Publications by authors named "Makoto Komiyama"

201 Publications

Stepwise Strategy for One-Pot Synthesis of Single-Stranded DNA Rings from Multiple Short Fragments.

Chembiochem 2021 Mar 26;22(6):1005-1011. Epub 2020 Nov 26.

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, P. R. China.

Cyclic rings of single-stranded (ss) DNA have various unique properties, but wider applications have been hampered by their poor availability. This paper reports a convenient one-pot method in which these rings are efficiently synthesized by using T4 DNA ligase through convergent cyclization of easily available short DNA fragments. The key to the present method is to separate all the splint oligonucleotides into several sets, and add each set sequentially at an appropriate interval to the solutions containing all the short DNA fragments. Compared with simple one-pot strategies involving simultaneous addition of all the splints at the beginning of the reaction, both the selectivity and the yields of target ssDNA rings are greatly improved. This convergent method is especially useful for preparing large-sized rings that are otherwise hard to obtain. By starting from six short DNA fragments (71-82 nt), prepared by a DNA synthesizer, a ssDNA ring of 452-nt size was synthesized in 35 mol % yield and in high selectivity. Satisfactorily pure DNA rings were obtainable simply by treating the crude products with exonuclease.
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http://dx.doi.org/10.1002/cbic.202000738DOI Listing
March 2021

Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand.

Nucleic Acids Res 2020 05;48(9):e54

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.

Rings of single-stranded RNA are promising for many practical applications, but the methods to prepare them in preparative scale have never been established. Previously, RNA circularization was achieved by T4 RNA ligase 2 (Rnl2, a dsRNA ligase) using splints, but the yield was low due to concurrent intermolecular polymerization. Here, various functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences. The key is to design a precursor RNA, which is highly activated for the efficient circularization without any splint. First, secondary structure of target RNA ring is simulated by Mfold, and then hypothetically cut at one site so that a few intramolecular base pairs are formed at the terminal. Simply by treating this RNA with Rnl2, the target ring was selectively and efficiently produced. Unexpectedly, circular RNA can be obtained in high yield (>90%), even when only 2 bp form in the 3'-OH side and no full match base pair forms in the 5'-phosphate side. Formation of polymeric by-products was further suppressed by diluting conventional Rnl2 buffer to abnormally low concentrations. Even at high-RNA concentrations (e.g. 50 μM), enormously high selectivity (>95%) was accomplished.
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http://dx.doi.org/10.1093/nar/gkaa181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229815PMC
May 2020

Facile Characterization of Topology of DNA Catenanes.

Biophys J 2020 04 15;118(7):1702-1708. Epub 2020 Feb 15.

College of Food Science and Engineering, Ocean University of China, Nucleic acids Chemistry and Biotechnology Laboratory, Shinan-qu, Qingdao, China; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China. Electronic address:

During the preparation of single-stranded DNA catenanes, topological isomers of different linking numbers (Lk) are intrinsically produced, and they must be separated from each other to construct sophisticated nanostructures accurately. In many previous studies, however, mixtures of these isomers were directly employed to construct nanostructures without sufficient characterization. Here, we present a method that easily and clearly characterizes the isomers by polyacrylamide gel electrophoresis. To the mixtures of topological isomers of [2]catenanes, two-strut oligonucleotides, which are complementary with a part of both rings, were added to connect the rings and fix the whole conformations of isomers. As a result, the order of migration rate was always Lk3 > Lk2 > Lk1, irrespective of gel concentration. Thus, all the topological isomers were unanimously characterized by only one polyacrylamide gel electrophoresis experiment. Well-characterized DNA catenanes are obtainable by this two-strut strategy, opening the way to more advanced nanotechnology.
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http://dx.doi.org/10.1016/j.bpj.2020.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136276PMC
April 2020

Effect of sulfated polysaccharides on the digestion of DNA by pepsin under simulated gastric juice in vitro.

Food Funct 2020 Feb;11(2):1790-1797

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.

