to form a microchannel. To connect the flow cell to a fluid system consisting of a syringe pump and a 6-way infusion valve, a nanoport (IDEX) was attached to the orifice.

The microchannels were rinsed with deionized water and lipid buffer (20 mM Tris-HCl [8.0] and 100 mM NaCl). Liposomes contain DOPC (1,2-dioleoyl-sn-glycero-phosphocholine), 0.5% biotinylated-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-(cap biotinyl)) and 8% mPEG 2000-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) , and all were purchased from Avanti-Polar Lipids. To form a lipid bilayer, the liposomes were deposited on the microchannel surface by incubation for 20 minutes, and then the free liposomes were washed with a lipid buffer. Any remaining uncoated surface was then passivated by injection of BSA buffer (40 mM Tris-HCl [8.0], 50 mM NaCl, 2 mM MgCl ₂ and 0.4% BSA). Then, 0.025 mg/ml streptavidin in BSA buffer was injected. After incubation for 10 minutes, unbound streptavidin was washed with BSA buffer, and λ-DNA molecules tagged with biotin at one end were immobilized on biotinylated lipids via biotin-streptavidin linkages. The flow cell was then connected to the fluidic system, placed in a TIRFM, and fluorescence was imaged.

As a result, in the presence of 0.1 nM FP-DBP in 1xTE (pH7.5) without anti-bleaching agent, λ-DNA molecules stained well, and end-bound DNA molecules in flow buffer were elongated but in the evanescent field when flow was stopped. was rewound (Fig. 4C). To determine whether the kinetic properties of DNA are altered by binding of FP-DBP, relative elongation (<x>/ L ) versus flow rate of DNA was measured in the presence or absence of 0.1 nM FP-DBP (Fig. 4D) . For DNA elongation, the DNA length was measured from the center of the barrier to the end of each DNA molecule, and DNA elongation as a function of flow rate was determined by the following worm-like chain model, using the duration length as the only parameter. Fitted: F=k _B T/P(1/4(1-<x>/ L ) ² -1/4+<x>/ L ). where F is the hydrodynamic force due to flow, <x> is the DNA elongation, L is the contour length of λ-DNA, k _B is the Boltzmann constant, T is the absolute temperature, P is the DNA duration, k _B The value of T is 4.1 pN nm at room temperature.

In the presence of 0.1 nM FP-DBP, the only fitting parameter, P, was 187 nm, and 53 nm in the absence of FP-DBP. As such, when FP-DBP is present, the P value is about 3.7 times that of the naked DNA value (~50 nm). These results indicate that, in the presence of free FP-DBP, the increase in flow rate elongates the DNA longer so that more FP-DBP can bind. In addition, in the DNA-binding motif of FP-DBP, an aromatic ring of tryptophan residues is inserted between DNA bases, indicating that as a result, binding of FP-DBP makes the DNA more rigid and increases the persistence length.

Figure 4A is a schematic diagram of a DNA curtain using a nano-trench barrier; Figure 4B is a diagram showing the structure of the FP-DBP consisting of a DNA binding motif and a fluorescent protein; 4C is a TIRFM image showing a DNA curtain stained with FP-DBP with and without buffer flow, respectively; FIG. 4D is a TIRFM image showing DNA elongation according to flow rate and a graph showing DNA relative elongation versus flow rate with and without free FP-DBP.

Experimental Example 3. Comparative analysis of photochemical properties of FP-DBP and YOYO-1

The photochemical properties of FP-DBP and YOYO-1 were compared at the same concentration (0.1 nM).

Specifically, as in Experimental Example 2, a DNA curtain was prepared and under continuous buffer flow, λ-DNA molecules moved along the lipid bilayer in the flow direction until it stopped in the nano-trench, where the biotinylated end was in the bilayer. Retained attached, the DNA molecules were stretched in flowing solution. DNA molecules were stained with 0.1 nM YOYO-1 or 0.1 nM FP-DBP in imaging buffer (1xTE [7.5] with 0.1 mg/ml BSA and 1 mM DTT). In YOYO-1 staining, an oxygen scavenger system (1.6% D-glucose and 0.5x gloxy) was included to inhibit YOYO-1-induced photobleaching. Images were recorded at a frame rate of 10 Hz. To measure photobleaching, images were collected at continuous laser illumination (488 nm, 6.7 W/mm ² ) until little fluorescence signal was detected. To quantify laser scattering in the barrier and autofluorescence, the fluorescence intensity at the barrier and the slide surface near the barrier, respectively, was measured. In ImageJ, the barrier or slide surface was confined by a fixed box and then the average fluorescence intensity within the box was obtained. To measure photobleaching, we used ImageJ to trap individual DNA molecules inside fixed boxes so that the fluorescence signal was completely selected from a single DNA. Then, the average fluorescence intensity within the box was extracted and the total background intensity was subtracted to obtain the actual fluorescence intensity. In the photobleaching curve, the average true intensity of 10 DNA molecules was plotted as a function of time. The photobleaching curve was fitted by a single exponential decay function.

