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. 2020 Feb 21;14(1):014115. doi: 10.1063/1.5142666

Molecular ring toss of circular BAC DNA using micropillar array for single-molecule studies

Daiki Dohi 1, Ken Hirano 2,3,2,3,a), Kyohei Terao 1,4,1,4,a)
PMCID: PMC7039730  PMID: 32128009

Abstract

This paper reports a method for trapping circular DNA molecules and imaging the dynamics with high spatial resolution using a micropillar-array device. We successfully trapped circular bacterial artificial chromosome DNA molecules at a micropillar-based “ring toss” in the laminar flow of a microchannel under a fluorescence microscope and demonstrated the imaging of their extension by flow and condensation process induced by spermine solution. DNA molecules were visualized in an extended loop conformation, allowing high spatial resolution, and the results showed that the dynamics is induced by the microfluidic control of the surrounding chemical environment. The method is expected to lead to the elucidation of the physical characteristics and the dynamics of circular DNA molecules.

INTRODUCTION

Fluorescence imaging of isolated single DNA molecules has greatly influenced the research fields of molecular biology, including transcription,1,2 DNA replication,3 epigenetic regulation,4,5 DNA recombination and repair,6–8 as well as their physical characterization as a flexible polymer.9 This technique yields spatial information directly and aids the visualization of the dynamics of DNA molecules and the biomolecules interacting with them, which are hardly obtained through typical bulk experiments. Furthermore, imaging of single molecules has been employed in clinical applications, such as diagnosis of genome-derived diseases.10,11

Several techniques have been developed to manipulate single DNA molecules under a fluorescence microscope for their observation, including that based on optical tweezers,12–18 magnetic tweezers,19 nanotweezers,20 nanochannels,21,22 electroosmosis,23,24 and dielectrophoresis.25 Typically, DNA molecules of tens of or a hundred and some tens of kbp are targeted, which are represented by lambda phage DNA (λDNA). Although there are several reports on the manipulation of relatively large DNA of Mbp size (e.g., yeast chromosomal DNA),12–14 target DNA is still focused on linear-type molecules.

In contrast to extensive studies about linear DNA, there is less of an understanding about the dynamics of circular DNA molecules. This is because a circular DNA molecule has no end; these ends are utilized for the manipulation of a linear DNA to immobilize the DNA on a solid surface through chemical modification.26 On the other hand, a circular DNA molecule has great potential to serve as model molecules of a circular flexible polymer in polymer physics,27 loop-form DNA existing in genomic DNA,28,29 and super-coiled DNA caused by the activity of DNA-binding proteins (e.g., RNA polymerase,30 topoisomerase,31 etc.). In particular, topoisomerases, which regulate the supercoiling and relaxing of DNA, are targets of anticancer drugs and antibiotics.32,33 In addition, a decrease in their activity is thought to be one of the causes of autism.34,35 The circular form DNA is expected to allow the visualization of the twist/untwist motion induced by the molecules and the dynamics of proteins interacting with them.

To realize circular DNA imaging, we propose a “molecular ring toss” technique to trap single circular DNA molecules without chemical modifications and to observe their dynamics with the molecules in an extended loop conformation for high spatial resolution, using a micropillar-based device. According to the demonstration, DNA condensation induced by polyamine solution was observed using fluorescence microscopy.

PRINCIPLE

Molecular ring toss

A circular DNA molecule has no end available for chemical modification, which lead us to trap it in a purely physical manner. We employed micropillars and a controlled laminar flow in a microchannel for the trapping and imaging processes (Fig. 1). Single circular DNA molecules having random-coil conformation are trapped at a micropillar-based “ring toss,” which is a stochastic process, and extended by the flow in the microchannel to have an extended loop conformation in a focal plane of a microscope for high resolution imaging. Switching the surrounding aqueous environment (e.g., pH, and chemical composition) by microfluidic control can bring about changes in the behavior of DNA molecules, providing for the in situ fluorescence observation of their dynamics. The method can allow us to observe not only the structural change of a circular DNA molecule itself, but also the dynamics of DNA-binding molecules on it. The trapping is easily parallelized with the array of pillars for efficient observation. This approach involves no chemical modifications and thus involves simple operative methods and observation.