The effect of sulfated polysaccharides on the digestion of dietary DNA by pepsin was studied using in vitro simulated gastric juice. The results showed that fucoidan (FUC), dextran sulfate (DS) and chondroitin sulfate (CS) could inhibit the digestion of DNA in a dose-dependent manner. Polysaccharides with high sulfate group content have stronger inhibition ability. Fluorescence spectroscopy results showed that polysaccharides could bind to pepsin, and transmission electron microscopy (TEM) confirmed that polysaccharides can interact with DNA, which not only is the main reason that polysaccharides inhibit the digestion of DNA by pepsin but also causes the digestion of DNA by DNase II to be inhibited. The finding suggests that the digestion of DNA should be reevaluated when eating foods rich in sulfated polysaccharides. This study enriched the known pharmacological properties of sulfated polysaccharides as pepsin inhibitors and provided inspiration for the use of sulfated polysaccharides as oligonucleotide drug delivery carriers.
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http://dx.doi.org/10.1039/c9fo02578bDOI Listing
February 2020

DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.

Biochemistry 2020 02 10;59(4):400-406. Epub 2020 Jan 10.

College of Food Science and Engineering , Ocean University of China , Qingdao 266003 , China.

DNA ligase ( DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.
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http://dx.doi.org/10.1021/acs.biochem.9b00866DOI Listing
February 2020

Colorimetric determination of mercury(II) ion based on DNA-assisted amalgamation: a comparison study on gold, silver and [email protected] Nanoplates.

Mikrochim Acta 2019 10 25;186(11):713. Epub 2019 Oct 25.

College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China.

Inspired by the increasing use of plasmonic gold and silver nanoplates as probes for diverse analytes, the research community often questions which metal nanoplates should be chosen for a given application. A comparative study was performed on the performance and physicochemical properties of three types of metal nanoplates for use in plasmonic detection of Hg(II) ion. Specifically, gold, silver and [email protected] nanoplates were studied. The established amalgamation method integrated into a detection scheme using nanoplates affords a unique yet straightforward signaling and extraction route for selective recognition of Hg(II) ion. Upon transformation of Hg(II) ion to metallic mercury, nanoplate amalgamation takes place instantly. This reshapes both the morphology and the optical characteristics of nanoplates. It is found that gold and [email protected] nanoplates enable highly selective quantitation of Hg(II) ion by using a DNA oligomer consisting of poly-deoxycytidine (poly(C)) as a masking agent against Ag(I) ion. The silver nanoplates, in turn, display the best sensitivity owing to the chemical instability. The induced surface plasmonic shifts (of up to 250 nm and color changes from red to green) allows for determination of Hg(II) over a wide range and with a limit of detection of ~10 nM. It is recommended that the gold and [email protected] nanoplates are used in relatively complex systems, while silver nanoplates are suited for simple matrices. Graphic abstract The amalgamation process integrated with metal (e.g., Au, Ag and [email protected]) nanoplates affords plasmonic detection of Hg(II) ion with the aid of a poly(c) DNA sequence as the masking agent for Ag(I) ion.
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http://dx.doi.org/10.1007/s00604-019-3873-zDOI Listing
October 2019

Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis.

Chembiochem 2020 03 5;21(6):785-788. Epub 2019 Dec 5.

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, P. R. China.

High-resolution melting (HRM) analysis has been improved and applied for the first time to quantitative analysis of enzymatic reactions. By using the relative ratios of peak intensities of substrates and products, the quantitativity of conventional HRM analysis has been improved to allow detailed kinetic analysis. As an example, the ligation of sticky ends through the action of T4 DNA ligase has been kinetically analyzed, with comprehensive data on substrate specificity and other properties having been obtained. For the first time, the kinetic parameters (k and apparent K ) of sticky-end ligation were obtained for both fully matched and mismatched sticky ends. The effect of ATP concentration on sticky-end ligation was also investigated. The improved HRM method should also be applicable to versatile DNA-transforming enzymes, because the only requirement is that the products have T values different enough from the substrates.
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http://dx.doi.org/10.1002/cbic.201900489DOI Listing
March 2020

Efficient Preparation of Large-Sized Rings of Single-Stranded DNA through One-Pot Ligation of Multiple Fragments.

