DNA In Aqueous Solution Research Articles

A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy. Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution. Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongated morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O. This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.

Taxol Anticancer Activity and DNA Binding

The interaction of taxol with DNA has major biological importance since it is shown the presence of higher concentration of taxol in the nucleus, than in the human lung tumor cell. Therefore, in this report we examine the interaction of taxol with calf-thymus DNA in aqueous solution at physiological pH, using constant DNA concentration (25 or 1.25 mM phosphate) and various taxol/DNA (phosphate) ratios 1/200 to 1/2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods are used to characterize the nature of drug-DNA interaction and to determine the taxol binding site, the binding constant, sequence selectivity, helix stability and biopolymer secondary structure in the taxol-DNA complexes in vitro. Structural analysis showed that taxol is an external DNA binder with no affinity towards DNA intercalation. The major target of taxol is A-T, G-C bases and the backbone PO(2) groups. Two bindings were observed for taxol-DNA complexes with K(1)= 1.4 x 10(4) M(-1) and K(2)=3.5 X 10(3) M(-1). The taxol-DNA interaction is associated with a partial helix stabilization and no major alterations of B-DNA structures.

Label-free detection and classification of DNA by surface vibration spectroscopy in conjugation with electrophoresis

We propose a method of in situ (in vitro) analysis of the chemical bonding state of DNA in aqueous solution by infrared absorption spectroscopy in the multiple internal reflection geometry (MIR-IRAS) in conjugation with electrophoresis. We demonstrate that MIR-IRAS is capable of detecting in vitro DNA hybridization. In our method, a Si prism, through which infrared lights penetrate and are also internally reflecting, also serves as the electrode for electrophoresis. We show that with a positive or negative potential being applied to the electrode (prism), DNA molecules are concentrated on or expelled from the electrode, respectively, leading to highly sensitive detection and manipulation of DNA in aqueous solution.

Surfactant encapsulated DNA: structure characterization and interaction with dye molecules in organic media

The recognition of electrostatically-bound DNA–didodecyldimethylammonium (DNA–DDDA) complex by three dye molecules, acridine orange (AO), ethidium bromide (EB) and 5,10,15,20-tetrakis(4- N-methylpyridyl) porphyrin tetra( p-toluenesulfonate) (TMPyP) in organic media was investigated through 1H NMR, UV–vis, and circular dichroism (CD) spectroscopies. When the organic solvent in which DNA–DDDA complex dissolves is changed from ethanol to chloroform, the adsorbed AO undergoes a reversible transformation from a monomer to a highly aggregated state at the interface between DNA and DDDA. EB also adsorbs at the interface between DNA and DDDA when EB interacts with the DNA–DDDA complex in organic media, but its existing state is independent of the used solvents. The third dye, TMPyP cation can intercalate into the G–C region while its anionic p-tosylate counterion remains unbound when it mixes with DNA complex in organic media. The complexes of DDDA with previously recognized DNA by the three dye molecules (DNA–dye), respectively, are also investigated. AO seems having changed its location from the grooves of DNA to the interface between DNA and DDDA after DNA–AO complex was electrostatically encapsulated with DDDA. The aggregation behavior of AO also shows a dependence on the polarity of the organic solvent. EB molecules are believed to intercalate into the base pairs of DNA in aqueous solution. The intercalation mode is still maintained after the encapsulation for DNA–EB in organic solvents, which is different from the situation between DNA–DDDA complex and EB. But in both cases, the existing states of EB are independent of the polarity of the organic solvents. Finally, TMPyP in the complex of DNA–TMPyP and DDDA is also judged to intercalate into the G–C region of DNA while its anionic p-tosylate counterion remains separated from DNA complex, which is similar to its interaction with DNA–DDDA complex in organic media. These data also strongly suggest that the intercalation state of TMPyP is more stable than its adsorption state in grooves when it is recognized with DNA. The present results are significant for the designs of both laser dye and conductive materials.