As a result, YOYO-1 severely damaged many DNA molecules within the first 80 seconds, and almost all DNA molecules were cleaved at 160 seconds (Fig. 1C). In contrast, DNA density in the curtain was not reduced under continuous laser illumination when stained with FP-DBP, indicating that FP-DBP did not cleave DNA even when photoactivated (Fig. 4E).

It was also observed that FP-DBP was photobleached, resulting in a gradual decrease in fluorescence. The relative photobleaching of FP-DBP and YOYO-1 was measured under continuous laser illumination. For this comparison, only intact DNA molecules were included in the comparison because of DNA photocleavage by YOYO-1. Under the same illuminance conditions, FP-DBP photobleached much slower than YOYO-1 (Fig. 4F). The measured photobleaching time of FP-DBP (τ_FP-DBP) was 42.4 seconds, which was 10 times longer than that of YOYO-1 (τ_YOYO-1), which was 4.3 seconds (Fig. 4F). In general, a fluorescent protein has a longer fluorescence lifetime when the fluorophore moiety is embedded inside the protein. Therefore, these results indicate that FP-DBP has better photochemical properties for fluorescence imaging of DNA molecules on the DNA curtain than YOYO-1, and enables DNA observation for a longer period of time.

1C is an image showing photocleavage of DNA molecules by YOYO-1 under continuous laser illumination when YOYO-1 is used.

4E is a TIRFM image of DNA molecules stained with FP-DBP under continuous illumination; 4F is a graph showing the photobleaching curve of the DNA curtain when stained with FP-DBP or YOYO-1.

Experimental Example 4. Analysis of Ease of Removal of FP-DBP from DNA

YOYO-1 is inserted into DNA and interferes with protein-DNA interactions, and even at high salt concentrations, it is difficult to remove YOYO-1 from DNA. In contrast, in the present invention, it was tested whether FP-DBP can be easily removed from DNA.

Specifically, DNA molecules were first stained with 0.1 nM FP-DBP in imaging buffer. Then, only basal buffer (1xTE [11.0] with 0.1 mg/ml BSA and 1 mM DTT) without FP-DBP was injected until the fluorescence disappeared, then the buffer was added with 0.1 nM FP-DBP and imaging buffer (pH 7.5). exchanged again.

As a result, as a high pH buffer (1xTE [11.0]) without FP-DBP was introduced, the fluorescence temporarily increased and then gradually decreased (Fig. 4G). The increase in fluorescence is due to the fact that the intrinsic fluorescence of eGFP increases as the solution becomes basic, which appears as a similar transient pH-dependent increase in background fluorescence that occurs in the absence of DNA (Fig. 4G). The gradual loss of fluorescence at high pH was due to the dissociation of FP-DBP from DNA, and then reinjection of FP-DBP at pH 7.5 resulted in the recovery of fluorescence upon recombination of FP-DBP with DNA, which was responsible for the pH exchange. shows the reversibility of binding to DNA ( FIG. 4G ). These results indicate that FP-DBP can be removed from DNA by high pH.

4G is an image showing the chymograph of FP-DBP according to pH change.

Experimental Example 5. Analysis of DNA binding site mapping of fluorescently labeled proteins using a DNA curtain formed of nano trenches

We tested whether the DNA curtain formed in the nanotrench could be applied to investigate protein-DNA interactions.

To this end, EcoRI ^E111Q , which is catalytically inactive as shown in Example 4, but retains very high affinity binding to its cognate sequence (K _d ~ fM), was purified.

Then, specifically, Qdot was tagged with an anti-FLAG antibody (Q10161 MP, Invitrogen). 3xFLAG-tagged EcoRI ^E111Q was conjugated with Qdot in a molar ratio of 1:10 so that one Qdot had at most one EcoRI ^E111Q . FP-DBP was washed from DNA with a high pH solution. A 1 nM solution of QDot-labeled EcoRI ^E111Q was injected into the DNA curtain in EcoRI buffer (40 mM Tris-HCl [8.0], 100 mM NaCl, 2 mM MgCl ₂ , 1.6% glucose and 1 mM DTT) (Fig. 5A) . After incubation in the flow cell for 15 minutes, unbound EcoRI ^E111Q was washed and labeled EcoRI ^E111Q molecules bound to DNA were observed. Then, EcoRI ^E111Q -Qdot was injected into the DNA curtain to test the effect of FP-DBP on protein binding to DNA in which DNA molecules were pre-stained with 0.1 nM FP-DBP.