Chem Asian J 2019 Oct 22;14(19):3251-3254. Epub 2019 Aug 22.

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China.

Circular single-stranded DNA (c-ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c-ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one-pot ligation method to efficiently prepare large c-ssDNA. By ligating several short fragments of linear single-stranded DNA (l-ssDNA) in one-pot by using T4 DNA ligase, longer l-ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large-sized single-stranded c-ssDNAs (e.g., 200-nt) were successfully synthesized in high selectivity and yield.
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http://dx.doi.org/10.1002/asia.201900963DOI Listing
October 2019

The staining efficiency of cyanine dyes for single-stranded DNA is enormously dependent on nucleotide composition.

Electrophoresis 2019 07 29;40(12-13):1708-1714. Epub 2019 Apr 29.

College of Food Science and Engineering, Ocean University of China, Qingdao, P. R. China.

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson-Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.
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http://dx.doi.org/10.1002/elps.201800445DOI Listing
July 2019

Isothermal double-cycle catalytic system using DNAzyme and RNase H for the highly selective one-pot detection of oligonucleotides.

Analyst 2019 Apr;144(8):2773-2779

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, P. R. China.

With the use of a double-cycle system involving two catalytic reactions by RNase H and DNAzyme, the signal of oligoDNAs has been specifically amplified in an isothermal mode. The precursor of DNAzyme was introduced to the system as a ring-structured and inactivated form, which involves the 6-nt RNA portion being complementary to target oligoDNA. In the presence of target oligoDNA, the RNA portion forms a DNA/RNA hetero-duplex and is cut by RNase H. This scission converts the precursor to catalytically active DNAzyme, which in turn disconnects the molecular beacon to produce the amplified signal. Because the covalent bonds were disconnected to provide discrete structural changes in both cycles, high sensitivity and specificity are obtained, indicating the strong potential of this double catalytic cycle method for versatile applications.
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http://dx.doi.org/10.1039/c8an02520gDOI Listing
April 2019

Topologically Constrained Formation of Stable Z-DNA from Normal Sequence under Physiological Conditions.

J Am Chem Soc 2019 05 26;141(19):7758-7764. Epub 2019 Apr 26.

College of Food Science and Engineering , Ocean University of China , No. 5 Yushan Road , Qingdao , People's Republic of China.

Z-DNA, a left-handed duplex, has been shown to form in vivo and regulate expression of the corresponding gene. However, its biological roles have not been satisfactorily understood, mainly because Z-DNA is easily converted to the thermodynamically favorable B-DNA. Here we present a new idea to form stable Z-DNA under normal physiological conditions and achieve detailed analysis on its fundamental features. Simply by mixing two complementary minicircles of single-stranded DNA with no chemical modification, the hybridization spontaneously induces topological constraint which twines one-half of the double-stranded DNA into stable Z-DNA. The formation of Z-conformation with high stability has been proved by using circular dichroism spectroscopy, Z-DNA-specific antibody binding assay, nuclease digestion, etc. Even at a concentration of MgCl as low as 0.5 mM, Z-DNA was successfully obtained, avoiding the use of high salt conditions, limited sequences, ancillary additives, or chemical modifications, criteria which have hampered Z-DNA research. The resultant Z-DNA has the potential to be used as a canonical standard sample in Z-DNA research. By using this approach, further developments of Z-DNA science and its applications become highly promising.
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http://dx.doi.org/10.1021/jacs.8b13855DOI Listing
May 2019

Affinity Isolation of Defined Genomic Fragments Cleaved by Nuclease S1-based Artificial Restriction DNA Cutter.

Curr Protoc Nucleic Acid Chem 2019 03 12;76(1):e76. Epub 2019 Feb 12.

Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan.

