DNA complexes with polycations useful for delivery of DNA into cells

Generation and physicochemical properties of complexes formed by high-molecular thymus DNA and plasmid DNA with synthetic polymers of (dimethyl amino)ethyl methacrylate, (diethyl amino)ethyl methacrylate, and poly(vinyl amine) were studied in solutions of different ionic strength using low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The complexes were tested for toxicity with T98G cell cultures. Condensation of DNA was shown to occur when the ratio of charged groups in the polycations and DNA exceeded unity. This condensation manifested itself as an increase in the optical density of DNA solutions. Condensation-associated changes in the dimensions of DNA molecules were determined, and phase diagrams of DNA-polycation systems were analyzed in the presence of NaCl. MTT analysis revealed no toxicity of these complexes.     

Hyperbranched polylysines modified with histidine and arginine: The optimization of their DNA compacting and endosomolytic properties

The DNA compacting and transfection properties of hyperbranched polylysines whose N-terminal amino groups were modified with histidine and arginine were studied. The histidine-modified hyperbranched polylysines were shown to provide higher efficacy of binding and transfection in comparison with unmodified or hyperbranched arginine-containing polylysines. This fact was explained by the intrinsic endosomolytic activity of the histidine-modified polymers. The dependence between the quantity of the amino acids that modified the terminal lysine residues in the hyperbranched polylysines, the efficacy of their DNA binding, and the transfection activity of the DNA complexes with the corresponding carriers was found. The possibility to increase the transfection activity of the DNA complexes with the hyperbranched polylysines by glycerin or the JTS-1 amphipathic nonapeptide was studied. At the same time, their simultaneous use was found to result in a transfection decrease.     

Analysis of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 complexes with DNA using a reversible DNA end-blocking system

The Lac repressor-operator interaction was used as a reversible DNA end-blocking system in conjunction with an IAsys biosensor instrument (Thermo Affinity Sensors), which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of mismatch repair proteins to move along the DNA. The MSH2-MSH6 complex bound to a mispaired base was found to be converted by ATP binding to a form that showed rapid sliding along the DNA and dissociation via the DNA ends and also showed slow, direct dissociation from the DNA. In contrast, the MSH2-MSH6 complex bound to a base pair containing DNA only showed direct dissociation from the DNA. The MLH1-PMS1 complex formed both mispair-dependent and mispair-independent ternary complexes with the MSH2-MSH6 complex on DNA. The mispair-independent ternary complexes were formed most efficiently on DNA molecules with free ends under conditions where ATP hydrolysis did not occur, and only exhibited direct dissociation from the DNA. The mispair-dependent ternary complexes were formed in the highest yield on DNA molecules with blocked ends, required ATP and magnesium for formation, and showed both dissociation via the DNA ends and direct dissociation from the DNA.     

Membrane activity and transfection ability of amphipathic polycations as a function of alkyl group size

Cationic membrane disruptive peptides such as melittin would appear to have attributes necessary for DNA delivery: DNA binding via electrostatic interactions and membrane lysis to enable cytoplasmic delivery. However, the relatively small overall charge of membrane disruptive peptides results in weak interactions with DNA. As a model of cationic membrane disruptive peptides, amphiphilic polyvinyl ethers were synthesized. The number of positively charged groups incorporated into these polymers is substantially greater than membrane-active peptides, which enables these polymers to form stable complexes with DNA. By varying the length of the hydrophobic groups incorporated into the polymer from one to four carbons, the dependence of membrane activity on side chain length was established. The ability of these polymers to transfect DNA in tissue culture was tested, and it was found that transfection efficiency is dependent upon the membrane disruptive activity of the polymer. Comparison of melittin and synthetic polymers suggests that transfection and toxicity appear to be dependent upon their affinity for DNA. This demonstration of relationships among membrane lysis, transfection, DNA binding, and polymer side-chain composition establishes a new class of transfection reagents and may guide in the design of polymers and formulations that will enable efficient in vivo transfection.     