Effects of UV irradiation on calf thymus DNA in aqueous solution. A Raman spectroscopic study

The Raman spectra of calf thymus DNA in aqueous solution before and after irradiation in two UV regions (UVA, 320–340 nm; UVA–UVB, 280–400 nm) were investigated. Damage to the molecular structure in part was found in the DNA solution after UVA–UVB irradiation. The bands near 811 (±7) and 832 (±7) cm−1 appeared in all Raman spectra after UV irradiation. This result indicates that DNA in solution has a mixture of A- and B-form conformations under UV irradiation. In this experiment, all the main characteristic Raman peaks appeared at almost the original position after UVA irradiation, except for some bands corresponding to deoxyribose phosphate. This indicated that serious changes to the molecular structure did not occur in the DNA solution after UVA irradiation. The spatial structures of the DNA main chain backbone and phosphoric acid backbone were almost unchanged, and the bands corresponding to adenine also appeared at almost the original sites after UVA–UVB irradiation. The bands at 949 and 1445 cm−1 corresponding to deoxyribose shifted and were obviously hypochromic, and the bands assigned to thymine showed a large shift and decline after 5 h of UVA–UVB irradiation. Hence the pyrimidine bases and deoxyribose were likely first damaged when the calf thymus DNA in aqueous solution was irradiated by UVA–UVB. Copyright © 2004 John Wiley & Sons, Ltd.

Raman Spectroscopic Analysis of the Effect of Ultraviolet Irradiation on Calf Thymus DNA

Raman spectroscopy was used for the first time to detect the effect of independent UVA (ultraviolet-A: 320-400 nm) and UVB (ultraviolet-B: 280-320 nm) irradiation on the calf thymus DNA in aqueous solution. After both UVA and UVB irradiation for 1 h or 3 h, the damage to the conformation of DNA was moderate, but the reduction of the B-form DNA component was obvious. Both UVA and UVB caused significant damage to the deoxyribose moiety and bases, among which the pyrimidine base pairs were more seriously affected. There appeared to be preferential damaging sites on DNA molecules caused by UVA and UVB irradiation. UVA irradiation caused more damage to the deoxyribose than UVB irradiation, while UVB irradiation caused more significant damage to the pyrimidine moiety than UVA irradiation. After UVB irradiation for 3 h, unstacking of the AT base pairs and the cytosine ring took place, severe damage to the thymine moiety occurred, and some base pairs were modified. Moreover, with either UVA or UVB irradiation for 3 h, the photoreactivation of DNA occurred. The damage to the DNA caused by UVB was immediate, while the damage caused by UVA was proportional to the irradiation duration. The experimental results partly indicate the formation of some cyclobutane pyrimidine dimers and (6-4) photoproducts.

In-situ Observation of DNA Hybridization in Aqueous Solution by Multiple Internal Reflection Infrared Absorption Spectroscopy

We have observed in-situ the hybridization (double helix formation) and denaturation (separation of double helix at elevated temperatures) of DNA in aqueous solution using infrared absorption spectroscopy (IRAS) in the multiple internal reflection (MIR) geometry. We demonstrated that conformational changes of DNA strands due to hybridization and denaturation are reflected in the infrared absorption spectra of the bases of DNA. Comparing with the results of ab-initio cluster calculation, we found that hybridization produces the specific C=O stretching vibration modes in the hydrogen-bonded bases, and also that the C=O stretching vibration modes of the bases of a single strand may be greatly influenced by its surrounding water molecules that interact with the bases.

Involvement of proton transfer in the reductive repair of DNA guanyl radicals by aniline derivatives

The most easily oxidized sites in DNA are the guanine bases, and major intermediates produced by the direct effect of ionizing radiation (ionization of the DNA itself) are electron deficient guanine species. By means of a radiation chemical method (gamma-irradiation of aqueous thiocyanate), we are able to produce these guanyl radicals in dilute aqueous solutions of plasmid DNA where the direct effect would otherwise be negligible. Stable modified guanine products are formed from these radicals. They can be detected in the plasmid conversion to strand breaks after a post-irradiation incubation with a DNA base excision endonuclease enzyme. If aniline compounds are also present, the yield of modified guanines is strongly attenuated. The mechanism responsible for this effect is electron donation from the aniline compound to the guanyl radical, and it is possible to derive rate constants for this reaction. Aniline compounds bearing electron withdrawing groups (e.g., 4-CF3) were found to be less reactive than those bearing electron donating groups (e.g., 4-CH3). At physiological pH values, the reduction of a guanyl radical involves the transfer of a proton as well as of an electron. The mild dependence of the rate constant on the driving force suggests that the electron is not transferred before the proton. Although the source of the proton is unclear, our observations emphasize the importance of an accompanying proton transfer in the reductive repair of oxidative damage to guanine bases which are located in a biologically active double stranded plasmid DNA substrate.

