Condensed DNA Particles Research Articles

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL) volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications.

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg2+-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils – that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg2+ – mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.

Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes

A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process; nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors.

Characterization of a Lipophilic Plasmid DNA Condensate Formed with a Cationic Peptide Fatty Acid Conjugate

In the presence of cationic ligands, DNA molecules can become aggregated into larger particles in a process known as condensation. DNA condensates are of interest as models for the dense packing found in naturally occurring structures such as phage heads and chromatin. They have found extensive application in DNA transfection and also provide convenient models with which to study DNA damage by the direct effect of ionizing radiation. Further, conjugates of cationic peptides with fatty acids may represent a class of attractive ligands for these areas because of their simple synthesis. When plasmid pUC18 is used as the DNA target and N-caproyl-penta-arginine amide (Cap-R(5)-NH(2)) is used as the ligand, the physical properties of the resulting mixtures were characterized using static and dynamic light scattering, sedimentation, dye exclusion, circular dichroism, nanoparticle tracking, and atomic force microscopy. Their chemical properties were assayed using solvent extraction and protection against hydroxyl radical attack and nuclease digestion. Titration of the plasmid with the Cap-R(5)-NH(2) ligand produced sharply defined changes in both chemical and physical properties, which was associated with the formation of condensed DNA particles in the 100-2000 nm size range. The caproyl group at the ligand's N-terminus produced a large increase in the partitioning of the resulting condensate from water into chloroform and in its binding to the neutral detergent Pluronic F-127. Both the physical and chemical data were all consistent with condensation of the plasmid by the ligand where the presence in the ligand of the caproyl group conferred an extensive lipophilic character upon the condensate.

Condensed DNA particles formed in a PCR with plasmid templates: An electron microscopy study

An electron microscopy study of large-sized DNA microparticles produced in PCR with different gene-specific primers and plasmid DNAs is described. DNA microspheres of two distinct types were revealed in the all studied samples, namely smooth moderately electron-dense microspheres, and highly electron-dense particles with large thorns and offshoots. Singular microspheres have the average diameter of 1 mum, and their aggregates were up to 3 mum in dimensions. In addition, rare so-called three-dimensional net-like structures with various size (up to several micrometers) were observed. They consisted of different amounts of DNA nanoparticles, having the special compact topology. In some studied samples the discs (nanodiscs) of several dozens nm in thickness and up to 3 mum in diameter were revealed. It was shown that the quantity of net-like structures and nanodiscs sharply increases in asymmetric PCR. We also observed DNA nanowires of different length and thickness, nanodots, nanoparticles in the form of shits of paper as well as electron-dense spherical nanoparticles of big size. Aqueous suspensions of DNA microparticles were heated at 94 degrees C for 5 min and analyzed by electron microscopy. It was shown that microspheres in heated suspensions underwent partial melting; they lost a part of DNA, therefore details of their structure (ultrastructure) can be recognized. At the some time numerous tangles of nanowires appeared. Molecular mechanisms of the DNA micro- and nanoparticles formation are discussed.

An improved large‐scale synthesis of PEG‐peptides for gene delivery

Polyethylene glycol (PEG)-peptides are under development as components of nonviral gene delivery systems. Several earlier reports have demonstrated that covalent attachment of PEG to the surface of peptide condensed DNA particles blocks non-specific biodistribution during gene targeting. In this study, we report an improved large-scale synthesis and purification of a DNA condensing PEG-peptide used for gene delivery. The new method takes advantage of low-pressure cation-exchange chromatography to isolate dimeric Cys-Trp-Lys(18). The dimeric peptide was reduced and directly conjugated with PEG-maleimide resulting in PEG-Cys-Trp-Lys(18). The PEG-peptide was purified by low-pressure chromatography affording 50 mumol (400 mg) quantities of PEG-peptide in >95% purity. The approach offers the advantage of avoiding preparative high-performance liquid chromatography (HPLC) purifications of polylysine peptides to increase yield and capacity.

Monomolecular condensation of DNA by cationic detergents

AbstractControlling the size of condensed DNA particles is a key determinant for their diffusion in vivo as well as for gene delivery to target cells. Towards this goal, DNA molecules were compacted individually by cationic thiol‐detergents into discrete nanometric entities. These particles were then stabilized by air‐induced dimerization of the detergent into a disulfide lipid on the template DNA. Using a tetradecane–cysteine–ornithine (C14COrn) detergent, a solution of 5.5 Kb plasmid DNA was thus converted into a monodisperse population of 35‐nm particles. The stability of the complexes, as well as their size, morphology and transfection efficiencies were investigated. Surprisingly, the electrophoretic mobility of the quasi‐neutral condensed DNA was found higher than that of the extended DNA polyanion. The diameters of particles resulting from the condensation of DNA of various sizes was measured by dynamic light scattering and found to vary as the cubic root of the DNA size. In an attempt to extend their biodistribution and to target tumour cells, we have prepared folate–poly(ethylene oxide)‐coated particles that were shown to bind to the cell‐surface folate receptor. Copyright © 2003 Society of Chemical Industry

