Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Binding and purification of plasmid DNA using multi-layered carbon nanotubes

We propose a new method for the separation of nucleic acids using multi-layered carbon nanotubes (CNTs) as an adsorbent. According to agarose gel electrophoresis, oxidized water-stable CNTs adsorb certain forms of nucleic acids, such as high molecular weight RNA, chromosomal DNA, linear and denatured forms of plasmid DNA. However, CNTs do not adsorb supercoiled form of plasmid DNA. Nucleic acids bound to CNTs can be readily removed by centrifugation whereas supercoiled plasmid DNA remains in solution. Upon the addition of divalent metal ions supercoiled plasmid DNA forms relatively stable complexes with CNTs due to chelation. Thus, new details about association of nucleic acids with CNTs were revealed and stoichiometry of the complexes was estimated. Our results can be used for fine purification of supercoiled plasmid DNA for gene therapy applications as well as manipulation of nucleic acids for biosensor design.     

Probing of DNA structure with osmium tetroxide. Effect of ligands

Fourteen OsO4 complexes with different ligands were tested as probes of DNA structure. Of these complexes, only OsO4-2,2'-bipyridine (Os-bipy), OsO4-bathophenanthrolinedisulfonic acid (Os-bpds) and OsO4-N,N,N',N'-tetramethylenediamine (Os-TMEN) site-specifically modified the ColE1 cruciform in a supercoiled plasmid pColIR215 at millimolar concentrations. Os-bipy, Os-bpds and Os-TMEN also displayed site-specific modification of the B-Z junctions in the supercoiled plasmid pRW751 containing (dC-dG)n inserts.     

Hydrolysis of DNA by a Dipeptides Containing Histidine

Three ligands which contain histidine and conjugated by a flexible linker, have been characterized and evaluated as DNA cleavage agents. The cleavage activity of metal complexes were evaluated by monitoring the conversion of supercoiled plasmid DNA (pUC19) (Form I) to nicked circular DNA (Form II) by agarose gel electrophoresis. The results showed that the cleavage activity of Cu(II) complexes was enhanced compared with histidine. Specially, at a high reaction concentration (0.2 mM), Cu(II) complexes can cleave the plasmid DNA with some selectivity.     

Oxygen radical-mediated DNA damage by redox-active Cr(III) complexes.

The mechanism of DNA damage induced by Cr(III) complexes is currently unknown even though it is considered to be the ultimate biologically active oxidation state of chromium. In this study, we have employed the Salmonella reversion assay to identify mutagenic Cr(III) complexes. Cyclic voltammetry was used to differentiate the redox kinetics between mutagenic and selected nonmutagenic Cr(III) species. Plasmid relaxation of supercoiled DNA was employed to show in vitro interactions with plasmid DNA and correlate the interactions with the electrochemical behavior and biological activity. The results of this study demonstrate that the mutagenic Cr(III) complexes identified in the Salmonella reversion assay display characteristics of reversibility and positive shifts of the Cr(III)/Cr(II) redox couple consistent with the ability of these Cr(III) complexes to serve as cyclical electron donors in a Fenton-like reaction. These same mutagenic complexes display an ability to relax supercoiled DNA in vitro, presumably by the induction of single-strand breaks. Nonmutagenic complexes were selected to test different ligands to determine how the ligand directs the activity of Cr(III) complexes. All nonmutagenic complexes tested thus far have shown classical irreversibility, more negative reduction potentials, and an inability to relax supercoiled plasmid DNA. These results suggest that the mechanism by which chromium complexes potentiate mutagenesis involves an oxygen radical as an active intermediate. These data also demonstrate the effect of associated ligands with regard to the ability of a metal to generate an active redox center.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to 'functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to `functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.     

Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis

Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 μmol/l ethidium bromide (EB) and then exposed to 110 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.     

