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Article Cite This: ACS Omega 2019, 4, 458−464
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Encapsulation of Long Genomic DNA into a Confinement of a Polyelectrolyte Microcapsule: A Single-Molecule Insight Anatoly Zinchenko,* Eisuke Inagaki, and Shizuaki Murata Graduate School of Environmental Studies, Nagoya University, Nagoya 464-8601, Japan
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S Supporting Information *
ABSTRACT: Encapsulation of nucleic acids is an important technology in gene delivery, construction of “artificial cells”, genome protection, and other fields. However, although there have been a number of protocols reported for encapsulation of short or oligomeric DNAs, encapsulation of genome-sized DNA containing hundreds of kilobase pairs is challenging because the length of such DNA is much longer compared to the size of a typical microcapsule. Here, we report a protocol for encapsulation of a ca. 60 μm contour length DNA into several micrometer-sized polyelectrolyte capsules. The encapsulation was carried out by (1) compaction of T4 DNA with multivalent cations, (2) entrapment of DNA condensates into micrometer-sized CaCO3 beads, (3) assembly of polyelectrolyte multilayers on a bead surface, and (4) dissolution of beads resulting in DNA unfolding and release. Fluorescence microscopy was used to monitor the process of long DNA encapsulation at the level of single-DNA molecules. The differences between long and short DNA encapsulation processes and morphologies of products are discussed.
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INTRODUCTION Encapsulation of nucleic acids is an important research area from both fundamental and applied points of view. DNA encapsulation is an attractive tool for genetic information storage,1,2 DNA protection,3,4 and transport.5 Being coupled with a suitable nucleic acid cargo release approach, DNA encapsulation represents an attractive strategy for gene delivery.6−8 During the past decade, several research groups successfully elaborated protocols for encapsulation of DNA macromolecules9−11 and DNA oligomers12 into polyelectrolyte capsules and studied their properties inside microconfinement. Release of encapsulated DNA cargo was achieved in a controlled way by suitable design of a capsule wall.9,12−14 Notably, most of these studies utilized relatively short DNA macromolecules at high concentrations that were, in most of cases, homogeneously distributed in the interior of polyelectrolyte capsules. Therefore, the features related to spatial localization of individual DNA chains could not be revealed. Encapsulation of substantially longer DNA molecules is a more challenging task due to issues related to DNA damage, adhesion to the interface of a confinement, etc. and to the best of our knowledge, has never been demonstrated so far. On the other hand, in vitro systems containing submegabase-sized DNA macromolecules inside a micrometersized confinement are highly relevant to the state of DNA in living cells, in which several meters long DNA is confined inside a tiny nucleus of several micrometers size. Such systems can be utilized for a better understanding of DNA structure and behavior in vivo.15 A number of “artificial cell” models © 2019 American Chemical Society
containing DNA in a microconfinement were elaborated on the basis of water-in-oil16−18 or water-in-water microdroplets,19 liposomes,20 and giant vesicles21−24 (Figure 1) to
Figure 1. In vitro “artificial cell” models. (A) DNA confined in waterin-oil droplet. (B) DNA confined in a giant vesicle. (C) DNA confined in a semipermeable polyelectrolyte capsule (this study).
compare DNA conformational behavior in a bulk solution and inside the microconfinement. These studies found a strong effect of the confinement on the DNA higher-order structure and its biological activity.25 However, due to the nonpermeable nature of the interface between a confinement and external solution, the composition of a fluid inside such containers cannot be changed after DNA encapsulation and the analysis of dynamic changes in DNA higher-order structure is not generally possible. Received: October 18, 2018 Accepted: December 25, 2018 Published: January 7, 2019 458
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Here, we propose an alternative model of “artificial cell” with a semipermeable boundary (Figure 1) that may allow a direct observation of long DNA behavior inside the microconfinement. We report an encapsulation protocol for a long DNA (ca. 166 kbp, ca. 60 μm contour length) into a polyelectrolyte capsule of ca. 5 μm diameter by introducing a crucial DNA compaction step and discuss DNA conformational changes during construction of such systems. By comparing encapsulation of long bacteriophage DNA and short salmon sperm DNA (ca. 300 bp), we also make clear a difference in the partitioning of long and short DNAs inside polyelectrolyte microcapsules.