Characterisation of the binding interaction between poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery vectors

A major factor limiting the development of non-viral gene delivery systems is the poor characterisation of polyelectrolyte complexes formed between cationic polymers and DNA. The present study uses the fluorescamine reagent to improve characterisation of poly(L-lysine) (pLL)/DNA complexes post-modified with a multivalent hydrophilic polymer by determining the availability of free amino groups. The results show that the fluorescamine reagent can be used to monitor the self-assembly reaction between pLL and DNA and the degree of surface modification of the resultant complexes with a hydrophilic polymer. This experimental approach should enable the preparation of fully defined complexes whose properties can be better related to their biological activity.     

HU binding to DNA: evidence for multiple complex formation and DNA bending

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.     

Surface-tethered DNA complexes for enhanced gene delivery

Overcoming the barriers to efficient gene transfer is a fundamental goal of biotechnology. A versatile approach to enhance the delivery of nonviral DNA involves complexation with cationic polymers, which can be designed to overcome the barriers to effective gene transfer. More recently, DNA release from a polymer substrate or scaffold has been shown to enhance gene transfer, likely by increasing DNA concentrations in the cell microenvironment. We propose a novel approach that combines these two strategies in which cationic polymer/DNA complexes are tethered to a substrate that supports cell adhesion. The cationic polymers package the DNA for efficient internalization and the surface tethering functions to maintain elevated concentrations in the cell microenvironment for cells adhered to the substrate. The cationic polymer polylysine (degree of polymerization equal to 19 or 150) was modified with biotin groups, which was confirmed by mass spectrometry and biochemical analysis. Complex formation of DNA with biotinylated-polylysine, or mixtures of biotinylated and nonbiotinylated polylysines, was confirmed by gel electrophoresis. Plasmid DNA encoding for the reporter gene beta-galactosidase was complexed with different mixtures of biotinylated and nonbiotinylated polylysine and incubated on neutravidin (nonglycosylated avidin)-coated surfaces. DNA surface densities ranging from 0.1 to 4.3 microg/cm2 were observed and found to be a function of the number of biotin groups, the molecular weight of the polylysine, and the amount of DNA. HEK293T or NIH/3T3 cells were then seeded onto the DNA-modified surfaces, and transfection was quantified at 48 and 96 h. Transfection by the DNA surfaces was observed with both cell lines, and expression levels up to 100 fold greater than bulk delivery of the complexes was obtained. Transfection was found to be a function of the surface DNA quantities and the number of tethers on the complex. Transfected cells were observed only in the region in which DNA complexes were tethered, suggesting that the location of transfected cells can be specifically controlled. Surface tethering of DNA represents a promising approach to enhancing gene transfer and spatially controlling gene delivery, which may have applications to a multitude of fields ranging from tissue engineering to functional genomics.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Salt-dependent co-operative interaction of histone H1 with linear DNA

The nature of the complexes formed between histone H1 and linear double-stranded DNA is dependent on ionic strength and on the H1 : DNA ratio. At an input ratio of less than about 60% (w/w) H1 : DNA, there is a sharp transition from non-co-operative to co-operative binding at a critical salt concentration that depends on the DNA size and is in the range 20 to 50 mM-NaCl. Above this critical ionic strength the H1 binds to only some of the DNA molecules leaving the rest free, as shown by sedimentation analysis. The ionic strength range over which this change in behaviour occurs is also that over which chromatin folding is induced. Above the salt concentration required for co-operative binding of H1 to DNA, but not below it, H1 molecules are in close proximity as shown by the formation of H1 polymers upon chemical cross-linking. The change in binding mode is not driven by the folding of the globular domain of H1, since this is already folded at low salt in the presence of DNA, as indicated by its resistance to tryptic digestion. The H1-DNA complexes at low salt, where H1 is bound distributively to all DNA molecules, contain thickened regions about 6 nm across interspersed with free DNA, as shown by electron microscopy. The complexes formed at higher salt through co-operative interactions are rods of relatively uniform width (11 to 15 nm) whose length is about 1.6 times shorter than that of the input DNA, or are circular if the DNA is long enough. They contain approximately 70% (w/w) H1 : DNA and several DNA molecules. These thick complexes can also be formed at low salt (15 mM-NaCl) when the H1 : DNA input ratio is sufficiently high (approximately 70%).     

