Denatured DNA Molecules Research Articles

Thermoresponsive DNA hydrogels through hydrophilic pre-polymer cross-linker and self-assembly

Denatured DNA molecules were used as building blocks to fabricate thermoresponsive hydrogels by cross-linking DNA strands with an isocyanate (NCO) end-capped star-shaped poly(EO-stat-PO) cross-linker. The NCO groups reacted with the amine groups of DNA’s bases through urea links while base pairing between the matching strands of DNA led to the self-assembly of DNA into a three-dimensional hydrogel network. Analysis of the ultrastructure by scanning force microscopy (SFM) revealed the temperature-mediated transition from the rod-to-coil structure as a result of the destabilization of double-stranded DNA (dsDNA) to form a single-stranded DNA (ssDNA), which eventually resulted in a DNA network due to base pairing, along with hairpin loops due to mismatches between nucleotides. Dynamic oscillatory and large deformation creep tests, as well as stress relaxation tests, revealed the formation of robust cross-linked gels: A ten-fold rise in the elastic modulus was observed for the covalent DNA gel compared to the physical gel at the identical DNA concentration. The thermoresponsiveness of the hydrogels was confirmed by a heating–cooling cycle while the non-porous internal morphology of the hydrogel was demonstrated through scanning electron microscopic analysis. Overall, this is the first study showing the fabrication of covalent DNA hydrogels using an NCO-end-capped hydrophilic prepolymer. Such gels have a high potential to be exploited in many fields to benefit from the structural properties of DNA molecules.

Low-frequency spectroscopy of four-photon scattering in aqueous solutions of biopolymers

The novel method of nonlinear laser spectroscopy -- low frequency spectroscopy of four-photon scattering of laser radiation was applied to detect a considerable growth of ortho-H2O spin isomer and also H2O2 molecule concentration in a hydrate layer at the interface between water and DNA, denatured DNA molecules and alpha-chymotrypsin. Spectra of rotational resonances of ortho/para-H2O spin isomers were observed in aqueous solutions of different biopolymers and also in distilled water in the range from zero to 100 cm(-1) with the spectral resolution of 0.05-0.1 cm(-1). The fitting of four-wave mixing spectra shows notable growth of the H2O2 concentration and rotational line's amplitude by a factor of approximately 3 in DNA solutions due to denaturizing. Besides, we studied the four-photon scattering spectra of alpha-chymotrypsin aqueous solutions at protein concentrations between 0 and 20 mg/cm(3) in the range of +/- 7 cm(-1). We found that the velocity of sound in the protein aqueous solution measured by the shift of the Mandelstam-Brillouin scattering spectrum components was a cubic dependence on the protein concentration and reached the value of about 3000 m/s at 20 mg/cm(3).

Low-frequency spectroscopy of four-photon scattering of laser radiation in aqueous solutions of biopolymers

Four-photon scattering (FPS) spectroscopy is used to record rotational resonances of H2O and H2O2 molecules in aqueous solutions of DNA and denatured DNA within a range of ±10 cm-1 with a spectral resolution of 3 GHz. The resonance contribution of rotational transitions of these molecules in solutions was found to be considerably larger than that in distilled water. This fact is interpreted as a manifestation of the specific properties of a hydrate layer at the interface between water and DNA or denatured DNA molecules. An analysis of the FPS spectra shows that the concentration of H2O2 molecules in the hydrate layer of the DNA molecule increases threefold after denaturation. In addition, the FPS spectra of aqueous solutions of α-chymotrypsin protein with concentrations of 0-20 mg cm-3 were measured in the spectral range of ±7 cm-1. It is found that the velocity of hypersound in the protein aqueous solution, which is measured by the shift of the Mandelstam—Brillouin scattering spectrum components, is a cubic function of the concentration and reaches 3000 m/s at 20 mg/cm3.

An Improved Alkaline Lysis Method for Minipreparation of Plasmid Dna

ABSTRACT This study is to improve the digestion pattern of miniprepped plasmid analyzed on gel. Frequently, some ambiguous DNA bands, which are suspected to be denatured DNA molecules, appear during electrophoresis of enzyme digested miniprepped plasmids. By employing Southern hybridization of two identical gels, one had been treated with denaruration-neutralization step and another without such treatment, we confirmed that many of these ambiguous DNA bands were single- stranded (SS) DNA molecules. The presence of SS DNA was due to the use of excess amount of NaOH during plasmid DNA purification with the conventional alkaline lysis method. We, therefore, modified the procedure and recommend that a half amount of NaOH (0.1N instead of 0.2N) should be used when isolating small quantity of plasmid DNA with the method.

Electron microscopy of the melting of sequenced DNA.