The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site-specific scission of DNA using a pair of pseudo-complementary peptide nucleic acids (pcPNAs) and S1 nuclease. When conjugated to biotin, one of the pcPNAs can be used to affinity purify the cleavage product. Here we report a detailed protocol to isolate defined kilobase-length DNA fragments that can be applied to plasmid or genomic DNA and is not limited by sequence. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpnc.76DOI Listing
March 2019

Artificial Restriction DNA Cutter Using Nuclease S1 for Site-Selective Scission of Genomic DNA.

Curr Protoc Nucleic Acid Chem 2019 03 5;76(1):e72. Epub 2019 Feb 5.

Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan.

By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpnc.72DOI Listing
March 2019

Two-Holder Strategy for Efficient and Selective Synthesis of Lk 1 ssDNA Catenane.

Molecules 2018 Sep 5;23(9). Epub 2018 Sep 5.

College of Food Science and Engineering, Ocean University of China, Nucleic Acids Chemistry and Biotechnology Laboratory, No. 5 Yushan Road, Shinan-qu, Qingdao 266003, China.

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18⁻46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson⁻Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures.
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http://dx.doi.org/10.3390/molecules23092270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225352PMC
September 2018

Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings.

Nucleic Acids Res 2018 12;46(22):e132

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.

When oligonucleotide bearing a hairpin near either its 3'- or 5'-end was treated with T4 DNA ligase, the intramolecular cyclization dominantly proceeded and its monomeric cyclic ring was obtained in extremely high selectivity. The selectivity was hardly dependent on the concentration of the oligonucleotide, and thus it could be added in one portion to the mixture at the beginning of the reaction. Without the hairpin, however, the formation of polymeric byproducts was dominant under the same conditions. Hairpin-bearing oligonucleotides primarily take the folded form, and the enzymatically reactive species (its open form) is minimal. As the result, the intermolecular reactions are efficiently suppressed due to both thermodynamic and kinetic factors. The 'terminal hairpin strategy' was applicable to large-scale preparation of a variety of DNA rings. The combination of this methodology with 'diluted buffer strategy', developed previously, is still more effective for the purpose. When large amount of l-DNA bearing a terminal hairpin (e.g. 40 μM) was treated in a diluted ligase buffer (0.1× buffer) with T4 DNA ligase, the DNA ring was prepared in 100% selectivity. Even at [l-DNA]0 = 100 μM in 0.1× buffer, the DNA ring was also obtained in pure form, simply by removing tiny quantity of linear byproducts by Exonuclease I.
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http://dx.doi.org/10.1093/nar/gky769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294566PMC
December 2018

Topology- and linking number-controlled synthesis of a closed 3 link chain of single-stranded DNA.

Chem Commun (Camb) 2018 Sep;54(72):10156-10159

College of Food Science and Engineering, Ocean University of China, Nucleic Acids Chemistry and Biotechnology Laboratory, No. 5 Yushan Road, Shinan-qu, Qingdao 266003, China.

In spite of remarkable progress in synthetic methodology, a closed three-link chain (one of the simplest but the most important topological isomers of [3]catenane) has never been prepared. Here we synthesized this isomer in high yield from three oligonucleotides which are designed to optimize various chemical and steric factors in their mutual hybridization.
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http://dx.doi.org/10.1039/c8cc04965cDOI Listing
September 2018

Effects of secondary structures of DNA templates on the quantification of qPCR.

J Biomol Struct Dyn 2019 Jul 18;37(11):2867-2874. Epub 2019 Jan 18.

a College of Food Science and Engineering , Ocean University of China , Qingdao , China.

