A Nanochannel Platform for Single DNA Studies: From Crowding,Protein DNA Interaction,to Sequencing of Genomic Information

The study of nanochannel-confined DNA is important from biotechnological and biophysical points of view. We produce nanochannels in elastomer with soft lithography and proton beam writing. Issues concerning DNA confined in such quasi one-dimensional channels are discussed. We describe DNA stretching via the control of channel diameter and buffer conditions and how the extension can be interpreted with theory and computer simulation. We then discuss the conformation of nano-confined DNA crowded by neutral polymers and like-charged proteins. As an example of a protein that has an affinity to DNA, the effect of heat-stable nucleoid-structuring protein, H-NS, on the folding and compaction of DNA is reviewed. Compaction of DNA by eukaryotic protamine and unpacking of pre-compacted DNA through an increase in salt concentration are discussed. We review results obtained with a novel, cross-channel device that allows the monitoring of the dynamic, conformational response of DNA after exposure to a ligand or protein and/or a change in buffer conditions in situ. As a biotechnological application, linearization of DNA by bottlebrush coating with a polypeptide copolymer is discussed. It is demonstrated that large-scale genomic organization can be sequenced using single DNA molecules on an array of elastomeric nanochannels. Overall, our results show that the effects of ligands and proteins on the conformation, folding, and condensation of DNA are not only related to classical controlling factors, such as osmotic pressure, charge, and binding, but that the interplay with confinement in a nanospace is of paramount importance.     

Genetic Analysis of Hexaploid Wheat (Triticum aestivum L.) Using the Complete Sequencing of Chloroplast DNA and Haplotype Analysis of the Wknox1 Gene

The aim of the presented study is a genetic characterization of the hexaploid wheat Triticum aestivum L. Two approaches were used for the genealogical study of hexaploid wheats—the complete sequencing of chloroplast DNA and PCR-based haplotype analysis of the fourth intron of Wknox1d and of the fifth-to-sixth-exon region of Wknox1b. The complete chloroplast DNA sequences of 13 hexaploid wheat samples were determined: Free-threshing—T. aestivum subsp. aestivum, one sample; T. aestivum subsp. compactum, two samples; T. aestivum subsp. sphaerococcum, one sample; T. aestivum subsp. carthlicoides, four samples. Hulled—T. aestivum subsp. spelta, three samples; T. aestivum subsp. vavilovii jakubz., two samples. The comparative analysis of complete cpDNA sequences of 20 hexaploid wheat samples (13 samples in this article plus 7 samples sequenced in this laboratory in 2018) was carried out. PCR-based haplotype analysis of the fourth intron of Wknox1d and of the fifth-to-sixth exon region of Wknox1b of all 20 hexaploid wheat samples was carried out. The 20 hexaploid wheat samples (13 samples in this article plus 7 samples in 2018) can be divided into two groups—T. aestivum subsp. spelta, three samples and T. aestivum subsp. vavilovii collected in Armenia, and the remaining 16 samples, including T. aestivum subsp. vavilovii collected in Europe (Sweden). If we take the cpDNA of Chinese Spring as a reference, 25 SNPs can be identified. Furthermore, 13–14 SNPs can be identified in T. aestivum subsp. spelta and subsp. vavilovii (Vav1). In the other samples up to 11 SNPs were detected. 22 SNPs are found in the intergenic regions, 2 found in introns, and 10 SNPs were found in the genes, of which seven are synonymous. PCR-based haplotype analysis of the fourth intron of Wknox1d and the fifth-to-sixth-exon region of Wknox1b provides an opportunity to make an assumption that hexaploid wheats T. aestivum subsp. macha var. palaeocolchicum and var. letshckumicum differ from other macha samples by the absence of a 42 bp insertion in the fourth intron of Wknox1d. One possible explanation for this observation would be that two Aegilops tauschii Coss. (A) and (B) participated in the formation of hexaploids through the D genome: Ae. tauschii (A)—macha (1–5, 7, 8, 10–12), and Ae. tauschii (B)—macha M6, M9, T. aestivum subsp. aestivum cv. ‘Chinese Spring’ and cv. ‘Red Doly’.     

Sequence-specific methyltransferase-induced labeling of DNA (SMILing DNA)

A new concept for sequence-specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S-adenosyl-L-methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase-catalyzed sequence-specific coupling with DNA. Sequence-specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.     