compact DNA particles from solution (Figure 2B). Layer-bylayer deposition method is then used to construct a multilayered film from cationic and anionic polyelectrolytes on the surface of the beads (Figure 2C). Finally, CaCO3 beads are dissolved by EDTA to release the DNA into the interior of a capsule (Figure 2D). Compaction and Entrapment of Long-Chain DNA Molecules into CaCO3 Microbeads. Conformational behavior of single-sub-megabase long DNA molecules can be easily monitored by fluorescence microscopy (FM). T4 DNA is a giant nucleic acid of ca. 60 μm contour length that, in aqueous solutions of 0.01−0.1 M ionic strength, adopts a random coil conformation of ca. 5 μm long-axis length and exhibits a free Brownian motion (Figure 3A,B). The dimension
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RESULTS AND DISCUSSION Figure 2 illustrates the procedure used for long DNA molecule encapsulation into a microcapsule of several micrometers
Figure 2. General strategy for long DNA encapsulation into a micrometer-sized polyelectrolyte microcapsule. (A) Compaction of DNA in a solution of multivalent cations. (B) Co-precipitation of precompacted DNA with CaCO3 resulting in entrapment of the compact DNA into CaCO3 bead. (C) Layer-by-layer deposition of polyelectrolyte multilayer on a surface of beads. (D) Dissolution of CaCO3 core by EDTA and release of DNA into a capsule.
Figure 3. T4 DNA compaction by multivalent cations. Fluorescence images of single T4 DNA molecules (10 μM in phosphates) in TE buffer solution (A, B) and in TE buffer solution containing 5 mM spermine (C, D). Snapshot series (B, D) show single-molecule motion T4 DNA in coil (B) and in globule (D) conformations observed in corresponding samples. The time interval between snapshots is 2−3 ms. Insets are DNA long-axis length distributions of unfolded and compact DNA molecules built by measurement of at least 100 individual DNA molecules.
diameter. This protocol is based on co-precipitation strategy used for short DNA and proteins but contains an important DNA compaction step (Figure 2A). Similar to a living cell, which stores DNA having length several orders of magnitude larger than the size of cell, the contour length of T4 DNA (ca. 60 μm) is much larger than the diameter of a typical polyelectrolyte capsule (ca. 10 μm). Previous protocols used for short DNA encapsulation, such as adsorption of DNA on the surface of a solid sacrificial template9,12 or diffusionadsorption of DNA in the interior of nanoporous beads,10 cannot be used because neither can long DNA diffuse into capsules’ interiors nor can it adsorb in a regular and reproducible way on the surface of a microtemplate. Therefore, long DNA was first compacted into globular condensates using a cationic binder (Figure 2A) and transferred into the reaction mixture of CaCl2 and Na2CO3. Reaction of CaCl2 and Na2CO3 yields CaCO3 nanoparticles assembling into metastable, several micrometer-sized nanoporous CaCO3 beads entrapping
of DNA coil is comparable with diameter of typical CaCO3 beads (5−10 μm) that are used for DNA encapsulation; therefore, to avoid DNA damage, compaction of DNA is necessary to drastically decrease DNA molecules’ dimension. Due to its long length, giant T4 DNA molecule is fragile and easily suffers damage under hydrodynamic stress,26 irradiation,27 etc.; thus, compaction of DNA plays an important role in protecting DNA during entrapment into CaCO3 beads. Negatively charged DNA molecules undergo compaction into high-density condensates upon addition of various multivalent cations that cause DNA charge neutralization.28 Among DNA condensing agents, spermine, a naturally 459
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occurring tetraamine, was routinely utilized for DNA compaction.29 Figure 3C,D shows FM images of compacted T4 DNA molecules after addition of spermine (5 mM) and snapshots of the Brownian motion of DNA globule. Comparison of DNA long-axis length distributions in Figure 3A,C shows that addition of spermine causes a drastic decrease of DNA molecular volume, which is on the order of 104−105 times.30 The actual size of compact DNA condensates is approximately 100 nm,31 i.e., significantly smaller than the typical diameter of CaCO3 beads; therefore, the precompacted DNA can be accurately entrapped into a sacrificial template. Mixing of CaCl2 and Na2CO3 solutions of submolar concentrations under vigorous stirring yields nanoparticles assembling into spherical vaterite (CaCO3) microbeads of several micrometers diameter. Addition of compact DNA globules into this solution should result in entrapment of DNA globules into growing CaCO3 beads. However, the presence of high concentrations of divalent Ca2+ cations in the reaction mixture may lead to the unfolding of compact DNA globules due to competition of di- and tetravalent cations for DNA binding32 whereas vigorous stirring may also cause severe mechanical damage of unfolded long DNA molecules by sheared