DNA biosensors based on self-assembled carbon nanotubes

DNA biosensors based on self-assembled multi-walled carbon nanotubes (MWNTs) were described in this paper, in which the probe DNA oligonucleotides were immobilized by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA oligonucleotides. Hybridization between the probe and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of the indicator of methylene blue. Our results demonstrate that the DNA biosensors based on self-assembled MWNTs had a higher hybridization efficiency compared to those based on random MWNTs. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.     

Direct facile screening of recombinant DNA vector constructs

Direct efficient facile screening of bacterial transformants with the goal of selecting, retrieving, and using recombinant DNA is exemplified by simple visual-based colorimetric inspections or fluorescent protein-based assays. We describe pRedScript, which introduces the constitutive expression of a very bright red fluorescent protein into transformants. On agar plates, red colonies are simply visualized in ambient white light in stark contrast to recombinant transformants that are white. In addition, the bright red fluorescence of the reporter protein can also be harnessed as a sensitive signal for screening bacterial promoters during the development of optimized fermentation conditions.     

Diepoxybutane interstrand cross-links induce DNA bending

The bifunctional alkylating agent 1,2,3,4-diepoxybutane (DEB) is thought to be a major contributor to the carcinogenicity of 1,3-butadiene, from which it is derived in vivo. DEB forms DNA interstrand cross-links primarily between distal deoxyguanosine residues at the duplex sequence 5′-GNC. In order for the short butanediol tether to span this distance, distortion of the DNA target has been postulated. We determined that the electrophoretic mobility of ligated DNA oligomers containing DEB cross-links was retarded in comparison with control, uncross-linked DNA. Our data are consistent with DNA bending of ∼34° per lesion towards the major groove.     

The absence of tertiary interactions in a self-assembled DNA crystal structure

DNA is a highly effective molecule for controlling nanometer-scale structure. The convenience of using DNA lies in the programmability of Watson-Crick base-paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky-ended cohesion. Recently, the tensegrity triangle motif has been used to self-assemble three-dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine-PO(4) tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation.     

Synthesis of 2'-amino-LNA purine nucleosides

The first reported synthesis of 2'-amino-LNA purine nucleosides via a transnucleosidation is accomplished enabling the preparation of oligonucleotides incorporating 2'-amino-LNA with all four natural bases.     

Force-induced structural transitions in cross-linked DNA films

We report on the preparation and characterization of wet-spun films of sodium DNA in which intermolecular cross-links were introduced following formaldehyde treatment. Raman scattering shows that the DNA in moderately cross-linked films is mainly in the B conformation. Stretching experiments show a transition from plastic to elastomeric behavior with increasing exposure to the cross-linking agent. Elastomeric DNA films are strongly disordered. X-ray diffraction shows that stretching of moderately cross-linked films under controlled high humidity conditions results in increased molecular orientation as well as the appearance of meridional reflections at 7.4-7.8 and 8.2 A. These reflections are not observed for any of the classical conformations associated with mixed sequence DNA, and may arise from extended base-pair stacking in a stretched DNA structure.     

Solution structure of a DNA duplex containing 8-hydroxy-2'-deoxyguanosine opposite deoxyguanosine

Deoxyguanosine residues are hydroxylated by reactive oxygen species at the C-8 position to form 8-hydroxy-2'-deoxyguanosine (8-OG), one of the most important mutagenic lesions in DNA. Though the spontaneous G:C to C:G transversions are rare events, the pathways leading to this mutation are not established. An 8-OG:G mispair, if not corrected by DNA repair enzymes, could lead to G:C to C:G transversions. NMR spectroscopy and restrained molecular dynamics calculations are used to refine the solution structure of the base mismatch formed by the 8-OG:G pair on a self complementary DNA dodecamer duplex d(CGCGAATT(8-O)GGCG)(2). The results reveal that the 8-OG base is inserted into the helix and forms Hoogsteen base-pairing with the G on the opposite strand. The 8-OG:G base-pairs are seen to be stabilized by two hydrogen bonding interactions, one between the H7 of the 8-OG and the O6 of the G, and a three-center hydrogen bonding between the O8 of the 8-OG and the imino and amino protons of the G. The 8-OG:G base-pairs are very well stacked between the Watson-Crick base-paired flanking bases. Both strands of the DNA duplex adopt right-handed conformations. All of the unmodified bases, including the G at the lesion site, adopt anti glycosidic torsion angles and form Watson-Crick base-pairs. At the lesion site, the 8-OG residues adopt syn conformations. The structural studies demonstrate that 8-OG(syn):G(anti) forms a stable pair in the interior of the duplex, providing a basis for the in vivo incorporation of G opposite 8-OG. Calculated helical parameters and backbone torsional angles, and the observed 31P chemical shifts, indicate that the structure of the duplex is perturbed near lesion sites, with the local unwinding of the double helix. The melting temperature of the 8-OG:G containing duplex is only 2.6 deg. C less than the t(m) of the unmodified duplex.     