FIG. 1.

FIG. 1.

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Molecular ring toss for fluorescence observation of single circular DNA molecules. (a) Circular DNA molecules are trapped at micropillars and extended by laminar flow in a microchannel (schematics). (b) Streamlines around a micropillar (simulation result). Lines are visualized as red dots present 10 μm and 2 μm on the upper streamside of a micropillar in the planes of the y-section.

Flow around a pillar

We checked the flow profile around a micropillar by numerical simulation using COMSOL Multiphysics 5.2. The dimensions of the microchannel model were set to have a channel height, pillar height, and pillar diameter of 10 μm, 5 μm, and 4 μm, respectively, and the pillar was placed on the top of the flow channel [Fig. 1(b)]. A DNA molecule in solution has random-coil conformation, whose hydraulic diameter was approximately 3.0–3.5 μm in our experiments. The value was estimated experimentally by the observation of its Brownian motion. This configuration means that the DNA must be extended to have a diameter larger than the micropillar before it is trapped.

The simulation result shows that there is a flow over the top of the micropillar and a horizontal flow passing around its sidewall of the pillar, which is expected to extend random-coil DNA [Fig. 1(b)]. A DNA molecule at a position of z = 6.5 μm height is carried by the flow splitting vertically. Additionally, a DNA in the plane of the center of the pillar (x = 0) is carried by the flow splitting horizontally. This indicates that a DNA flowing along the line (0, y, 6.5) is extended in the y plane to cover the upper part of the micropillar and trapped like a “ring toss” stochastically. After being trapped, a DNA molecule is extended by the flow along the y axis. The extensional flow has been extensively reported in the literature in the case of a linear DNA molecule.36,37 Circular DNA can also be extended by hydrodynamic force against its entropic elasticity; it exhibits loop conformation as schematically shown in Fig. 1(a).

MATERIALS AND METHODS

Design and fabrication

The flow channel is 6.4 mm and 27 mm in length and width, respectively; its height ranges from 10 to 64 μm [Fig. 2(a)]. Rectangular posts are arranged to prevent adhesion between the top and bottom of the flow channel. The channel has 2 inlets and 1 outlet, between which an array of micropillars is fabricated. The height of a micropillar is 5 μm, and its diameter ranges from 2.2 to 7.4 μm [Fig. 2(b)].

FIG. 2.

FIG. 2.

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Device design for a molecular ring toss. (a) Channel and micropillar-array dimensions. (b) Cross section of the channel. The dimensions show the design values. (c) Experimental setup. Solution replacement from solution A to B in the micropillar-array area by changing the balance of the flow rate. Flow rate: Inlet 1 > Inlet 2 (left) and Inlet 1 < Inlet 2 (right).

The balance between the flow from inlets 1 and 2 determines whether the solution is introduced into the pillar area; a higher flow rate causes the solution to fill in the pillar area [schematically shown in Fig. 2(c)]. This allows simple and quick replacement of the solution surrounding the trapped DNA molecules for single-molecule studies based on in situ observations.

The microfluidic device was fabricated with standard PDMS molding. The mold was patterned with photoresist SU-8 (Microchem Corp.) to have 2-layer microstructures, by which the PDMS microchannel with micropillars was fabricated. Subsequently, holes were punched in the device for the inlets and outlet and bonded to a glass substrate (thickness: 0.12–0.17 mm) through an O2 plasma treatment.

DNA trapping

The 2 inlets were connected with 2 syringe pumps (Legato, KDScientific Inc.) using Teflon tubes [Fig. 2(c)]. The device was placed on the stage of an inverted fluorescence microscope (IX-71, Olympus Corp.), where images were obtained through an EM-CCD camera (ImagEM, Hamamatsu Photonics K.K.).

BAC (Bacterial Artificial Chromosome) DNA (RP11-518C18, 208 kbp, 0.02 ng/ml) was stained with 0.1 μM YOYO-1 (Thermo Fischer Scientific Inc.) in a 10 mM MOPS buffer (pH 7.4). In the DNA trapping experiment, the DNA sample and pure water were introduced from Inlets 1 and 2, respectively. The average flow velocity at the micropillar area was adjusted to be approximately 150 μm/s. We estimated the flow velocity experimentally by measuring the transportation velocity of free DNA molecules between pillars. After trapping the DNA molecules, we obtained florescence images and evaluated the trap efficiency.