Preparation of monomethyl poly(ethylene glycol)-g-chitosan copolymers with various degrees of substitution: Their ability to encapsulate and condense plasmid DNA

Chitosan (CS) has great potential as a nonvirus gene delivery vector, but its application is limited because of poor water solubility. Monomethyl poly(ethylene glycol) (mPEG)-graft-CS copolymers were synthesized by the reaction of mPEG–aldehyde (oxidized mPEG) with amino groups on CS chains; they showed enhanced solubility in water. Copolymers with various mPEG degrees of substitution (DS) and CS molecular weights were obtained, and their capabilities of DNA encapsulation were compared through gel retardation assay and particle size and ζ potential measurements. The effects of different ratios of primary amines on CS to the phosphate groups on DNA (N/P ratios), DS, and molecular weights on particle size and encapsulation efficiency were investigated. The results show that high N/P ratios and proper DS were necessary for the formation of well-distributed complex particles. Among all of these samples, mPEG (3.55)–CS (50 kDa)/DNA complexes [where the parentheses following mPEG indicate DS (%), and the parentheses following CS indicate the molecular weight of CS] raised the ζ potential from negative to positive most quickly, yielded the smallest particle size, and were retarded in agarose gel at the lowest N/P ratio; this indicated the best efficiency of DNA encapsulation. On the contrary, mPEG (0.80)–CS (50 kDa)/DNA complexes raised the ζ potential to positive most slowly, fluctuated around the value 0 from N/P ratios of 15 : 1 to 30 : 1, and were retarded in agarose gel at the highest N/P ratio; this indicated the lowest efficiency of encapsulating plasmids. Copolymers with desirable efficiencies of DNA encapsulation could be promising gene carriers. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

DNA promotes the activation of oxygen molecules by binuclear cobalt(II) compounds

We have observed that binuclear cobalt(II) compounds with H(HPTP), Co₂(HPTP)(CH₃COO)(ClO₄)₂ and Co₂(HPTP)Cl(ClO₄)₂, react with oxygen molecules when DNA is present in the solution, cleaving the DNA (H(HPTP) denotes N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,3-diaminopropane-2- ol).     

Incorporation of 2'-deoxy-2'-isonucleoside 5'-triphosphates (iNTPs) into DNA by A- and B-family DNA polymerases with different recognition mechanisms

Recently, α-L-threofuranosyl nucleoside 3'-triphosphates (tNTPs) have been reported to be incorporated into DNA by DNA polymerases. Isonucleosides especially the 2'-deoxy-2'-isonucleosides, would be considered regioisomers of α-L-threofuranosyl nucleosides. Therefore, we investigated the synthesis of 2'-deoxy-2'-isonucleoside 5'-triphosphates (iNTPs) having the four natural nucleobases and their incorporation into primer-template duplexes consisting of oligonucleotides containing natural 2'-deoxyribonucleosides and 2'-deoxy-2'-isonucleosides by using primer-extension reactions. We found that Klenow fragment (exo-; an A-family DNA polymerase) has strict recognition of the shape of nucleoside 5'-triphosphates and Therminator (a B-family DNA polymerase) has strict recognition of the shape of primer-template complexes, especially two base pairs upstream of the primer 3' terminus.     

Structure‐Specific Recognition of Friedreich's Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives

Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)_n repeats form non‐B‐DNA structures, including triple helix H‐DNA and higher‐order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non‐B‐DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)_n binding by triplex‐stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex‐DNA‐cleaving BQQ‐1,10‐phenanthroline (BQQ‐OP) compounds. We also examined the ability of these compounds to act as structural probes for H‐DNA formation within higher‐order structures at pathological frataxin sequences in plasmids. DNA‐complex‐formation analyses with a gel‐mobility‐shift assay and sequence‐specific probing of H‐DNA‐forming (GAA)_n sequences by single‐strand oligonucleotides and triplex‐directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)_n repeats under physiologically relevant conditions.     

A comprehensive study of the interactions between DNA and poly(3,4-ethylenedioxythiophene)

The interaction between poly(3,4-ethylenedioxythiophene), a conducting polymer with excellent electrical and electrochemical properties, and plasmid DNA has been investigated using electrophoresis, UV-visible and CD spectroscopy, and quantum mechanical calculations. Analyses of mixtures with different DNA:polymer mass ratios indicate that, in all cases, interactions form immediately and induce structural alterations in DNA. Furthermore, the existence of interactions between poly(3,4-ethylenedioxythiophene) and specific nucleotides sequences has been evidenced by adding restriction enzymes to the mixtures. In contrast, interactions between DNA and poly(3-methylthiophene), a similar polyheterocyclic conducting polymer but without hydrogen bonding acceptors, are weak or do not exist. These results suggest that, in addition to non-specific electrostatic interactions between the charged phosphate groups of DNA and the positively charged fragments of the conducing polymers, specific hydrogen bonding interactions play a crucial role. The ability of 3,4-ethylenedioxythiophene units to form hydrogen bonds with the methylated analogues of DNA bases has been examined in different environments using MP2/6-31G(d) and MP2/6-311++G(d,p) calculations. Results indicate that, in environments with low polarity, the formed interactions are significantly stronger than those reached by unsubstituted thiophene and similar to those established by pyrrole. However, in polar environments (aqueous solution) 3,4-ethylenedioxythiophene provides stronger interactions with nucleic acids than both thiophene and pyrrole. These theoretical results are fully consistent with experimental observations.     