flow.33 To avoid T4 DNA decompaction and damage, DNA globules compacted by spermine were introduced to a solution of growing CaCO3 beads 10−15 s after CaCl2 and Na2CO3 solutions mixing and further growth of CaCO3 was conducted without stirring. To gain insight into the difference between short and long DNA molecule encapsulation, we also performed encapsulation of short double-stranded DNA from salmon sperm (ca. 300 bp) under the same conditions. In contrast to predominately monomolecular compaction of long DNA molecules, compaction of short DNA by spermine results in random multimolecular condensation and gradual time-dependent growing of such DNA condensates (Supporting Information, Figure S1). CaCO3 beads containing long and short DNAs were visualized by bright-field and fluorescence microscopies (Figure 4A,B). In both cases, beads of ca. 10 μm size and spherical morphology were formed and contained nucleic acid cargo successfully loaded into beads. The partitioning of DNA in beads was strongly affected by DNA length. Sperminepretreated T4 DNA in beads was mostly observed as compact globules of ca. 0.7−1.0 μm size (Figure 4A). The number of DNA globules per bead varied in a range between 0 and ca. 5 globules. A rough estimation of an average number of T4 DNA molecules per CaCO3 bead based on 10 μm diameter and 1.6 g/cm3 density34 gives ca. 8 globules pear bead. The estimated value is somewhat higher than that found experimentally that can be caused by T4 DNA multimolecular condensation or aggregation during compaction with spermine. T4 DNA globules were preferentially positioned closer to the outer interface of beads (Figure 4C), which might be due to that fact that T4 DNA globules were added to a solution of growing beads after the core part had been formed. A very different state and distribution of entrapped DNA inside beads was observed for short DNA molecules (Figure 4B). DNA was found in the form of aggregates of different sizes and morphologies appearing at different densities. This highly inhomogeneous DNA partitioning is due to the formation and coexistence of DNA condensates of different sizes during compaction with spermine (Supporting Information, Figure S1).
Figure 4. Entrapment of DNA into CaCO3 beads. Images of T4 DNA (A) and salmon sperm DNA (B) labeled with YOYO fluorescent dye and entrapped into CaCO3 beads. The images of beads dispersed in Milli-Q water were recorded as superpositions of bright-field images and fluorescence images acquired through a blue excitation filter B-2A (Nikon) with longpass emission. Quasi-3D profiles of light and fluorescent intensities of representative beads are given for beads marked with an asterisk (*). Diameter distributions of beads were obtained by measuring 100 beads. (C) Preferential position of compacted T4 DNA globule entrapped into CaCO3 beads shown as a histogram of DNA globule relative position between the center of a bead (position 0.0) and an edge (position +1.0).
The entrapment efficiency of long and short DNAs into CaCO3 beads was quantified by fluorescence spectroscopy measurements of a DNA-bound fluorescent dye YOYO in solutions before and after DNA entrapment. Fluorescence spectra on Figure 5 show that the intensity of YOYO fluorescence in solution decreased drastically after DNA entrapment into beads and separation, indicating that the entrapment of both T4 DNA and salmon sperm DNA was almost quantitative. Construction of Capsule Walls by LbL Deposition of Polyelectrolytes. In the next step, the (PSS/PAH)2/PSS polyelectrolyte multilayers were assembled on a surface of beads loaded with DNA. The composition of the multilayer was chosen to get higher polyanion contents to gain an overall negative change and provide efficient repulsion between the encapsulated DNA and capsule wall in the target capsule. Cationic PAH was covalently labeled with Texas Red fluorescent dye added to the original PAH solution at 0.1% molar ratio to visualize the multilayer during microscopic observations. Figure 6A shows fluorescence images of beads containing T4 DNA (green fluorescence) coated with five layers of polyelectrolytes (red fluorescence). Deposition of multilayer did not affect T4 DNA conformational state in beads: T4 DNA globules were observed inside coated beads similar to the beads without polyelectrolyte multilayers, indicating that no DNA conformational changes happened during multilayer deposition. It should be mentioned that the size of pores in CaCO3 beads (20−60 nm35) is much smaller compared with the typical diameter of a compact condensate of T4 DNA (ca. 100 nm31); therefore, unfolding or diffusion of DNA 460
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Figure 5. Entrappment efficiency of DNA into CaCO3 beads. Fluorescence spectra of solutions containing DNA (0.6 μM), YOYO (0.06 μM), and spermine (300 μM) before and after co-precipitation with CaCO3 beads. (A) and (B) show spectral changes for salmon sperm DNA and T4 DNA, respectively.