AbstractDifferential melting curves (DMCs) of DNAs pA03 and pBR322 in solutions of different ionic strength (0.02 and 0.2M Na+) were obtained. A previously developed procedure of glyxal fixation of partially denatured DNA molecules at temperatures within the melting range was used to construct electron‐microscopic melting maps for pBR322 and pAO3 plasmid DNA and for the replicative form of bacteriophage ϕX174 DNA, allowing the melting of these DNA molecules to be followed in solutions of low (0.1 × SSC) and high (1 × SSC) ionic strength. In spite of the fact that the melting was at nonequilibrium at the low ionic strength, the melting maps for the two kinds of solutions practically coincided. Experimental data are compared with theoretical calculations based on the Fixman‐Freire algorithm. The conclusion is that the melting pattern of these DNAs is, on the whole, correctly described by the theory, although there are appreciable differences between the theoretical and experimental differential melting curves. We have also determined the relation between the melting temperature of a region and its GC content, with allowances made for the boundary conditions of melting in 0.1 × SSC and 1 × SSC solutions, and have analyzed the theoretical shape of peaks of the DMCs.

Alignment of partially denatured DNA molecules

An algorithm for aligning partially denatured DNA molecules was applied to various collections of molecules, using a Hewlett Packard desk-top calculator. The algorithm consists of two stages. The first is analogous to existing methods in that molecules are incorporated in a predetermined order into a reference map. The second refines the map via iteration which terminates upon convergence, resulting in a final map. Molecule order-dependence and the effects of algorithmic parameters on computation time and reproducibility of denaturation maps were studied. Differences between reference and final maps varied, as did differences between final maps derived from various molecule orders. Iteration consistently led to increased area overlap between histograms, hence, order-independence. Histogram quality is expressed in terms of a parameter which measures the correlation of all molecules of a population with their partial denaturation map. A preferential molecule order is suggested. Application to a unique (non-permuted) population showed that molecular, statistical and experimental factors inherent to all populations are crucial for the outcome of alignments. The present algorithm negotiates these more successfully than a pairwise cross-correlation alignment procedure used before. However, if these factors are too large, resolution is impaired but not necessarily due to algorithm quality. It is concluded that the present algorithm is operationally simple, generates maps with a high degree of reproducibility, produces optimal correlation of all molecules with their partial denaturation map, is time-saving and yields improved resolution compared to other methods. It also produces reliable results as judged from marker mapping capability.

Locations of frequently opening regions on natural DNAs and their relation to functional loci.

AbstractThe probability of each base pair being in open state under premelting conditions was calculated and plotted against the base number for nine DNA genomes whose entire nucleotide sequences have been determined. The base‐pair opening profiles thus obtained were compared with the functional maps. Origins of DNA replication fall on frequently opening regions for five of six DNA genomes with known initiation sites of replication. Coincidence of frequently opening regions with promoters and terminators of transcription is most prominent for DNAs with block heterogeneity in sequence such as papovavirus DNAs, a Colicin El‐related plasmid pAO3, pBR322, and Transposon 3 (Tn 3) DNAs, while this coincidence is less apparent in DNAs with random sequence such as the replicative form of ϕX174 and G4 phage DNAs. In fd phage DNA, about half the promoters are located in frequently opening regions, although the strength of a promoter is not correlated with the frequency of opening of base pairs. Correlation of frequently opening regions and gene boundaries is not so obvious as claimed previously. As a whole, coincidence of functional loci and frequently opening regions defined in base‐pair opening profiles is far from perfect but much better than that previously inferred from the conventional running average of local A + T content, theoretical denaturation mapping, or electron microscopy of partially denatured DNA molecules.

Analysis of interruptions in the phosphodiester backbone of herpes simplex virus DNA

Herpes simplex virus (HSV) DNA sediments homogeneously on a neutral sucrose gradient but heterogeneously on an alkaline sucrose gradient. To test whether the presence in HSV DNA of preexisting nicks and gaps or ribose was responsible for the heterogeneous alkaline sedimentation pattern, HSV DNA was analyzed by the sensitive method of agarose gel electrophoresis under two denaturing conditions: glyoxal treatment and denaturation by methylmercuric hydroxide. Treatment with glyoxal or methylmercury, unlike alkali denaturation, leaves ribose intact. HSV DNA yielded a broad distribution of denatured DNA molecules. The results indicate that few, if any, ribose residues were present in mature HSV DNA and that the heterogeneous alkaline sedimentation pattern of HSV DNA can be explained by the presence of nicks and gaps in the DNA.

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [ 3H]leucinelabeled or covalently bonded [ 3H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (⋍ 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [ 14C]TdR, then frozen and stored at − 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.