A thiol-inducible and quick-response DNA cross-linking agent

Three new 2,4-dinitrobenzenesulfonyl derivatives 1–3 were successfully prepared for the first time using a simple process. They were efficiently triggered by thiols (glutathione and l-cysteine) to release the corresponding phenol derivatives (4–6) within 5?min. The quick response of 1–3 toward thiols was determined by ¹H NMR and HPLC. Moreover, our results indicated that 1 could induce DNA cross-linking in the presence of glutathione, probably due to the quinone methide formation of phenol intermediate 4 followed by departure of 2,4-dinitrobenzenesulfonyl group.     

Temporal assessment of ribose treatment on self-assembled articular cartilage constructs

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties.     

The role of plasmid constructs containing the SV40 DNA nuclear-targeting sequence in cationic lipid-mediated DNA delivery

One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.     

Multinuclear magnetic resonance studies of monomers and dimers containing 2'-fluoro-2'-deoxyadenosine

A series of 2′-fluorinated adenosine compounds, dAfl, dAflp, pdAfl, dAfl-A, A-dAfl, and dAfl-dAfl, have been investigated by nmr spectroscopies. The ¹H-, ¹⁹F-, and ³¹P-nmr data provide structural information from different parts of these moleucles. The pK_a of the phosphate group of these two 2′-fluoro-2′-deoxyadenosine monophosphates was found to be the same as that of hte parent adenosine monophosphate. As for the pentose conformation, the ³E population is greatly increased as a result of the fluorine substitution at the C_2′ position. However, the populations of conformers of gg (C_4′-C_5′) and g′g′ (C_5′-O_5′) and the average angle ?′(C_3′-O_3′) of the 2′-fluoro compounds remain unchanged as compared to the natural riboadenosine monomer and dimer (A-A). Thefefore, the backbone conformation of the 2′-fluoro-2′-deoxy-adenosine, its monophosphates and dimers, resembles that of RNA. The extent of base-base overlapping in these 2′-fluoro-2′-deoxy-adenosine-containing dimers is also found to be similar to or even greater than A-A. Thus, the conformations of these compounds can be considered as those in the RNA family. These fluorocompounds also serve as models for a careful study on the ¹⁹F-nmr in nucleic acid. The ¹⁹F chemical-shift values are sensitive to the environment of the fluorine atom such as ionic structure of the neighboring group(s) (phosphate of base), solvation, and ring-ruccent anisotropic effect from the base(s). Qualitatively, the change of the ¹⁹F chemical-shift values (up to 2 ppm) is much larger than that of ¹H-nmr (up to 0.5 ppm) in the dimers. Using dAfl·poly(U), poly(dAfl)·poly(dAfl), and poly(dAfl)·poly(U) helix–coil transition as model systems, the linewidth of ¹⁹F in dAfl- residues reflects effectively the mobility of the unit in the nucleic acid complex as calibrated by uv data and by ¹H-nmr. Therefore, application of ¹⁹F-nmr spectroscopy on fluorine-substituted nucleic acid can also be used to detect nucleic acid-nucleic acid interaction in complicated systems.     

Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA

Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA.     