In the current design of quantitative polymerase chain reaction (qPCR) systems, the sequences of primers are the primary concerns. The secondary structures of DNA templates have not been much considered, although they should be also critically important. In this paper, various hairpins with different stem lengths and loop sizes are placed near primer-binding sites, and their effects on the amplification efficiency of qPCR are systematically investigated. When a hairpin is formed either in the inside of the amplicon or in its outside, the amplification is notably suppressed. The magnitudes of suppression increase with the increase in stem length and the decrease in loop size, and are especially significant for the hairpins formed inside the amplicon. With very long stems (e.g., 20-bp), the effect is still more drastic, and no targeted amplification products are formed. On the basis of melting temperature () measurements, these suppression effects of hairpins have been mostly ascribed to competitive inhibition of primer binding to the template. It has been concluded that, in order to design precise and reliable qPCR systems, at least 60-bp sequences around primer-binding sites, both inside and outside the amplicons, must be analyzed to confirm that stable secondary structures are not formed in the vicinity of primer-binding sites. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2018.1498804DOI Listing
July 2019

Fabrication of aqueous nanodispersion from natural DNA and chitosan as eminent carriers for water-insoluble bioactives.

Int J Biol Macromol 2018 Oct 24;118(Pt A):263-270. Epub 2018 May 24.

College of food Science and Engineering, Ocean University of China, Qingdao 266003, PR China; World Premier International (WPI) Research Centre for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.

For high-valued application of natural DNA as raw materials, we prepared nanocarriers by using salmon sperm DNA and chitosan to encapsulate water-insoluble bioactives. Here, water dispersible astaxanthin/DNA/chitosan nano-aggregates (ADC-NAs) were prepared by co-assemble evaporation method. The key point for preparing well formed ADC-NAs was specifically discussed. The resultant ADC-NAs were spherical with 100-300 nm diameter measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and their homogeneous dispersions were sufficiently stable at room temperature. One important feature of these nanocarriers is enormously high loading amount of cargo (about 40 wt%). According to the UV-Vis spectra of the nanosuspension, we deduced that astaxanthin was encapsulated as uniquely structured J-aggregates. Fourier transform infra-red (FTIR) spectroscopy proved fabrication was successfully and astaxanthin was embedding in DNA/chitosan nanocarriers. Cytotoxicity was examined in vitro using cell culture in L929 cell lines. When necessary, these nano-aggregates can be degraded by DNase I. Homogeneous dispersions of other non-charged guest molecules are also prepared by using DNA/chitosan nanocarriers. These dispersions are cheaply and easily obtainable from naturally occurring DNA and chitosan, and should be useful for versatile applications.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.05.054DOI Listing
October 2018

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination.

Biochemistry 2018 05 10;57(20):2908-2912. Epub 2018 May 10.

Life Science Center of Tsukuba Advanced Research Alliance , University of Tsukuba , 1-1-1 Tennoudai , Tsukuba , Ibaraki 305-8577 , Japan.

Scission of the human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, in proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel one-pot method for the site-specific scission of DNA by using a biotinylated pcPNA/S1 nuclease combination and isolation of a desired fragment by streptavidin-coated magnetic beads. The proof of concept was initially demonstrated for the clipping of plasmid DNA and isolation of the required fragment. Our method was then successfully applied for the isolation of a fragment from the cell-derived human genome.
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http://dx.doi.org/10.1021/acs.biochem.8b00202DOI Listing
May 2018

Inhibition of nonenzymatic depurination of nucleic acids by polycations.

FEBS Open Bio 2017 11 22;7(11):1707-1714. Epub 2017 Sep 22.

College of Food Science and Engineering Ocean University of China Qingdao China.

DNA base depurination is one of the most common forms of DNA damage and , and the suppression of depurination is very important for versatile applications of DNA in biotechnology and medicine. In this work, it was shown that the polycations chitosan (Cho) and spermine (Spm) strongly inhibit DNA depurination through the formation of polyion complexes with DNA molecules. The intramolecular electrostatic interaction of positively charged polycations with DNA efficiently suppresses the protonation of purine groups, which is the key step of depurination. Importantly, the optimal pH for Cho's inhibition of depurination is significantly different from that of Spm. Cho is very effective in the inhibition of depurination in highly acidic media (pH: 1.5-3), whereas Spm is found to suppress the chemical reaction near neutral pH, as well as in acidic solutions. This remarkable pH specificity of the two biorelevant polycations is attributed to the difference in the values of the amino groups. The relevance of our results with the biological roles of biogenic polycations is also discussed.
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http://dx.doi.org/10.1002/2211-5463.12308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666391PMC
November 2017

Applications of PNA-Based Artificial Restriction DNA Cutters.