Adenovirus DNA: III. Separation of the complementary strands of adenovirus types 2, 7 and 12 DNA molecules

The binding of homo- and oo-polyribonucleotides to the denatured DNA molecules of adenovirus types 2, 7 and 12, members of three different oncogenic and transforming human virus groups, was studied by analytical equilibrium centrifugation in cesium chloride density gradients. Denatured adenovirus 2 DNA binds to poly(G) and poly(U) at pH 7.2, and to poly(C) at pH 5, but not to poly(A); satisfactory separation of the complementary DNA strands as complexes with homopolymers was not achieved. Copolymer poly(U,G) preparations with suitable ratios of U to G bind to both strands of adenovirus 2, 7 and 12 DNA molecules and produce increases in buoyant density of 50 to 80 mg/cm 3. Each of the complementary strands of adenoviruses 2, 7 and 12 DNA bind poly(U,G) to different extents, producing differences in buoyant density of 10 to 17 mg/cm 3 and thus permitting strand separation. Adenovirus 2 heavy and light DNA strands were separated preparatively as intact DNA molecules sedimenting at 35 s as determined by analytical zone centrifugation in alkali. Each strand was uncoutaminated by its complement as shown by self-annealing experiments and by hybridization with virus-specific RNA

Effect of ionic strength on DNA hydration during thermal helix-coil transition

The longitudinal T 1 and transversal T 2 magnetic relaxation times of DNA solutions were measured at various ionic strength at elevated and lowered temperatures. From the dependence of T 1 and T 2 values on temperature the non-rotational hydration of DNA molecules in the native and denatured state and also during helix-coil transition was calculated. Dehydration of native DNA molecules was observed at elevated temperature. A higher ionic strength of DNA solutions is favourable for stronger dehydration at low temperatures. At temperatures close to the T m value, determined spectrophotometrically at 260 nm (73.5, 89.5 and 99° for DNA at 0.02, 0.15 and 1 M NaCl, respectively), the DNA molecules seem to be completely dehydrated. At lowered temperatures rehydration of denatured DNA molecules was observed. However, at each temperature the hydration of denatured DNA has an average value of only 42 % of that observed for native DNA.

Heterogeneity of base composition of DNA extracted from human leukaemic leucocytes

The DNA extracted from human leukaemic leucocytes forms a band (density=1·699 g/cm 3) asymmetric to the denser side in a CsCl gradient at equilibrium in the analytical ultracentrifuge. Such asymmetry is present to some extent also in every normal mammalian DNA. It is due to heterogeneity of DNA base composition, as shown by its persistence after treatments (renaturation, digestion with Mustelus canis liver deoxyribonuclease) upon which the asymmetry should disappear if it were due to denatured DNA molecules. By CsCl preparative centrifugation of leukaemic DNA, a DNA fraction displaying a density of 1·711 g/cm 3 has been isolated from the denser side of the band. This fraction is formed by native double-stranded DNA, as shown by the density shift and the increase in optical density upon thermal denaturation. The extent of asymmetry of DNA band seems to be greater and more variable in leukaemic DNA (particularly in the case of chronic myeloid leukaemia) than in normal mammalian DNA. The interpretation of this finding is discussed.

Spin-echo technique study of the non-rotational hydration of deoxyribonucleic acid.

The non-rotational hydration of DNA and its correlation time have been investigated by the NMR spin-echo technique. Relaxation times T1 and T2 were measured at 20° and the dependence of the values of T1 and T2 on the DNA concentration was established. The theory of this phenomenon is discussed. The non-rotational amount of water was shown to be dependent on the molecular weight of the DNA, ranging from about 8 to 10% with molecular weights of 6·106 to 7·7·106. The non-rotational hydration represents only a part of the total water present in hydration shells. Denaturation of the DNA decreases the non-rotational hydration 1.45 times as compared with native DNA simultaneously resulting in an increase in the correlation time, τc, by a factor of 1.45. Denatured DNA molecules have distinctly less freedom of movement than double-helix DNA which has a high degree of structural organization.

SENSITIZATION OF PURINE-STARVED BACTERIA TO X-RAYS.

Purine-requiring strains of E. coli grown on deficient levels of natural purines develop a 3-fold increase in sensitivity to the lethal effects of x rays. There is no concomittant change in uv sensitivity. Exponential growth of the purine-restricted cultures ceases prematurely but is otherwise normal, and viability remains unimpaired for several hours thereafter. The transition from the normal to the x-ray-sensitized state is an all-or-none phenomenon which follows exponential kinetics, with a half-time of 1618 min. Cells begin to exhibit sensitization almost immediately in log-phase cultures, and after a minimal latent period of 1 hour in lag-phase. The sensitized state is completely reversible by purine supplementation. Sensitization and its reversal both appear to antedate cell replication. It is postulated that purine exhaustion may arrest the replication of new DNA strands after strand separation has occurred, producing partly denatured DNA molecules of high intrinsic radiosensitivity. (auth)