As a result, EcoRI ^E111Q molecules were well bound to λ-DNA in the curtain, displaying vertically aligned fluorescent spots ( FIG. 5B ). Chemographs of EcoRI ^E111Q binding to individual DNA molecules showed several distinct lines of fluorescent spots corresponding to EcoRI binding sites of λ-DNA (Fig. 5C). Binding of DNA to proteins was distinguished from nonspecific binding to surfaces by the disappearance of fluorescence spots when flow was turned off due to DNA rewinding with EcoRI ^E111Q molecules bound in the evanescent field (Fig. 5C). To further analyze the binding sites of EcoRI ^E111Q in λ-DNA, individual fluorescence spots were fitted with a two-dimensional Gaussian function and histograms for EcoRI ^E111Q binding sites were constructed using centroid coordinates (Fig. 5D). Histograms for joint position distributions were built in Origin 2017 (Origin Lab) and the distribution errors were obtained by bootstrapping 70% confidence intervals using Matlab (Mathworks). The histogram showed five distinct peaks of 4.4 kbp, 9.8 kbp, 17.9 kbp, 23.9 kbp, and 29.5 kbp, which are known as 3.53 kbp, 9.334 kbp, 16.755 kbp, 22.398 kbp, and 27.276 kbp of EcoRI of λ-DNA. coincided with the binding site. These results indicate that protein binding sites can be mapped using the DNA curtain formed by the nanotrench.

Figure 5A is a diagram schematically showing that EcoRI ^E111Q bound to quantum dots is bound to a DNA molecule; 5B is a TIRFM image of DNA molecules stained with FP-DBP (top), a fluorescent speckle image showing a quantum dot-tagged EcoRI ^E111Q (middle), and an image of DNA and EcoRI ^E111Q superimposed (bottom); Figure 5C is an image showing the chymograph of a single DNA molecule to which EcoRI ^E111Q is bound; and FIG. 5D is a histogram showing the binding site of EcoRI ^E111Q bound to λ-DNA.

Claims (14)

solid support; a non-straight nanotrench having a depth of 100 nm to 5 μm and a width of 50 nm to 1 μm etched on the solid support; fat bilayer; and one or more single molecules bound to the fat bilayer by a linker. The single molecule array for imaging according to claim 1, wherein the solid support is made of fused silica. The single molecule array for imaging of claim 1 , wherein the solid support forms a flow cell. The single molecule array for imaging according to claim 1, wherein the single molecule is a nucleic acid molecule. The single molecule array for imaging according to claim 1, wherein the nano trenches are etched by a dry etching method. The single molecule array for imaging according to claim 1, wherein the linker comprises one or more proteins. The single molecule array for imaging according to claim 1, wherein the fluorescent protein is bound to the single molecule. The single molecule array for imaging according to claim 7, wherein the fluorescent protein is a Fluorescent Protein-DNA Binding Peptide (FP-DBP) comprising a DNA binding motif and a fluorescent protein. The single molecule array for imaging of claim 1 , wherein the single molecules are aligned along the nano trenches. The single molecule array for imaging of claim 1 , wherein the single molecule is stretched on the fat bilayer under the flow of hydrodynamic force, electrophoretic force, or a combination thereof. The single molecule array for imaging of claim 1, wherein the one or more single molecules are each bound to the fat bilayer by a separate linker. etching non-straight nano trenches having a depth of 100 nm to 5 μm and a width of 50 nm to 1 μm on a solid support;
forming a fat bilayer on the solid support;
attaching a single molecule to the fat bilayer by a linker;
elongating the single molecule on the fat bilayer under the flow of a hydrodynamic force, an electrophoretic force, or a combination thereof; and
A method for producing a single molecule array for imaging, comprising the step of binding a fluorescent protein to the single molecule. etching non-straight nano trenches having a depth of 100 nm to 5 μm and a width of 50 nm to 1 μm on a solid support;
forming a fat bilayer on the solid support;
attaching a single molecule to the fat bilayer by a linker;
elongating the single molecule on the fat bilayer under the flow of a hydrodynamic force, an electrophoretic force, or a combination thereof;
binding a fluorescent protein to the single molecule; and
A method for analyzing the interaction of a single molecule with a protein to be analyzed, comprising the step of binding the protein to be analyzed to the single molecule. etching non-straight nano trenches having a depth of 100 nm to 5 μm and a width of 50 nm to 1 μm on a solid support;
forming a fat bilayer on the solid support;
attaching a single molecule to the fat bilayer by a linker;
elongating the single molecule on the fat bilayer under the flow of a hydrodynamic force, an electrophoretic force, or a combination thereof;
binding a fluorescent protein to the single molecule; and
A protein binding site mapping method comprising the step of binding a protein to be analyzed to the single molecule.

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