Constitutively active Artemis nuclease recognizes structures containing single-stranded DNA configurations

The Artemis nuclease recognizes and endonucleolytically cleaves at single-stranded to double-stranded DNA (ss/dsDNA) boundaries. It is also a key enzyme in the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. Previously, a truncated form, Artemis-413, was developed that is constitutively active both in vitro and in vivo. Here, we use this constitutively active form of Artemis to detect DNA structures with ss/dsDNA boundaries that arise under topological stress. Topoisomerases prevent abnormal levels of torsional stress through modulation of positive and negative supercoiling. We show that overexpression of Artemis-413 in yeast cells carrying genetic mutations that ablate topoisomerase activity have an increased frequency of DNA double-strand breaks (DSBs). Based on the biochemical activity of Artemis, this suggests an increase in ss/dsDNA-containing structures upon increased torsional stress, with DSBs arising due to Artemis cutting at these ss/dsDNA structures. Camptothecin targets topoisomerase IB (Top1), and cells treated with camptothecin show increased DSBs. We find that expression of Artemis-413 in camptothecin-treated cells leads to a reduction in DSBs, the opposite of what we find with topoisomerase genetic mutations. This contrast between outcomes not only confirms that topoisomerase mutation and topoisomerase poisoning have distinct effects on cells, but also demonstrates the usefulness of Artemis-413 to study changes in DNA structure.     

Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

The results presented in this report demonstrate that an 18–20 hour exposure/³H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin Bi and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18–20 hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18–20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10^–3M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 × 10^–4M when the 18–20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 × 10^–5M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10^–5M when the 18–20 hour exposure/labeling period was used.Abbreviations 2AAF 2-acetylaminofluorene - AB₁ aflatoxin B₁ - BENZ benzidine - DEB diepoxybutane - DEN diethylnitrosamine - DMN dimethylnitrosamine - EMS ethyl methanesulfonate - MITC mitomycin C - MMS methyl methanesulfonate - NG mean net nuclear grain counts - NM nitrogen mustard - 4NQO 4-nitroquinoline-N-oxide     

Design,synthesis, and characterization of nucleosomes containing site-specific DNA damage

How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.     

Isolation and analysis of recombinant DNA molecules containing yeast DNA.

2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Escherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.     

Synthesis, photophysical characterisation and metal ion binding properties of new ligands containing anthracene chromophores

Two new fluorescent chemosensors for heavy metal ions have been synthesised and their photophysical properties have been investigated. They present a pyridyl-thioether-based binding site and the anthracene moiety as a chromophore. In the experimental conditions used, no evidence is found for the formation of complexes with Pb²⁺, Zn²⁺, Cd²⁺, and Ag⁺ ions. On the contrary, in acetonitrile solutions both ligands strongly bind Cu²⁺ and Hg²⁺ cations according to a 1:1 and a 1:2 (metal:ligand) stoichiometry. In these complexes, the intense luminescence typical of anthracene derivatives is almost completely quenched and this phenomenon can be mainly attributed to an intraligand electron transfer process from the anthracene chromophore to the complexed pyridine. These results are of interest for the development of new chemosensors for the design of efficient electronic tongues for the detection of transition metal ions.     

OLFinder-a program which disentangles DNA sequences containing heterozygous indels

The presence of heterozygous indels in a DNA sequence usually results in the sequence being discarded. If the sequence trace is of high enough quality, however, it will contain enough information to reconstruct the two constituent sequences with very little ambiguity. Solutions already exist using comparisons with a known reference sequence, but this is often unavailable for nonmodel organisms or novel DNA regions. I present a program which determines the sizes and positions of heterozygous indels in a DNA sequence and reconstructs the two constituent haploid sequences. No external data such as a reference sequence or other prior knowledge are required. Simulation suggests an accuracy of >99% from a single read, with errors being eliminable by the inclusion of a second sequencing read, such as one using a reverse primer. Diploid sequences can be fully reconstructed across any number of heterozygous indels, with two overlapping sequencing reads almost always sufficient to infer the entire DNA sequence. This eliminates the need for costly and laborious cloning, and allows data to be used which would otherwise be discarded. With no more laboratory work than is needed to produce two normal sequencing reads, two aligned haploid sequences can be produced quickly and accurately and with extensive phasing information.     

Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.