Molecules 2017 Sep 21;22(10). Epub 2017 Sep 21.

World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.

More than ten years ago, artificial restriction DNA cutters were developed by combining two pseudo-complementary peptide nucleic acid (pcPNA) strands with either Ce(IV)/EDTA or S1 nuclease. They have remarkably high site-specificity and can cut only one predetermined site in the human genome. In this article, recent progress of these man-made tools have been reviewed. By cutting the human genome site-selectively, desired fragments can be clipped from either the termini of chromosomes (telomeres) or from the middle of genome. These fragments should provide important information on the biological functions of complicated genome system. DNA/RNA hybrid duplexes, which are formed in living cells, are also site-selectively hydrolyzed by these cutters. In order to further facilitate the applications of the artificial DNA cutters, various chemical modifications have been attempted. One of the most important successes is preparation of PNA derivatives which can form double-duplex invasion complex even under high salt conditions. This is important for in vivo applications, since the inside of living cells is abundant of metal ions. Furthermore, site-selective DNA cutters which require only one PNA strand, in place of a pair of pcPNA strands, are developed. This progress has opened a way to new fields of PNA-based biochemistry and biotechnology.
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http://dx.doi.org/10.3390/molecules22101586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151779PMC
September 2017

Site-Selective RNA Activation by Acridine-Modified Oligodeoxynucleotides in Metal-Ion Catalyzed Hydrolysis: A Comprehensive Study.

ACS Omega 2017 Sep 1;2(9):5370-5377. Epub 2017 Sep 1.

International Center for Materials Nanoarchitechtonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.

Various types of acridine were conjugated to DNA and used for site-selective RNA scission together with another unmodified DNA and a Lu(III) ion. The target phosphodiester linkage in the substrate RNA was selectively and efficiently activated, and was hydrolyzed by the free Lu(III) ion. Among the investigated 14 conjugates, the conjugate bearing 9-amino-2-isopropoxy-6-nitroacridine was the best RNA-activator. Systematic evaluation of the RNA-activating ability of the acridines showed that (1) the acridines act as an acid catalyst within the RNA activation, (2) the amino-group at the 9-position of acridine is essential to modulate the acidity of acridine, (3) the electron-withdrawing group at the 3-position further enhances the acid catalysis, and (4) the substituent at the 2-position sterically modulates the orientation of acridine-intercalation favorably for the catalysis. Moreover, it is revealed that the opposite base of acridine does not inhibit direct interaction of acridine with the target phosphodiester linkage.
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http://dx.doi.org/10.1021/acsomega.7b00966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644747PMC
September 2017

Suppression of Myogenic Differentiation of Mammalian Cells Caused by Fluidity of a Liquid-Liquid Interface.

ACS Appl Mater Interfaces 2017 Sep 1;9(36):30553-30560. Epub 2017 Sep 1.

International Research Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS) , 1-1, Namiki, Tsukuba, Ibaraki 305-0044, Japan.

There is growing evidence to suggest that the prevailing physical microenvironment and mechanical stress regulate cellular functions, including adhesion, proliferation, and differentiation. Moreover, the physical microenvironment determines the stem-cell lineage depending on stiffness of the substrate relative to biological tissues as well as the stress relaxation properties of the viscoelastic substrates used for cell culture. However, there is little known regarding the biological effects of a fluid substrate, where viscoelastic stress is essentially absent. Here, we demonstrate the regulation of myogenic differentiation on fluid substrates by using a liquid-liquid interface as a scaffold. C2C12 myoblast cells were cultured using water-perfluorocarbon (PFC) interfaces as the fluid microenvironment. We found that, for controlled in vitro culture at water-PFC interfaces, expression of myogenin, myogenic regulatory factors (MRF) family gene, is remarkably attenuated even when myogenic differentiation was induced by reducing levels of growth factors, although MyoD was expressed at the usual level (MyoD up-regulates myogenin under an elastic and/or viscoelastic environment). These results strongly suggest that this unique regulation of myogenic differentiation can be attributed to the fluid microenvironment of the interfacial culture medium. This interfacial culture system represents a powerful tool for investigation of the mechanisms by which physical properties regulate cellular adhesion and proliferation as well as their differentiation. Furthermore, we successfully transferred the cells cultured at such interfaces using Langmuir-Blodgett (LB) techniques. The combination of the interfacial culture system with the LB approach enables investigation of the effects of mechanical compression on cell functions.
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http://dx.doi.org/10.1021/acsami.7b11445DOI Listing
September 2017