四环素-铜（Ⅱ）配合物与DNA相互作用的吸收光谱研究

用紫外光谱方法研究了四环素(TC)-Cu(Ⅱ)配合物与DNA的相互作用．吸收光谱研究表明，DNA能与四环素(TC)及Cu(Ⅱ)形成的配合物发生反应，配合物与DNA的作用方式随着配合物类型及DNA浓度的不同而不尽相同：当四环素与铜形成1：1型配合物时，较低浓度的DNA能与配合物以嵌插方式相互作用，而较高浓度的DNA与该配合物除了发生嵌插作用外，还存在另外的作用方式；当四环素与铜形成1：2型配合物时，DNA与该配合物则主要以嵌插方式相互作用，并且这两种配合物与DNA的嵌插作用均是通过四环素配体插入的．     

Site-selective DNA hydrolysis by Ce(IV)-EDTA with the use of one oligonucleotide additive bearing two monophosphates

Two deoxyuridine derivatives each bearing a monophosphate group at the 5-position with a C3 linker, were incorporated into an oligonucleotide. By using this modified oligonucleotide, a bulge was formed at a predetermined position in a DNA substrate, and two monophosphate groups were placed at both junctions of the bulge. Upon treatment of the mixture with Ce(IV)-EDTA at pH 7.0, the phosphodiester linkages at the bulge site were selectively and efficiently hydrolyzed. The monophosphate groups introduced into the bulge site greatly accelerated site-selective DNA scission. Compared with the previously reported two-additive system, which combines two oligonucleotide additives each with a monophosphate at their termini, the present one-additive system is simpler and more convenient. Furthermore, site-selective DNA hydrolysis by using this one-additive system is successful even at high reaction temperatures (e.g., 55 degrees C). This reflects the thermodynamic stability of the duplexes formed between the substrate and the additive DNA.     

Molecular Systematics of Genus Atractylodes (Compositae,Cardueae): Evidence from Internal Transcribed Spacer (ITS) and trnL-F Sequences

To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis.     

Mitochondrial DNA Copy Number and Developmental Origins of Health and Disease (DOHaD)

Mitochondrial dysfunction is known to contribute to mitochondrial diseases, as well as to a variety of aging-based pathologies. Mitochondria have their own genomes (mitochondrial DNA (mtDNA)) and the abnormalities, such as point mutations, deletions, and copy number variations, are involved in mitochondrial dysfunction. In recent years, several epidemiological studies and animal experiments have supported the Developmental Origin of Health and Disease (DOHaD) theory, which states that the environment during fetal life influences the predisposition to disease and the risk of morbidity in adulthood. Mitochondria play a central role in energy production, as well as in various cellular functions, such as apoptosis, lipid metabolism, and calcium metabolism. In terms of the DOHaD theory, mtDNA copy number may be a mediator of health and disease. This paper summarizes the results of recent epidemiological studies on the relationship between environmental factors and mtDNA copy number during pregnancy from the perspective of DOHaD theory. The results of these studies suggest a hypothesis that mtDNA copy number may reflect environmental influences during fetal life and possibly serve as a surrogate marker of health risks in adulthood.     

Efficiency of ITS Sequences for DNA Barcoding in Passiflora (Passifloraceae)

DNA barcoding is a technique for discriminating and identifying species using short, variable, and standardized DNA regions. Here, we tested for the first time the performance of plastid and nuclear regions as DNA barcodes in Passiflora. This genus is a largely variable, with more than 900 species of high ecological, commercial, and ornamental importance. We analyzed 1034 accessions of 222 species representing the four subgenera of Passiflora and evaluated the effectiveness of five plastid regions and three nuclear datasets currently employed as DNA barcodes in plants using barcoding gap, applied similarity-, and tree-based methods. The plastid regions were able to identify less than 45% of species, whereas the nuclear datasets were efficient for more than 50% using “best match” and “best close match” methods of TaxonDNA software. All subgenera presented higher interspecific pairwise distances and did not fully overlap with the intraspecific distance, and similarity-based methods showed better results than tree-based methods. The nuclear ribosomal internal transcribed spacer 1 (ITS1) region presented a higher discrimination power than the other datasets and also showed other desirable characteristics as a DNA barcode for this genus. Therefore, we suggest that this region should be used as a starting point to identify Passiflora species.     

Identification of the Biosynthetic Gene Cluster for Himeic Acid A: A Ubiquitin‐Activating Enzyme (E1) Inhibitor in Aspergillus japonicus MF275

Himeic acid A, which is produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by Agrobacterium‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the him biosynthetic gene cluster for himeic acid in A. japonicus MF275 has been identified.