Inhibition of recA-mediated strand exchange by adducts of azacytosine-containing DNA and the EcoRII methylase

Wild type Escherichia coli cells containing elevated levels of DNA (cytosine-5)methyltransferases have increased sensitivity to the toxic effects of 5-azacytidine. The methyltransferases form tight binding complexes with azacytosine in DNA which could interfere with the recA recBCD repair pathway which is largely responsible for cell survival after treatment with the drug. We therefore determined if these complexes interfered with recA-mediated strand exchange in vitro. 32P-Labeled DNA fragments containing a single EcoRII site, with cytosine in the (-) strand replaced by 5-azacytosine, were prepared. We investigated the effect of the EcoRII methyltransferase on recA-mediated strand exchange with homologous M13 DNA by electrophoresis in agarose gels. In the absence of the methylase the rate and extent of strand exchange of azacytosine-containing DNA is the same as control DNA. In the presence of the methyltransferase strand exchange is inhibited, but some incorporation of duplexes into recA-single-stranded DNA (ssDNA) complexes still occurs. The formation of these complexes is dependent on the length of the fragment 3' to the methylase binding site on the strand complementary to the ssDNA. The greater the length the greater the number of complexes that form. S-Adenosyl-L-methionine, which enhances binding of the methyltransferase to azacytosine-containing DNA, causes an increase in the inhibition of strand exchange and an increase in the number of inactive complexes formed. The complexes can be dissociated with guanidinium chloride which denatures the methyltransferase and leads to release of the (+) strand. The (-) strand remains associated with the ssDNA. This result implies that a plectonemic joint is formed between recA-ssDNA complexes and azacytosine-containing DNA-methyltransferase complexes. However, branch migration in these complexes is inhibited. Denaturation of the methyltransferase allows branch migration to proceed to completion, releasing the (+) strand.     

Retrovirus-polymer complexes: study of the factors affecting the dose response of transduction

We have previously shown that complexes of Polybrene (PB), chondroitin sulfate C (CSC), and retrovirus transduce cells more efficiently than uncomplexed virus because the complexes are large and sediment, reaching the cells more rapidly than by diffusion. Transduction reaches a peak at equal weight concentrations of CSC and PB and declines when the dose of PB is higher or lower than CSC. We hypothesized that the nonlinear dose response of transduction was a complex function of the molecular characteristics of the polymers, cell viability, and the number of viruses incorporated into the complexes. To test this hypothesis, we formed complexes using an amphotropic retrovirus and several pairs of oppositely charged polymers and used them to transduce murine fibroblasts. We examined the effect of the type and concentration of polymers used on cell viability, the size and charge of the complexes, the number of viruses incorporated into the complexes, and virus binding and transduction. Transduction was enhanced (2.5- to 5.5-fold) regardless of which polymers were used and was maximized when the number of positive charge groups was in slight excess (15-28%) of the number of negative charge groups. Higher doses of cationic polymer were cytotoxic, whereas complexes formed with lower doses were smaller, contained fewer viruses, and sedimented more slowly. These results show that the dose response of transduction by virus-polymer complexes is nonlinear because excess cationic polymer is cytotoxic, whereas excess anionic polymer reduces the number of active viruses that are delivered to the cells.     

DNA complexing with polyamidoamine dendrimers: implications for transfection.

DNA and polyamidamine (PAMAM) dendrimers form complexes on the basis of the electrostatic interactions between negatively charged phosphate groups of the nucleic acid and protonated (positively charged) amino groups of the polymers. Charge neutralization of both components and subsequent increases of the net positive charge of the complex result in changes in the physicochemistry and biological properties of the complexes. The formation of soluble, low-density and insoluble, high-density complexes was analyzed using UV light absorption and measurements of radioactive labeled DNA. Formation of high molecular weight and high-density complexes depended mainly on the DNA concentration and was enhanced by increasing the dendrimer-DNA charge ratio. Electrostatic charge related effects (attraction or repulsion of charged particles) appeared to be modulated by the generation of dendrimer (size of the polymer). With the progressive increases in the dendrimer-DNA charge ratio (above 20), an increase in the amount of low-density, soluble complexes was observed. Functional analysis revealed that the great majority (>90%) of transfection is carried by low-density, soluble, complexes which only represent approximately 10-20% of total complexed DNA. The ability of the dendrimer to complex and form aggregates with DNA is crucial for efficient transfection and the function of the complexed DNA.     