Allosteric control of nanomechanical DNA origami pinching devices for enhanced target binding.

Chem Commun (Camb) 2017 Jul;53(59):8276-8279

Department of Chemistry and Materials Engineering, Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan.

Significant enhancement of single-molecular binding to a miRNA target and bidentate and asymmetric conjugation of two distinct thiolated DNA strands to single gold nanoparticles (AuNPs) were visibly demonstrated, by introducing two groups of ligands into our nanomechanical DNA origami devices (DNA pliers) to construct allosterically controllable systems.
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http://dx.doi.org/10.1039/c7cc03991cDOI Listing
July 2017

Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration.

Nucleic Acids Res 2017 Sep;45(15):e139

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.

Preparation of large amount of single-stranded circular DNA in high selectivity is crucial for further developments of nanotechnology and other DNA sciences. Herein, a simple but practically useful methodology to prepare DNA rings has been presented. One of the essential factors is to use highly diluted T4 ligase buffer for ligase reactions. This strategy is based on our unexpected finding that, in diluted T4 buffers, intermolecular polymerization of DNA fragments is greatly suppressed with respect to their intramolecular cyclization. This promotion of cyclization is attributable to abnormally low concentration of Mg2+ ion (0.5-1.0 mM) but not ATP in the media for T4 ligase reactions. The second essential factor is to add DNA substrate intermittently to the mixture and maintain its temporal concentration low. By combining these two factors, single-stranded DNA rings of various sizes (31-74 nt) were obtained in high selectivity (89 mol% for 66-nt DNA) and in satisfactorily high productivity (∼0.2 mg/ml). A linear 72-nt DNA was converted to the corresponding DNA ring in nearly 100% selectivity. The superiority of this new method was further substantiated by the fact that small-sized DNA rings (31-42 nt), which were otherwise hardly obtainable, were successfully prepared in reasonable yields.
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http://dx.doi.org/10.1093/nar/gkx553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587803PMC
September 2017

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes.

Int J Anal Chem 2016 8;2016:3216523. Epub 2016 Jun 8.

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan; Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan.

Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear Tb(III) complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the Tb(III) ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these Tb(III) complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.
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http://dx.doi.org/10.1155/2016/3216523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916314PMC
July 2016

Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination.

Nucleosides Nucleotides Nucleic Acids 2016 May 8;35(5):233-44. Epub 2016 Apr 8.

a University of Tsukuba , Tsukuba , Japan.

By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.
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http://dx.doi.org/10.1080/15257770.2015.1131294DOI Listing
May 2016

Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.

Authors:
Makoto Komiyama

Artif DNA PNA XNA 2014 Dec;5(3):e1112457

a Life Science Center of Tsukuba Advanced Research Alliance; University of Tsukuba ; Tsukuba , Ibaraki , Japan.