The Role of a Microscopic Colloidally Stabilized Phase in Solubilizing Oligoamine-Condensed DNA Complexes

DNA complexes of spermine and spermidine become resolubilized at very high concentrations of the oligoamine. It has been postulated that high oligoamine concentrations shift the DNA from the globule back to the coil phase. The present study indicates that DNA resolubilization at high concentrations of spermine and spermidine is explained by formation of small particles of condensed DNA that cannot be precipitated by centrifugation. The fact that DNA stays condensed during resolubilization was confirmed using a relatively new condensation assay and three independent microscopic techniques. A considerable portion of DNA was found to be in particles with diameter <100 nm. Formation of such small particles is likely to be caused by colloidal forces. The ability to form small, condensed DNA particles in solutions that contain high concentrations of oligocation should aid in the design of synthetic DNA vectors for gene transfer and gene therapy and in the handling of DNA for diagnostic studies.

Intracellular delivery of nanometric DNA particles via the folate receptor.

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.

Development of plasmid and oligonucleotide nanometric particles.

Nucleic acids delivery vectors have shown promising therapeutic potential in model systems. However, comparable clinical success is delayed essentially because of their poor biodistribution and of their ineffective intracellular trafficking. The size of condensed DNA particles is a key determinant for in vivo diffusion, as well as for gene delivery to the cell nucleus. Towards this goal, we have developed cationic thiol-detergents that individually compact plasmid DNA molecules into anionic particles. These particles are then 'stabilized' by air-induced dimerization of the detergent into a disulfide lipid on the template DNA. The particles all measure approximately 30 nm, which corresponds to the volume of a single molecule of plasmid DNA. The gel electrophoretic mobility of the anionic particles was found to be higher than that of the plasmid DNA itself. Similarly, particles formed with a 31-mer oligonucleotide measured 19 nm. Improved in vivo diffusion, as well as improved intracellular trafficking may be inferred from the faster migration of the complexes. Moreover, the size of the particles remains compatible with nuclear pore crossing. Finally, in an attempt to improve the biodistribution of these particles, we have coated the monomolecular particles with a poly(ethylene glycol) corona.

Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles and transfect cells in culture.

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.

Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.

We have evaluated the effect of NaCl concentration on the mode of binding of poly-L-lysine to DNA and the resulting structural and functional features of the condensed DNA particles using DNA precipitation, DNase I resistance, electron microscopy, and receptor-mediated gene transfer assays. At a high concentration of NaCl and in the presence of excess DNA, poly-L-lysine interacted with DNA cooperatively, fully condensing some of the DNA and leaving the rest of the DNA unbound. At low NaCl concentrations, poly-L-lysine molecules interacted with DNA in a noncooperative fashion, i.e. they bind randomly to the whole population of DNA molecules. Cooperative binding of poly-L-lysine to DNA occurred over a narrow range of NaCl concentrations, and the specific salt concentration depended on the length of the poly-L-lysine. The ability of condensed DNA to withstand digestion by DNase I was correlated with the structural features of the condensed DNA as determined by electron microscopy. Using our condensation procedure, cooperative binding of poly-L-lysine to DNA is a necessary prerequisite for the preparation of condensed DNA having a spherical shape and a diameter of 15-30 nm. Condensed DNA, containing galactosylated poly-L-lysine, was evaluated further for the extent and specificity of receptor-mediated gene transfer into HuH-7 human hepatoma cells via the asialoglycoprotein receptor. Efficient receptor-mediated transfection occurred only when condensed DNA complexes had a spherical shape with a diameter of 15-30 nm; asialofetuin, a natural ligand for the asialoglycoprotein receptor, inhibited this process by up to 90%. Our results support the importance of appropriate DNA condensation for the uptake and ultimate expression of DNA in hepatic cells.

Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.

Circular dichroism and fluorescence-detected circular dichroism of deoxyribonucleic acid and poly[d(A-C).cntdot.d(G-T)] in ethanolic solutions: a new method for estimating circular intensity differential scattering

A method is presented for determining the circular dichroism (CD) of systems whose CD spectra contain contributions from CD differential scattering. The technique is shown to detect light over 4 pi steradians, and thus, for the first time, a complete correction for scattering is possible. The method is applied to ethanol-condensed DNA and poly[d(A-C).d(G-T)]. From the results obtained, the former are proposed to have an A-type secondary structure. The condensed polynucleotide particles are shown to exhibit behavior similar to that of cholesteric liquid crystals. CD difference spectra, obtained from the scattering corrections and showing the contributions to different sections of the scattering envelope, are displayed. It is asserted that these scattering patterns contain information about the tertiary structure of the condensed DNA particles studied.