Interaction cetyltrimethylammonium bromide–DNA investigated by dielectric spectroscopy

Abstract The complex dielectric permittivity of aqueous solutions of low-molecular weight DNA (0.5–1 kbp) in the presence of the cationic surfactant cetyltrimethylammonium bromide (CTAB) has been measured in the frequency range from 0.1 to 100 MHz, at the temperature of 25 °C. The DNA concentration was 0.64 × 10 −3 M in terms of phosphates and the CTAB concentration was varied from 1.3 × 10 −5 to 9 × 10 −4 M. The dielectric data have been analyzed in terms of a single dielectric dispersion and discussed on the basis of a model involving the fluctuation of counter-ions along short segments of the DNA chains. Viscosity and surface tension measurements on DNA/CTAB solutions are also reported. The results show that CTAB binds as a single monomer to the DNA phosphate sites and that there is a release of one Na + ion per CTAB bound to a phosphate site. Appreciable modification of the DNA conformation subsequent to the formation of the complex is also evidenced.

Encapsulation of DNA by Cationic Diblock Copolymer Vesicles

Encapsulation of dsDNA fragments (contour length 54 nm) by the cationic diblock copolymer poly(butadiene-b-N-methyl-4-vinyl pyridinium) [PBd-b-P4VPQ] has been studied with phase contrast, polarized light, and fluorescence microscopies, as well as scanning electron microscopy. Encapsulation was achieved with a single emulsion technique. For this purpose, an aqueous DNA solution is emulsified in an organic solvent (toluene) and stabilized by the amphiphilic diblock copolymer. The PBd block forms an interfacial brush, whereas the cationic P4VPQ block complexes with DNA. A subsequent change of the quality of the organic solvent results in a collapse of the PBd brush and the formation of a capsule. Inside the capsules, the DNA is compacted as shown by the appearance of birefringent textures under crossed polarizers and the increase in fluorescence intensity of labeled DNA. The capsules can also be dispersed in an aqueous medium to form vesicles, provided they are stabilized with an osmotic agent [poly(ethylene glycol)] in the external phase. It is shown that the DNA is released from the vesicles once the osmotic pressure drops below 10(5) N/m(2) or if the ionic strength of the supporting medium exceeds 0.1 M. The method has also proven to be efficient to encapsulate pUC18 plasmid in submicrometer-sized vesicles, and the general applicability of the method has been demonstrated by the preparation of the charge inverse system: cationic poly(ethylene imine) encapsulated by the anionic diblock poly(styrene-b-acrylic acid).

Mouse NEIL1 Protein Is Specific for Excision of 2,6-Diamino-4-hydroxy-5-formamidopyrimidine and 4,6-Diamino-5-formamidopyrimidine from Oxidatively Damaged DNA

A functional homologue of human DNA glycosylase NEIL1 (hNEIL1) in mouse has recently been cloned, isolated, characterized, and named mouse NEIL1 (mNEIL1). This enzyme exhibited specificity for excision of oxidatively modified pyrimidine bases such as thymine glycol, 5,6-dihydrouracil, and 5-hydroxypyrimidines, using oligonucleotides with a single base lesion incorporated at a specific site. It also acted upon AP sites; however, no significant excision of 8-hydroxyguanine was observed [Rosenquist, T. A., Zaika, E., Fernandes, A. S., Zharkov, D. O., Miller, H., and Grollman, A. P. (2003) DNA Repair 2, 581-591]. We investigated the substrate specificity and excision kinetics of mNEIL1 for excision of oxidatively modified bases from high-molecular weight DNA with multiple lesions, which were generated by exposure of DNA in aqueous solution to ionizing radiation. Among a large number of pyrimidine- and purine-derived lesions detected and quantified in DNA, only purine-derived lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine were significantly excised. This finding establishes that mNEIL1 and its functional homologue hNEIL1 possess common substrates, namely, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine. Measurement of excision kinetics showed that mNEIL1 possesses equal specificity for these two formamidopyrimidines. This enzyme also excised thymine-derived lesions thymine glycol and 5-hydroxy-5-methylhydantoin, albeit at a much lower rate. A comparison of the specificity and excision kinetics of mNEIL1 with other DNA glycosylases shows that this enzyme is as efficient as those DNA glycosylases, which specifically remove the formamidopyrimidines from DNA.