Recently, completely chemistry-based tools for site-selective scission of DNA (ARCUT) have been prepared by combining 2 strands of pseudo-complementary PNA (pcPNA: site-selective activator) and a Ce(IV)-EDTA complex (molecular scissors). Its site-specificity is sufficient to cut the whole human genome at one predetermined site. In this first-generation ARCUT, however, there still remain several problems to be solved for wider applications. This review presents recent approaches to solve these problems. They are divided into (i) covalent modification of pcPNA with other functional groups and (ii) new strategies using conventional PNA, in place of pcPNA, as site-selective activator. Among various chemical modifications, conjugation with positively-charged nuclear localization signal peptide is especially effective. Furthermore, unimolecular activators, a single strand of which successfully activates the target site in DNA for site-selective scission, have been also developed. As the result of these modifications, the site-selective scission by Ce(IV)-EDTA was achieved promptly even under high salt conditions which are otherwise unfavourable for double-duplex invasion. Furthermore, it has been shown that "molecular crowding effect," which characterizes the inside of living cells, enormously promotes the invasion, and thus the invasion seems to proceed effectively and spontaneously in the cells. Strong potential of pcPNA for further applications in vivo and in vitro has been confirmed.
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http://dx.doi.org/10.1080/1949095X.2015.1112457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5329899PMC
December 2014

Baseline Correction of Diffuse Reflection Near-Infrared Spectra Using Searching Region Standard Normal Variate (SRSNV).

Appl Spectrosc 2015 Dec;69(12):1432-41

An alternative baseline correction method for diffuse reflection near-infrared (NIR) spectra, searching region standard normal variate (SRSNV), was proposed. Standard normal variate (SNV) is an effective pretreatment method for baseline correction of diffuse reflection NIR spectra of powder and granular samples; however, its baseline correction performance depends on the NIR region used for SNV calculation. To search for an optimal NIR region for baseline correction using SNV, SRSNV employs moving window partial least squares regression (MWPLSR), and an optimal NIR region is identified based on the root mean square error (RMSE) of cross-validation of the partial least squares regression (PLSR) models with the first latent variable (LV). The performance of SRSNV was evaluated using diffuse reflection NIR spectra of mixture samples consisting of wheat flour and granular glucose (0-100% glucose at 5% intervals). From the obtained NIR spectra of the mixture in the 10 000-4000 cm(-1) region at 4 cm intervals (1501 spectral channels), a series of spectral windows consisting of 80 spectral channels was constructed, and then SNV spectra were calculated for each spectral window. Using these SNV spectra, a series of PLSR models with the first LV for glucose concentration was built. A plot of RMSE versus the spectral window position obtained using the PLSR models revealed that the 8680–8364 cm(-1) region was optimal for baseline correction using SNV. In the SNV spectra calculated using the 8680–8364 cm(-1) region (SRSNV spectra), a remarkable relative intensity change between a band due to wheat flour at 8500 cm(-1) and that due to glucose at 8364 cm(-1) was observed owing to successful baseline correction using SNV. A PLSR model with the first LV based on the SRSNV spectra yielded a determination coefficient (R2) of 0.999 and an RMSE of 0.70%, while a PLSR model with three LVs based on SNV spectra calculated in the full spectral region gave an R2 of 0.995 and an RMSE of 2.29%. Additional evaluation of SRSNV was carried out using diffuse reflection NIR spectra of marzipan and corn samples, and PLSR models based on SRSNV spectra showed good prediction results. These evaluation results indicate that SRSNV is effective in baseline correction of diffuse reflection NIR spectra and provides regression models with good prediction accuracy.
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http://dx.doi.org/10.1366/15-07905DOI Listing
December 2015

Clipping of Telomere from Human Chromosomes Using a Chemistry-Based Artificial Restriction DNA Cutter.

Curr Protoc Nucleic Acid Chem 2015 Jun 2;61:6.13.1-6.13.13. Epub 2015 Jun 2.

Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan.

The detection of individual telomere lengths of human chromosomes can provide crucial information on genome stability, cancer, and telomere-related diseases. However, current methods to measure telomere length entail shortcomings that have limited their use. Recently, we have developed a method for detection of individual telomere lengths (DITL) that uses a chemistry-based DNA-cutting approach. The most beneficial feature of the DITL approach is to cleave the sequence adjacent to the telomere followed by resolution of the telomere length at the nucleotide level of a single chromosome. In this unit, a protocol for successful detection of individual telomere lengths from individual chromosomes is described in detail.
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http://dx.doi.org/10.1002/0471142700.nc0613s61DOI Listing
June 2015