"External chromophores" and intensive bands in circular dichroism spectra of liquid-crystalline microphases of nucleic acids

From the analysis of CD-spectra of liquid crystalline microphases formed from DNA molecules in complexes with "external chromophores" requirements are formulated for the compounds to be used in revealing peculiarities of spatial structure of nucleic acids liquid crystalline microphases. These requirements satisfied it is possible to record the CD-spectra having intensive bands in the regions of chromophores absorption. These bands prove the helical pattern of the spatial structure of nucleic acid liquid crystalline microphase.     

Two modes of longe-range orientation of DNA bases realized upon compaction.

Formation of compact particles from linear DNA-anthracycline complexes is accompanied by appearance of intense bands in the CD spectra in the region of absorption of DNA bases (UV-region) and in the region of absorption of anthracycline chromophores (visible region). The intense (positive or negative) bands in the region of anthracycline absorption demonstrate an ordered helical location of anthracycline molecules on the DNA template. This fact, in its turn, is related to formation of the DNA superstructure in PEG-containing water-salt solutions with a long-range orientation of nitrogen bases. Possible types of DNA superstructures and the relation between the local- and the long-range order of bases in the DNA superstructure are discussed.     

Reversible covalent modification of DNA

The reaction of bromomethylbenzoyl esters of choline and dimethylaminoethanol with DNA and model compounds led predominantly to phosphotriester formation. In model compounds the phosphotriester formation was verified by uv spectrometry. The bromomethylbenzoyl cationic esters reacted with DNA at room temperature at neutral pH values. The amount of the reagent chromophores was assessed semiquantitatively by spectrophotometry. The maximum binding appeared to be stoichiometric, i.e., one residue per phosphorus. The binding of one mole of reagent per phosphorus was confirmed by electron spectroscopic measurements of the phosphorus atom electron emission of maximally modified DNA. The modified DNA showed altered CD spectra indicating that the reagent chromophores are arranged in an orderly fashion affording a strong (Δ_? > 4), positive, apparently extrinsic CD band at ~240 nm; a double helical array is proposed. The introduced chromophores were readily removed by heat treatment or by treatment with nucleophiles at neutral pH values at moderate temperatures (<37 °C); no measurable fraction of the DNA became dialyzable. A decrease in viscosity accompanied the reversal, indicative of some chain breaking. The modified DNA's show higher T_m values than the native DNA and some display a higher and some a lower degree of cooperativity in their melting curves. No chemically detectable amounts of base alkylation, depurination, or depyrimidination were found when dialyzates of treated DNA and hydrolyzed samples of modified DNA were examined. However, presumptive evidence for some base alkylation by these novel alkylating agents was found utilizing Salmonella typhimurium tester strains sensitive to reversion by alkylation. No comparable binding of benzoylcholine, a nonalkylating analogue, by DNA was seen under conditions utilized here.     

Anomalous optical activity induced during condensation of various spatial forms of double-stranded circular DNA

Formation of dispersed phases from complexes of (closed circular DNA (c.c. DNA)--antibiotics (drugs) in PEG-containing solutions has been studied. It is shown that under definite concentrations of bound antibiotics relatively intense bands in the CD spectra of dispersed phases in the absorption region of DNA and antibiotics chromophores appear. The properties of liquid crystalline phases formed from the complexes of linear DNA with antibiotics were compared to those of dispersed phases formed from c. c. DNA. Such comparison demonstrates existence of some differences in the optical properties of the phases formed from linear and c.c. DNA molecules. For example a change of the bands sign in the CD spectra of dispersed phases formed from the complexes (c.c. DNA--antibiotics), which is the case with all the substances studied, does not exist in the case of liquid crystalline phases formed from the complexes of (linear DNA--antibiotics). It was shown that a change of the bands sign in the CD spectra correlates with a change of the sign of superhelical twist of closed circular DNA molecules.     

Irreversible disassembly of chiral macrodomains in thylakoids due to photoinhibition

We investigated the effect of photoinhibitory illumination on the chiral macroorganization of the chromophores in spinach thylakoid membranes. By measuring circular dichroism (CD), we found that prolonged (15 min) illumination of membranes with intense white light led to irreversible diminishment of the main CD bands originating from the chiral macroorganization of the chromophores. The irreversible decrease of the main CD bands showed a nearly linear correlation with the extent of photoinhibition which was determined by chlorophyll fluorescence induction. CD measurements also revealed that the excitonic CD bands, which are given rise by short-range interactions between the chromophores inside the complexes or particles, were largely insensitive to the photoinhibitory illumination of the membranes. These data show that, whereas photoinhibitory treatment has no perceptible effect on the molecular architecture of the bulk of the pigment–protein complexes, it leads to a disorganization of their macroarray, and an irreversible disassembly of the chirally organized macrodomains.     

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.     

Isolation of DNA from single microsurgically excised bands of polytene chromosomes of Chironomus

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 m. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).     

Sites of gene activity and of inactive genes in polytene chromosomes of diptera

DNA has been found between bands, in puffs, and in RNA-rich lamellae of bands as dispersed DNA double helices coiled to form secondary helices with a diameter and period of about 400Å. In the bands and heterochromatic regions the secondary helix is further coiled or folded into compact masses. RNA-containing granules are present in interbands and RNA-rich lamellae as well as in puffs. The dispersed DNA of puffs, interbands and the RNA-rich lamellae of bands may all represent the part of the genome that is active in the salivary gland nucleus, while the compact DNA of bands as well as heterochromatic parts may comprise inactive genes.     