Selective purification of supercoiled p53-encoding pDNA with l-methionine–agarose matrix

The p53 tumor suppressor gene has been widely explored for gene therapy as an alternative to the common treatments. Recently, the supercoiled conformation of a p53-encoding plasmid proved to be more efficient in cell transfection and protein expression than the open circular conformation. To successfully isolate this isoform, several chromatographic techniques have been used, namely affinity chromatography with amino acids as ligands. However, the study of new matrices and ligands with higher specificity and robustness for supercoiled plasmid purification is still required. The present work explores for the first time a new matrix of l-methionine–agarose to efficiently purify the supercoiled p53-encoding plasmid. The binding/elution conditions, such as salt concentration and temperature, were manipulated and combined to attain the best strategy. Therefore, the supercoiled plasmid isoform was purified from a clarified lysate by using a decreasing stepwise gradient comprising 2.35 and 1.7 M ammonium sulfate in 10 mM Tris–HCl, pH 8.0, and finally 10 mM Tris–HCl, pH 8.0, at 5 °C. After accomplishing the purification process, we performed several tests to assess the quality of the supercoiled plasmid, revealing that the amounts of proteins, gDNA, RNA, and endotoxins were significantly reduced or undetectable in the final formulation.     

Preparation of plasmid DNA by sequential enzymatic digestion.

A new method for the preparation of plasmid DNA from Escherichia coli, sequential enzymatic digestion, is described. The method is based on sequential and selective enzymatic digestion of all components of E. coli except for the supercoiled plasmid DNA. The key enzymes are exonuclease I and exonuclease III that specifically hydrolyze linear chromosomal DNA and are unable to attack supercoiled plasmid DNA under controlled conditions. Isolated plasmid DNA can be sequenced and digested with restriction enzymes.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to functionalize their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented     

Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities

The selective adsorption of supercoiled plasmid, open-circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes. Genomic DNA and open-circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid. As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid. The advantage of gyrolite adsorbents is their lower unit price and ability to selectively adsorb DNA impurities without binding supercoiled plasmid under certain conditions. The effects of ionic strength, temperature, chelating agent, divalent cation, and lyotropic salts on adsorption of highly purified plasmid are studied to understand the forces that bind DNA to gyrolite, a structure with hydrophilic and hydrophobic characteristics. The results indicate that DNA binding is governed by hydrogen bonding, electrostatic bridging with divalent cations, shielding of electrostatic repulsion, hydrophobic adsorption, and disruption of integral surface water layer on gyrolite. On the basis of results from a range of Hofmeister series salts, strongly hydrated anions may enhance DNA adsorption by promoting hydrophobic interactions between DNA and gyrolite. Conversely, the very weakly hydrated chaotrope I(-) may enhance adsorption by strongly associating with hydrophobic siloxanes of gyrolite, thereby disrupting an integral water layer, which competes for hydrogen bonding sites.     

High frequency excision of Ty elements during transformation of yeast.

Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.     

Developmental-stage-specific plasmid supercoiling in Chlamydia trachomatis

Chlamydia trachomatis elementary body (EB) and reticulate body (RB) developmental stages have polymorphic plasmid DNA. Several plasmid forms separated by gel electrophoresis were identified as topoisomers by treatment with topoisomerase I. Among these topoisomers was one form unique to EBs and one form unique to RBs. The unique EB plasmid topoisomer was characterized as highly supercoiled, on the basis of band migrations by gel electrophoresis and its appearance by electron microscopy. The unusual physical state of this topoisomer was probably mediated, in part, by DNA-specific structural proteins. The unique RB plasmid topoisomer was a supercoiled form of lower superhelical density than the other identified topoisomers. Developmental-stage-specific differences in super-helical density of plasmid DNA suggest cause-and-effect relationships between DNA topology and metabolic activity in RBs and metabolic quiescence in EBs.     

Circular dichroism investigation of the effect of plasmid DNA structure on retention in histidine chromatography

The effect of temperature-induced changes in secondary and tertiary structures of plasmid DNA (pDNA) and on the retention behaviour of open circular (oc) and supercoiled (sc) isoforms in histidine-agarose chromatography was investigated by Circular dichroism (CD) spectroscopy. Chromatographic experiments performed with three plasmids (2.7, 6.1 and 7.4 kbp) and with a decreasing ammonium sulphate gradient (2.3--2.0 M) showed that the retention of sc pDNA increased as temperature decreased from 24 to 5 °C. Such behaviour was attributed to the temperature-induced removal of negative superhelical turns in sc pDNA which is accompanied by a decrease in the number of dissociated base pairs responsible for interaction with the histidine ligands. CD spectroscopy showed that temperature has an important effect on plasmid secondary structure if adenine-rich inserts are present in the plasmid structure. Chromatographic experiments also suggested that base composition could also be responsible for the induction of specific interactions with histidine ligands.     

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.     

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair

As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.