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.     

Interaction of DNA with DNA binding proteins. II. Displacement of Escherichia coli DNA unwinding protein and the condensed structure of DNA complexed with protein HD.

Three DNA binding proteins from Escherichia coli cells have been complexed with single-stranded phage fd DNA. Electron microscopy reveals granular substructures in the complexes formed with protein HD. In complexes of DNA unwinding protein with fd DNA both protein HD and phage-coded gene 5 protein partially displace the unwinding protein which results in the formation of structures characteristic for the DNA complexes formed with either protein HD or gene 5 protein alone. Combination of protein HD with double-stranded phage T7 DNA leads to a progressive folding and condensing of the genome. The structures observed are discussed in relation to current concepts of the packing of DNA in protein complexes.     

DNA binding compatibility of the Streptococcus pneumoniae SsbA and SsbB proteins

Background

Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multiple SsbAs and SsbBs onto single-stranded DNA was investigated.

Methodology/Principal Findings

The complexes that were formed by the SsbA and SsbB proteins on dT_n oligomers of defined lengths were examined by polyacrylamide gel electrophoresis. Complexes containing either two SsbAs or two SsbBs, or mixed complexes containing one SsbA and one SsbB, could be formed readily, provided the dT_n oligomer was long enough to satisfy the full binding mode capacities of each of the bound proteins under the particular solution conditions. Complexes containing two SsbAs or two SsbBs could also be formed on shorter dT_n oligomers via a “shared-strand binding” mechanism in which one or both proteins were bound using only a portion of their potential binding capacity. Mixed complexes were not formed on these shorter oligomers, however, indicating that SsbA and SsbB were incompatible for shared-strand binding. Additional experiments suggested that this shared-strand binding incompatibility may be due in part to differences in the structure of a loop region on the outer surface of the subunits of the SsbA and SsbB proteins.

Conclusion/Significance

These results indicate that the SsbA and SsbB proteins may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required. The apparent compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.     

Creating a unique environment for selecting reactive enzymes with DNA: 'Sticky' binding of oligocation-grafted polymers to DNA

To provide colloidally stable polyplexes formed between pDNA and cationic polymers, cationic polymers have been modified with hydrophilic polymers to form a hydrophilic shell. Block copolymers of cationic and hydrophilic polymers and cationic polymers grafted with hydrophilic polymers are representative designs of such polymers. Here, we report a new design of cationic polymers and oligocationic peptide-grafted polymers. We synthesized 15 kinds of graft copolymers by varying the number of cationic charges of the peptides and their grafting density. We found that graft copolymers with less cationic peptides and less grafting density formed colloidally stable polyplexes. Interestingly, the less cationic graft copolymers bind to excess amounts of pDNA. We also found that the graft copolymers showed selectivity toward reactive enzymes affording the reaction of pDNA with nucleases, while suppressing both the replication of DNA by DNA polymerase and gene expression. The suppression of the replication and expression is considered to result from the high capacity of the graft copolymers for binding with pDNA. The polynucleotides produced by DNA polymerase or RNA polymerase would be captured by the graft copolymers to impede these enzymatic reactions.     

The ORFa protein, the putative transposase of maize transposable element Ac, has a basic DNA binding domain.

Ac encodes the 807 amino acid ORFa protein which binds specifically to multiple AAACGG motifs that are subterminally located in both ends of Ac. The wild-type ORFa protein and a number of deletion and amino acid exchange mutants were expressed in Escherichia coli, renatured and used for mobility shift assays. At least 136 amino acids from the N-terminus and 537 C-terminal amino acids may be removed from the ORFa protein without destroying the DNA binding domain, whereas a protein starting at amino acid 189 is DNA binding deficient. Certain basic amino acids between positions 190 and 200 are essential for DNA binding, as their substitution with uncharged amino acids leads to the loss of this function. The DNA binding domain of ORFa protein has an overall basic character, but no obvious sequence homology to any other known DNA binding protein. The homologies to the major open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila are found between the C-terminal two thirds of the three proteins. The ORFa protein forms discrete complexes with target DNA that appear, depending on the protein concentration, as a 'ladder' of bands on gels, indicating the occupation of target DNA by multiple ORFa protein molecules.