Imaging DNA condensation

DNA condensation induced by spermine was demonstrated as a single-molecule study. Bovine Serum Albumin (BSA) was coated on the channel surface by a 15-min incubation of the DNA molecules in a 1 mg/ml BSA solution to prevent them from adhering to the substrate surface. DNA sample solution and 70–1200 μM spermine (Wako Pure Chemical Industries, Ltd.) in a 50 mM MOPS buffer were introduced from Inlets 1 and 2, respectively. In DNA trapping, the flow rate of the DNA sample solution was set to 1000 μl/min, whereas that of the spermine solution was 150 μl/min. After DNA trapping, the flow rates from Inlets 1 and 2 were switched for treating the trapped DNA molecules with spermine.

RESULTS AND DISCUSSION

DNA trapping

Circular DNA molecules were successfully trapped at micropillar-based “ring tosses” and extended to have a loop conformation, thus allowing for their observation with high spatial resolution (Fig. 3, Multimedia view). DNA molecules were trapped at micropillars with various diameters ranging from 2.2 to 7.4 μm. The diameters and the trapped DNA molecules are shown in Fig. 4. The diameters were slightly smaller than the design values because of the expansion of the SU-8 mold structures in the exposure process. Smallest micropillars fabricated in our design had a conical shape of the height and base diameter of approximately 2.2 μm and 2 μm, respectively, whereas the micropillars of the larger diameters were successfully fabricated as cylindrical structures. The pillars of the diameter smaller than 2 μm were not fabricated thoroughly due to its high aspect ratio. The results show that the change of micropillar diameters results in different loop conformations, suggesting the possibility for analyzing the relationship between the conformation of DNA and DNA dynamics, including the activity of DNA-binding molecules on DNA loops of different curvature radii.

FIG. 3.

FIG. 3.

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Time series of trapping a single circular DNA molecule (208 kbp BAC DNA). Multimedia view: https://doi.org/10.1063/1.5142666.1 Download video file (1.9MB, mp4) DOI: 10.1063/1.5142666.1

FIG. 4.

FIG. 4.

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Micropillars of various diameters and the trapped DNA molecules. The pillars of 2.2 μm diameter were deformed due to fabrication problem, which had a conical shape. The diameter corresponds the base bottom.

Trap efficiency was evaluated as the percentage of (number of micropillars trapping DNA)/(total number of micropillars). The efficiency is in the range of 0.17%–8.61% [Fig. 5(a)]. The result indicates that the smaller the micropillar traps, the more the DNA molecules trapped in them. However, the efficiency is notably low in 2.2 μm micropillars (0.17%) that have a conical shape, owing to limitations in the fabrication process. The result suggests that the shape and size of the micropillars are critical factors for the efficient trapping of DNA molecules. From the viewpoint of imaging, the fluorescence of the micropillars resulting from the adsorption of DNA molecules, which sometimes occurred in our experiments, is liable to limit the observation of trapped DNA molecules. In such cases, surface treatment and adequate storage of the device would improve the situation.

FIG. 5.

FIG. 5.

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Relationship between the trap efficiency of DNA molecules and the (a) pillar diameter and (b) channel height.

We also evaluated the dependency of the channel height in the range of 10–64 μm, where the average flow velocity was set to 150 μm/s [Fig. 5(b)]. The result shows that high channels are associated with a low trapping efficiency. This must be because of the decrease in DNA molecules flowing towards micropillars. Thus, we used devices with 10-μm-height microchannels and 5-μm-height micropillars of 3–4 μm diameter in the following experiments.