Figure 6. LbL deposition of polyelectrolyte multilayer on the surface of DNA-entrapped beads. Typical fluorescence microscopy images of T4 DNA (A) and salmon sperm DNA (B) labeled with YOYO fluorescent dye (green) and encapsulated into CaCO3 beads after deposition of five polyelectrolyte layers ((PSS/PAH)2/PSS) on beads’ surface. Red fluorescence is from PAH labeled with Texas Red used for multilayer construction. The images of beads dispersed in Milli-Q water were recorded through a blue excitation filter B-2A (Nikon) with longpass emission. Quasi-3D profiles of fluorescent intensities are shown for representative beads marked with asterisk (*). Figure 7. DNA in polyelectrolyte capsules. Typical fluorescence microscopy image profiles of T4 DNA (A) and salmon sperm DNA (B) labeled with YOYO fluorescent dye (green) and encapsulated into multilayered capsule ((PSS/PAH)2/PSS) after dissolution of sacrificial CaCO3 core with EDTA. The images of capsules dispersed in TE buffer solution (pH 8) were recorded through a blue excitation filter B-2A (Nikon) with longpass emission. Quasi-3D profiles of fluorescent intensities are shown for representative beads marked with an asterisk (*). Red fluorescence is from PAH labeled with Texas Red used for multilayer construction. Histograms show diameter distributions of DNA-containing capsules. (C) Schematic illustration of plausible T4 DNA (green) state in polyelectrolyte capsules according to fluorescence microscopy observations.
condensates inside beads cannot occur. Figure 6B shows corresponding FM images of beads containing short DNA with an uneven distribution of fluorescence intensity, indicating persistence of DNA in aggregated state. DNA Release into Hollow Capsules. Finally, to dissolve CaCO3 core and release DNA molecules into capsules, DNAentrapped beads with deposited multilayers were dialyzed against 0.1 M EDTA solution. Figure 7 shows fluorescence microscopy images of core-free capsules containing long (Figure 7A) and short (Figure 7B) DNA molecules. Removal of the core resulted in substantial shrinking of the polyelectrolyte capsule by ca. 1.5 times compared with the diameter of CaCO3 template (Figure 5A,B). For example, the average diameter of capsules with T4 DNA decreased from ca. 12 to 7 μm. Shrinking of capsules containing a small number of layers can be attributed to a loosely entangled structure of polyelectrolyte molecules in multilayers deposited at relatively high ionic strength that can undergo electrostatically driven rearrangement under lower salt conditions. Shrinking/swelling of capsules is a complex function of many solution parameters as well as multilayer assembly history. About 10−30% shrinking of similar capsules was observed in previous studies
as a result of core removal36 or annealing37,38 due to postcompensation of remaining free charges. No compact globules of T4 DNA seen in Figure 6A were observed inside the hollow capsules. Instead, DNA fibers (green) typical for DNA in unfolded conformations were found in most capsules (Figure 7A), indicating that dissolution of CaCO3 core triggered decompaction of DNA globules into coils. In most capsules, T4 DNA attached to the walls of capsules and stretched toward the center of capsules where 461
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high fluorescence from DNA was observed (Figure 7A). Careful observation of DNA localization and partitioning in capsules let us propose the following scenario of DNA unfolding inside the capsules. As illustrated by Figure 7C, it is suggested that, despite the overall negative charge of the inner polyelectrolyte layer, the inner interface of capsules contains cationic fragments of second-layer PAH with uncompensated cationic charges, which results in electrostatic attachment of T4 DNA coil to the capsule wall. This is in a good agreement with earlier studies that acknowledged the interpenetration of multilayer components over length scales exceeding the thickness of a single layer39 as well as matrixlike structure of a capsule wall suggesting protrusion of individual polyelectrolyte chains into the interior of the capsule.34 At the same time, the gross negative charge of capsules containing the excess of anionic PSS causes repulsive interaction between the encapsulated DNA and capsule wall, resulting in localization of the remaining free DNA in the ca. 2 μm middle part of the confinement far from the like-charged multilayer. Smaller capsules with diameters of 3−4 μm possessed the same essential features as the larger ones: attachment of DNA to the wall and partial concentration in the middle of capsule (data not shown). It should be noted that the contour length of T4 DNA (ca. 60 μm) is significantly larger than the diameter of capsules (ca. 5 μm); thus, even a single-DNA molecule can demonstrate such morphological features. However, the results in Figure 4A that made clear encapsulation of multiple T4 DNA globules per CaCO3 bead suggest that DNA structures shown in Figure 7A are formed by several DNA chains. In contrast, localization of short DNA in capsules (Figure 7B) after dissolution of the sacrificial core was strikingly different from that of long T4 DNA. The entire interior of the capsules was filled with DNA. In this case, electrostatic interactions of short DNA and cationic domains in multilayers are also possible, causing absorption of a small fraction of short DNA on a capsule wall. Uniform distribution of short DNA inside capsules is entropically driven, overcoming the effect of the electrostatic repulsion of DNA from negatively charged walls, and the concentration of short DNA in the center of capsules does not occur. Although no large aggregates of DNA were observed in the hollow capsules, the distribution of fluorescent signal from DNA appeared slightly heterogenous with a higher intensity in the vicinity of the multilayered wall. Appearance of the heterogeneity in short DNA localization can be again attributed to the existence of polycationic fragments on the inner surface of the multilayers that bind electrostatically with short DNA forming complexes near capsule walls. As shown above, the length of DNA molecule plays an important role and determines the DNA partitioning scenario in a microcapsule.