Use of Oligonucleotide Conjugates in Creating Self-Assembling Supramolecular Systems

Abstract

The chemistry of two types of oligonucleotide conjugates containing novel chromophores are described. One, containing a stilbenedicarboxamide bridge, generates unusually stable hairpin structures that are useful in assessing rates of electron transfer through the π system of a DNA double helix. The other, containing gold nanoparticle conjugates, provides a highly selective system for detecting nucleotide sequences in oligonucleotides.     

Intramolecular DNA melting between stable helical segments: melting theory and metastable states.

Melting of DNA in a segment bounded at both ends by regions of greater stability during electrophoresis in denaturing gradient gels show complex properties, not accommodated with standard melting theory. Compact bands of some DNA molecules become anomalously broadened at the retardation level in a denaturing gradient, or double bands may appear in a uniform denaturant concentration. These properties are associated only with molecules for which the distribution of stability calculated by the Poland-Fixman-Freire algorithms indicates that the region of lowest stability does not extend to an end of the molecule. Retention of helicity at the ends is shown by the difference in the effect of base substitution in the end domains and in the least stable domain. Both the appearance of double bands and band broadening can be explained by invoking a hypothetical metastable intermediate in melting, which is converted into the equilibrium melted form at a relatively slow rate, depending on both denaturant concentration and field strength. A kinetic model permits plausible rate constants to be inferred from the patterns. Despite the increased band width, sequence variants with base changes in the least stable domain result in readily detectable band shifts in the gradient.     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.     

别藻蓝蛋白藻蓝胆素发色团分子构象研究

主要研究了蓝绿藻污棕席藻(Phormidium luridum)别藻蓝蛋白在不同 pH值条件下的吸收光谱和共振拉曼光谱.发现低聚化的结果导致了三聚体别藻蓝蛋白 650nm 特征吸收峰的消失和一些共振拉曼带强度和位置的移动.结果表明在低 pH 值作用下的低聚化的别藻蓝蛋白中藻蓝胆素发色团分子的构象和自由胆素分子类似,比三聚体的别藻蓝蛋白的发色团分子更趋于卷曲,折叠的构象态.而三聚体的别藻蓝蛋白,主要的拉曼带 1645cm^-1是其发色团分子构象处于更线性延展的标志,其光谱行为和吸收光谱 A_vis/A_uv所表征的发色团分子构象的结果相一致.     

Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26.

Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.     

Design and synthesis of efficient fluorescent dyes for incorporation into DNA backbone and biomolecule detection

We report here the design and synthesis of a series of pi-conjugated fluorescent dyes with D-A-D (D, donor; A, acceptor), D-pi-D, A-pi-A, and D-pi-A for applications as the signaling motif in biological-synthetic hybrid foldamers for DNA detection. The Horner-Wadsworth-Emmons (HWE) reaction and Knoevenagel condensation were demonstrated as the optimum ways for construction of long pi-conjugated systems. Such rodlike chromophores have distinct advantages, as their fluorescence properties are not quenched by the presence of DNA. To be incorporated into the backbone of DNA, the chromophores need to be reasonably soluble in organic solvent for solid-phase synthesis, and therefore a strategy of using flexible tetraethylene glycol (TEG) linkers at either end of these rodlike dyes was developed. The presence of TEG facilitates the protection of the chain-growing hydroxyl group with DMTrCl (dimethoxytrityl chloride) as well as the activation of the coupling step with phosphoramidite chemistry on an automated DNA synthesizer. To form fluorescence resonance energy transfer (FRET) pairs, six synthetic chromophores with blue to red fluorescence have been developed, and those with orthogonal fluorescent emission were chosen for incorporation into DNA-chromophore hybrid foldamers.     

Enhancement of fluorescence quenching and exciplex formation in DNA major groove by double incorporation of modified fluorescent deoxyuridines

5-(1-Naphthalenylethynyl)-2'-deoxyuridine ((N)U) and 5-[(4-cyano-1-naphthalenyl)ethynyl]-2'-deoxyuridine ((CN)U) were synthesized and incorporated into oligodeoxynucleotides. Fluorescence emissions of modified duplexes containing double (N)U were efficiently quenched depending upon the sequence pattern of the naphthalenes in DNA major groove, as compared to the duplex possessing single (N)U. When one of the naphthalene moieties has a cyano substituent, the exciplex emission from the chromophores in DNA major groove was observed at longer wavelength.     

Is DNA really a double helix?

Do the two chains of the DNA molecule coil round one another plectonemically ? If so, what is the approximate value of Lk (the linking number) for any closed, circular DNA molecule? Experiments using gel electrophoresis have shown that supercoiled DNA molecules usually migrate in a series of discrete bands. The only tenable explanation for this quantized behavior is that the molecules in one band all have the same value of Lk and that this value differs by unity from that of the adjacent bands. Various experiments in which circular DNA is unwound by known amounts show that (given this assumption) Lk for relaxed DNA is very roughly equal to $\text{N}\text{10}$ (where N is the number of base-pairs), as expected from the classical double helix.The original model for the double helix was right-handed. The experimental evidence for this feature is suggestive but not yet completely compelling.     

Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy.

The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments.     

油桐尺蠖核型多角体病毒（羊楼洞株）基因组特性

用多种限制性内切酶单酶切、双酶切分析了油桐尺蠖核型多角体病毒（羊楼洞株）基因组DNA,同时用[α－32p）－dATP对几种酶的酶切产物进行末端标记。结果表明,此株病毒基因组约129kb,组成比较单一。与国内其它分离株基因组大小及酶切电泳图谱均有较大差别。     

Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties.

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detection of various targets. The chromophores in these dyes, a thiazole orange-thiazole blue heterodimer (TOTAB), two different thiazole orange-ethidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium heterodimer (FED), are in each case linked through polymethyleneamine linkers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 614 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no sequence selectivity. In comparison with direct 488 nm excitation of the constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DNA-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.     

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.