Demonstration of DNA dynamics imaging

We investigated the relationship between the flow velocity and length of the trapped DNA to evaluate the extension of DNA induced by flow (Fig. 6). Trapped DNA molecules were successfully extended by a viscous drag against the DNA entropic elasticity.36 The length of a trapped DNA was 82.0 ± 2.9 μm (mean ± SD) under the flow velocity of 220 μm/s, which increased gradually from 80 μm in the flow of 50 μm/s to 92 μm in 250 μm/s. The contour length of the BAC DNA is estimated to be 71.3 μm from the following assumption about natural B-form DNA: 1 kbp = 3.4 nm; however, we used the fluorescence intercalator YOYO-1, which extends DNA to a length approximately 1.35 times longer than that of the unstained DNA, depending on the ratio of the intercalator concentration to the number of nucleotides.38 Thus, extended DNA in our results is estimated to have a length less than the actual contour length. Additionally, this could be within the range, without the structural transition of DNA molecules.39

FIG. 6.

FIG. 6.

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Extension of trapped DNA molecules. (a) Fluorescence images of a DNA molecule at a flow velocity of 50–250 μm/s. (b) Changes of the contour length of trapped DNA molecules in accordance with flow velocity. The 4 plots show those of each single DNA molecules.

Using the ring toss device, we performed the imaging of circular DNA dynamics by observing DNA condensation induced by spermine (150 μM spermine/50 mM MOPS buffer pH 7.4, Fig. 7(a), Multimedia view), which is a polyamine40 with the ability to condense a random-coil DNA molecule into a compact granule41,42 and to suppress transcription.43 The result shows the successful imaging of the condensation progress and nucleation at the DNA position farthest from a micropillar. This condensation shows the same mode as observed in the case of a linear DNA molecule.44 In a uniform flow, tension in a DNA molecule is minimum around its free end, because of which condensation starts there.44 The result implies that the loop-form DNA in an extensional flow also has minimum tension at this point, allowing relatively diffusive motion to start nucleation. The speed of the change of condensed DNA length (i.e., distance between the pillar surface and the DNA position farthest from it) was evaluated as a condensation rate with different spermine concentrations [Fig. 7(b)]. The rate was obtained with linear approximation of the time course of the length. This demonstration illustrates the capability of the ring toss technique for real time imaging of DNA dynamics induced by various chemical conditions surrounding DNA molecules. Detailed analysis of the time course of the condensation in various flow and chemical conditions will provide further insight into the condensation dynamics of circular DNA molecules.

FIG. 7.

FIG. 7.

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DNA condensation. (a) Time series of condensation of a circular DNA molecule induced by 150 μM spermine solution. White arrows indicate the nucleation point of DNA condensation. (b) Relationship between the condensation rate and spermine concentration. Multimedia view: https://doi.org/10.1063/1.5142666.2Download video file (685.1KB, mp4) DOI: 10.1063/1.5142666.2

This technique allows for the imaging of single circular DNA molecules with high spatial resolution in a simple and controlled manner and will elucidate its physical characteristics and the dynamics induced by various molecules, such as DNA-binding proteins.

CONCLUSIONS

We developed a method to observe single circular DNA molecules with high spatial resolution using a “molecular ring toss” microfluidic device. Circular DNA molecules (BAC DNA) were successfully trapped at micropillars under a fluorescence microscope. Trap efficiency depends on the pillar diameter and channel height. We found that smaller cylindrical structures are associated with higher trapping efficiencies. We also evaluated the relationship between the flow rate and the DNA length and verified the extension near full length by hydrodynamic drag force. Imaging of circular DNA dynamics was demonstrated by observing DNA condensation induced by spermine solution. The results suggest that the device can control the environment surrounding a circular DNA molecule and visualize the dynamic motion of biological events with high spatial resolution. It is thus expected to aid single-molecule studies of circular DNA based on their in situ observations.

ACKNOWLEDGMENTS

This work was partly supported by PRESTO (No. JPMJPR14FB) of the Japan Science and Technology Agency (JST), JSPS KAKENHI (Nos. 15K13324, 16H06077, 18K18837, and 18K06175) of the Ministry of the Education, Culture, Sports, Science and Technology (MEXT), and the matching fund of AIST-Shikoku and Kagawa University. This work was partly conducted at the Kagawa University Nano-Processing Facility, supported by the “Nanotechnology Platform Program” of the MEXT.

Contributor Information

Ken Hirano, Email: .

Kyohei Terao, Email: .

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