wall is an important future challenge for manipulation of DNA inside microconfinement. Further development of such a system is promising for construction of “artificial cell” models for better understanding of DNA behavior in a living cell.
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EXPERIMENTAL SECTION Materials. T4 G7 DNA (ca. 166 kbp, ca. 60 μm contour length) was purchased from Nippon Gene Co. Ltd. (Japan). Salmon sperm DNA (ca. 300 bp) was purchased from Wako Pure Chemical Industries, Ltd. (Japan). The concentration of DNA is given in phosphate groups. Fluorescent dye YOYO-1 (1,1′-(4,4,7,7-tetramethyl-4,7-diazaundeca-methylene)-bis-4[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]quinolinium tetraiodide) was provided by Molecular Probes (Invitrogen, Japan). NaCl, CaCl2·2H2O, and Na2CO3 were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Spermine tetrachloride, disodium dihydrogen ethylendiaminetetraacetate dihydrate (EDTA), and ethanol were purchased from Nacalai Tesque Inc. (Japan). Sodium polystyrene sulfonate (PSSNa, Mw ∼15 000) and poly(diallyldimethylammonium chloride) (PAH, Mw ∼75 000) were purchased from Aldrich. Milli-Q water purified by Simplicity UV apparatus (Millipore, Japan) was used in all experiments. Methods. Brightfield and Fluorescence Microscopies. Brightfield and fluorescence microscopy observations were performed on an Eclipse TE 2000-U microscope (Nikon Instruments Inc.) equipped with a blue excitation B-2A filter (Nikon Instruments Inc.) with longpass emission (excitation 450−490 nm, cutoff wavelength 500 nm, and barrier filter wavelength 515 nm) and oil-immersed ×100 or ×40 objective lens. YOYO-labeled T4 DNA molecules were illuminated with a mercury lamp, and fluorescent images were observed and recorded on a EB-CCD monochrome camera using an Argus 10 image processor system (Hamamatsu Photonics, Japan). Fluorescence images of beads containing DNA and multilayers and capsules were recorded on a Nikon DS-Ri1 digital camera and analyzed using a Micron Optics image-analysis system and NIS-Elements BR 3.1 software. The focal plane of beads’ and capsules’ images was chosen at approximately the center of the bead/capsule height. The size of DNA molecules and beads was measured using ImageJ 1.52i software (NIH). The apparent long-axis length of the T4 DNA molecule in solution was measured as the longest distance in the outline of DNA molecule fluorescence images of single-DNA chain. The diameter of beads and capsules was measured at approximately the center of bead/capsule height. Fluorescence Spectroscopy. Fluorescent spectra of T4 DNA and salmon sperm DNA labeled with YOYO-1 were recorded on an FP-6600 spectrofluorimeter (Jasco, Japan) in 1 cm optical path quartz cells at room temperature. Sample Preparations. Fluorescence Labeling of PAH with Texas Red Fluorescent Dye. First, 10 mL of 10 g/L PAH solution (pH 8.6) was mixed with a 250 μL solution of Texas Red-X succinimidyl ester in dimethyl sulfoxide for 1 h. The resulted solution was dialyzed two times against 1 mM NaCl solution (0.5 L) using membrane tubing with molecular weight cutoff (MWCOs) of 12 000−14 000 Da. After dialysis, the labeled PAH was stored in fridge at 4 °C. DNA Entrapment into CaCO3 Microparticles. To 1 mL solution of T4 DNA (10 μM) in TE buffer, a 10 μL solution of YOYO-1 fluorescent dye (100 μM) was added and gently mixed. Next, a 50 μL solution of spermine (0.1 M) was added
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CONCLUSIONS We successfully constructed a system confining 60 μm counter length DNA in a 5−10 μm diameter polyelectrolyte capsule by introducing an important DNA compaction step before its encapsulation. This protocol enables encapsulation of both long and short DNAs with almost quantitative entrapping efficiency. Being entrapped inside sacrificial CaCO3 beads, long DNA preserves the conformation that it exhibited before encapsulation. After dissolution of beads, DNA unfolds adhering to the capsule wall and partly concentrating in the central part of the capsule. Gaining control over electrostatic properties of capsules to prevent DNA adhesion to the capsule 462