Influence of anoxia on the induction of mutations by phenylalanine radicals during γ-irradiation of plasmid DNA in aqueous solution

When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during γ-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the γ-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.

Urea effect on Cu 2+-induced DNA structural transitions in solution

Cu 2+ ion interaction with DNA in aqueous solutions containing urea (0–5 M) was studied by IR spectroscopy. It was shown that upon the Cu 2+ ion binding DNA transition into a compact form occurs. This transition is of positive cooperativity. We suppose that the mechanism of Cu 2+-induced DNA compaction in solutions containing urea is not completely electrostatic. Urea addition to the DNA solution decreases the Cu 2+ ion concentration required to induce DNA compaction. As the urea content in solution rises, the binding constant of Cu 2+ ions interacting with DNA increases, going through the maximum in the case of 2 M solution; further increase of the urea content in solutions leads to decrease of the binding constant. DNA transition into the compact form under the Cu 2+ ion action is determined not only by the effects of the solution dielectric permeability but by the solvation effects; when changes of the dielectric permeability are small the solvation effects may prevail. Urea addition to the DNA solution also decreases cooperativity of the DNA compaction process. Perhaps, cooperativity of the DNA transition into the compact state depends on the ordered spatial structure of water adjacent to the macromolecule and decreases on the structure destruction.

Structure of solid-supported lipid–DNA–metal complexes investigated by energy dispersive X-ray diffraction

When mixing aqueous solutions of DNA and dioloeyl-phosphatidylcholine multilamellar liposomes in the presence of Mn 2+ ions, a multilamellar liquid-crystalline phase is formed in which DNA monolayers are comprised between opposing lipid bilayers. Here, we report on a new experimental procedure resulting in the formation of solid-supported DOPC–DNA–Mn 2+ complexes in the biologically relevant excess water condition. The supramolecular structure of solid-supported complexes, characterized by means of Energy Dispersive X-ray Diffraction, exhibits very similar structural properties with respect to the solution structure of the complexes as revealed by synchrotron Small Angle X-ray Scattering measurements.

Micellization of Cationic Gemini Surfactants with Various Counterions and Their Interaction with DNA in Aqueous Solution

The micellization of six cationic gemini surfactants with various counterions, [C12H25(CH3)2N(CH2)6N(CH3)2C12H25]X2, designated as C12C6C12X2, with X = F-, Cl-, Br-, Ac-, NO3-, and 1/2SO42- in aqueous solutions has been investigated by isothermal titration microcalorimetry (ITC) and conductivity measurements. The interaction of these surfactants with DNA in aqueous solutions has also been investigated by isothermal titration microcalorimetry. The critical micelle concentration (CMC) and the degree of micellar ionization (α), the critical aggregation concentration (CAC), the saturation concentration (C2) of the aggregation, and the associated thermodynamic parameters were determined. The nature of the counterion significantly affects the processes of both micellization and aggregation. The trends for aggregation basically follow the Hofmeister (lyotropic) series, but the pattern of the variation of the enthalpy of aggregation often revealed a more complex behavior. Among the counterions examined, SO42- is ...

Spectroscopic studies on the interaction of Safranine T with DNA in β-cyclodextrin and carboxymethyl-β-cyclodextrin

The interaction of Safranine T (ST) with cyclodextrins (CDs) including β-cyclodextrin (β-CD) and carboxymethyl-β-cyclodextrin (CM-β-CD) and DNA in aqueous solution is studied by UV–vis absorption and steady-state fluorescence technique. The interactive model of ST with double stranded DNA has been investigated by means of the inclusive action of β-CD and CM-β-CD. Based on the changes of absorption, fluorescence and resonance light scattering (RLS) spectra, the intrinsic binding constant (Kap) and the binding site number (n) of ST with DNA and the inclusion complexes ST–CD with DNA are obtained in the case of 20mmol/L Tris–HCl buffer solution (pH 7.2). According to the experimental results, it can be inferred that the interactive model of dimer ST with DNA is “electrostatic binding”, while the monomer ST with DNA is “intercalative binding”.

Salivary gland genetic vaccination: a scalable technology for promoting distal mucosal immunity and heightened systemic immune responses

Use of plasmid DNA for vaccination has been demonstrated quite successfully in small rodents. However, some of the many challenges of DNA vaccine development are the relatively low performance obtained in larger animals and a generally weak mucosal immune response. Vaccination through salivary gland (SG) cannulation and delivery of aqueous solutions of DNA is one potential solution. The scalability of SG DNA vaccination was tested in multiple animal models; antigen specific titers above 10,000 were demonstrated in dogs and rats. Immune responses were also present at a variety of mucosal sites. In conclusion, our data demonstrate that DNA vaccination to the SG presents a unique and advantageous method for eliciting systemic and mucosal immune responses.

Magnetic relaxation dispersion of lithium ion in solutions of DNA.

The magnetic field dependence of the nuclear spin-lattice relaxation rate constant defines the magnetic relaxation dispersion (MRD) and provides a direct characterization of the molecular dynamics that cause fluctuations in the magnetic couplings in the system and may also indicate the dimensional constraints on the motion. The counterion cloud surrounding a linear polyelectrolyte ion, such as DNA in solution, provides an interesting opportunity for ion confinement that helps in understanding the thermodynamics and the dynamics of the interactions between the polyion and other solutes. The MRD profiles of lithium ion and tetramethylammonium ion were recorded in dilute aqueous solutions of native calf thymus DNA, which provides a long, charged rod that reorients slowly. The 7Li ion relaxes through the nuclear electric quadrupole coupling and the proton-lithium dipole-dipole coupling; the protons of the tetramethylammonium ion relax by dipole-dipole coupling. MRD profiles of the 7Li+ ion are dominated by transient interactions with the DNA that yield a linear dependence of the spin-lattice relaxation rate constant on the logarithm of the Larmor frequency. This magnetic field dependence is consistent with diffusive ion motions that modulate two spatial coordinates that characterize the relaxation couplings in the vicinity of the polyion. Motions around the rod and fluctuations in the ion distance from the rod are consistent with these constraints for lithium. The magnetic field dependence of the tetramethylammonium ion proton relaxation rate constant is weak, but also approximately a linear function of the logarithm of the Larmor frequency, which implies that the field dependence is caused in part by local order in the DNA solution.

AZT-DNA interaction.

Oxidative DNA damage has been reported in fetal tissues by exposure to 3'-azido-3'-deoxythymidine (AZT). AZT has been used effectively for the treatment of human immunodeficiency virus-1 (HIV-1) and AIDS. It showed in vitro to block the nucleoside-binding site of the viral reverse transcriptase and to inhibit DNA replication by chain termination. It incorporates into both nuclear and mitochondrial DNA and is shown to cause cancer in vivo and in vitro. This study was designed to examine the interaction of AZT with DNA in aqueous solution at physiological condition, using different drug/DNA (phosphate) molar ratios (r) of 1/80 to 1/2 and constant DNA concentration of 12.5 mM (phosphate). Capillary electrophoresis, FTIR, and UV-visible difference spectroscopic methods and molecular modeling were used to determine the drug binding sites, the binding constants and the effects of the AZT complexation on DNA conformation. Structural analysis showed that AZT binds to DNA through G-C and A-T base pairs and the backbone PO(2) groups with two binding constants of K(1) = 2.60 x 10(5) M(-1) and K(2) = 1.20 x 10(5) M(-1). The drug distributions are 50% with G-C, 15% with A-T and 35% with the backbone phosphate group. AZT-DNA interaction is associated with a partial B- to A-DNA conformational transition.

The effects of ionizing radiation on DNA: The role of thiols as radioprotectors

Electron loss from N-(2-mercaptopropionyl) glycine (PSH) gave an EPR detectable radical anion, PS- SP −. When the PSH derivative was frozen in aqueous DNA solutions to 77 K and exposed to ionizing radiation, normal damage to the DNA was detected by EPR spectroscopy. However, on annealing above 77 K, central EPR features for the DNA base radical cations and anions gave central features assigned to PS- SP − σ*-radical anions, together with outer features for 5-6-dihydro-5-thymyl radicals, TH . It is proposed that on freezing, the PSH molecules are constrained into a glassy region around the DNA, and that, on annealing, electron donation gives PS radicals, with loss of quanine radical-cations, G + . The PS radicals were not detectable, but on reaction with another PSH molecule, gave good EPR spectra for PS- SP − radical-anions. These results indicate that PSH had little effect on the yield of the other base radicals C − /T − . Also, growth of TH radicals, formed from protonated thymine radical-anions, T − , were detected. We conclude that the primary effect of PSH is to capture the G + centers, and thus could either prevent or repair radiation damage to DNA.