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and the sample was incubated for at least 15 min. Into 10 mL of Milli-Q water, 3 mL of 0.67 M Na2CO3 and 3 mL of 0.67 M CaCl2 solutions were added and rapidly mixed under vigorous stirring (500 rpm) for 10−15 s. Solution of labeled DNA with spermine was quickly added to the solution of Na2CO3 and CaCl2, stirring was stopped, and the precipitate was allowed to settle down for 10 min. The precipitate was separated by decantation, washed twice with Milli-Q water (50 mL) and once with ethanol (30 mL), and dried at 60−70 °C in a convection oven for 3 h. Drying of DNA-entrapped beads is important to avoid their recrystallization from mesoporous vaterite to monocrystal calcite phase accompanied with a release of encapsulated cargo.34 Thermal denaturation of long bacteriophage DNA in aqueous solutions takes place between 60 and 80 °C;40 however, in the presence of multivalent cations, the resistance of DNA toward thermal denaturation significantly increases.41 The obtained CaCO3 beads containing encapsulated DNA were stored in a dry box at a relative humidity of 10−20%. Entrapment of salmon sperm DNA (ca. 300 bp) into beads was performed using the same protocol as that described above for T4 DNA. Construction of a PSS-PAH Multilayered Capsule. A multilayer of polyelectrolytes was sequentially deposited on CaCO3 beads according to the standard layer-by-layer (LbL) deposition procedure.42 PSSNa and PAH were dissolved in 0.5 M NaCl solution at 1 g/L concentrations. Solution of PAH labeled with Texas Red fluorescent dye was added to PAH solution at a molar ratio of 1:1000. CaCO3 beads (0.1 g) containing DNA were dispersed into 0.5 mL of 0.1 M NaCl solution; 9.5 mL of PSSNa (0.1 g/L) solution was added, vigorously mixed, and incubated for 5 min. The resulted beads were separated by centrifugation at 1000 rpm for 3 min and washed twice with 10 mL of 0.1 M NaCl solution followed by separation by centrifuging to remove nonbound polyelectrolytes. Alternating adsorption of PSSNa and PAH layers followed by washing with 10 mL of 0.1 M NaCl solution was repeated to deposit five-layered ((PSS/PAH)2/PSS) multilayer film on beads’ surfaces using 10 mL of 1 g/L solution of polyelectrolytes. To dissolve CaCO3 core, microbeads with deposited multilayers were separated and resuspended in 2 mL of distilled water and dialyzed twice against 50 mL of 0.1 M EDTA solution (pH 8.0) using a slide-A-Lyzer MINI dialysis device (MWCO 20 000 Da). The resulted suspension of capsules was further dialyzed two times against 50 mL of distilled water, one time against 50 mL of TE buffer (pH 8.0), and finally stored in a fridge at 4 °C.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Anatoly Zinchenko: 0000-0002-9257-0881 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a KAKENHI grant 17K05611 from Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT). Prof. Kenichi Yoshikawa (Doshisha University, Japan), Prof. Tatsuo Akitaya (Asahigawa Medical University, Japan), and Dr. Kanta Tsumoto (Mie University, Japan) are acknowledged for helpful discussions.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02865. FM analysis of salmon sperm DNA compaction by spermine; Figure S1. Salmon sperm DNA (ca. 300 bp) compaction by spermine. Fluorescence images of salmon sperm DNA in TE buffer solution containing 5 mM of spermine 30 s (A) and 120 s (B) after adding spermine (PDF) 463
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DOI: 10.1021/acsomega.8b02865 ACS Omega 2019, 4, 458−464
