In-vitro and in-vivo difference in gene delivery by lithocholic acid-polyethyleneimine conjugate.

Polyethyleneimine (PEI) is widely used for the delivery of nucleic acids, but its clinical application is limited due to high cytotoxicity and instability in biological fluids. To overcome these challenges, linear PEI (2.5 kDa) was modified with lithocholic acid (LCA) to produce a LCA-PEI conjugate (lp), and its complex with plasmid DNA (pDNA) was covered with hyaluronic acid (HA). Ternary complexes of pDNA, lp, and HA ("DlpH") were prepared in different ratios and tested in cells and tumor-bearing mice for gene transfection efficiency. DlpH with a relatively high lp/pDNA ratio (Hi-DlpH) was more resistant to DNase and heparin treatment and showed more efficient gene transfection than DlpH with a lower lp/pDNA ratio (Lo-DlpH) in vitro. In contrast, Hi- and Lo-DlpH showed distinct transfection efficiency in vivo in a tumor-size dependent manner, where Hi-DlpH showed relatively high gene transfection in tumors of <300 mm3 but performed poorly in tumors of >500 mm3 and Lo-DlpH did the opposite. Tumor-associated macrophages, which increase with tumor growth and preferentially intercept Hi-DlpH, may account for the poor performance of Hi-DlpH in relatively large tumors. Accordingly, suggestions are made for future in vitro screening of new gene formulations to better predict their in vivo performances.

Structure and Properties of DNA Molecules Over The Full Range of Biologically Relevant Supercoiling States.

Topology affects physical and biological properties of DNA and impacts fundamental cellular processes, such as gene expression, genome replication, chromosome structure and segregation. In all organisms DNA topology is carefully modulated and the supercoiling degree of defined genome regions may change according to physiological and environmental conditions. Elucidation of structural properties of DNA molecules with different topology may thus help to better understand genome functions. Whereas a number of structural studies have been published on highly negatively supercoiled DNA molecules, only preliminary observations of highly positively supercoiled are available, and a description of DNA structural properties over the full range of supercoiling degree is lacking. Atomic Force Microscopy (AFM) is a powerful tool to study DNA structure at single molecule level. We here report a comprehensive analysis by AFM of DNA plasmid molecules with defined supercoiling degree, covering the full spectrum of biologically relevant topologies, under different observation conditions. Our data, supported by statistical and biochemical analyses, revealed striking differences in the behavior of positive and negative plasmid molecules.

Effect of shear stress on structure and function of polyplex micelles from poly(ethylene glycol)-poly(l-lysine) block copolymers as systemic gene delivery carrier.

Structural stability of polyplex micelles (PMs), prepared from plasmid DNA (pDNA) and poly(ethylene glycol)-b-poly(l-lysine) block catiomer (PEG-PLys), was evaluated in terms of their resistance against shear stress. When exposed to shear stress at magnitudes typically present in the blood stream, structural deterioration was observed in PMs owing to the partial removal of PEG-PLys strands. Eventually, impaired PEG coverage of the polyplex core led to accelerated degradation by nucleases, implying that structural deterioration by shear stress in blood stream may be a major cause of rapid clearance of PMs from blood circulation. To address this issue, introduction of disulfide crosslinking into the PM core was shown to be an efficient strategy, which successfully mitigated unfavorable effects of shear stress. Furthermore, improved in vivo blood retention profile and subsequently enhanced antitumor efficacy in systemic treatment of pancreatic adenocarcinoma were confirmed for the crosslinked PMs loaded with pDNA encoding an anti-angiogenic protein, suggesting that high stability under the shear stress during blood circulation may be a critical factor in systemically applicable gene delivery systems.

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters.

BACKGROUND: Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood. METHODS: The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM). RESULTS: In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance. CONCLUSIONS: The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one. GENERAL SIGNIFICANCE: The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.

Functionalized Multi-Wall Carbon Nanotubes Enhance Transfection and Expression Efficiency of Plasmid DNA in Fish Cells.

DNA vaccines are considered to be the most promising method against infectious diseases in the aquaculture industry. In the present study, we investigated the potency of ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) in enhancing the transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus kidney (CIK) cells. Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense DNA in varying degrees. Scanning electron microscope (SEM) images shows that CIK cells show a great affinity for MWCNTs-NH3⁺ and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be "needle-pricking the cells". Transmission electron microscope (TEM) images confirmed that MWCNTs-NH3⁺ penetrate the cell membranes and are widely dispersed in the CIK cell. Real-time PCR was used to detect the transfection efficiency through the expression of the outer capsid protein (VP5). The results showed that the MWCNTs-NH3⁺:DNA complexes are able to transfect CIK cells effectively at different charge ratio than naked DNA. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. All these results indicated that MWCNTs-NH3⁺:DNA complexes could be suitable for developing DNA vaccine for the control of virus infection in the aquaculture industry.

A model for chromosome organization during the cell cycle in live E. coli.

Bacterial chromosomal DNA is a highly compact nucleoid. The organization of this nucleoid is poorly understood due to limitations in the methods used to monitor the complexities of DNA organization in live bacteria. Here, we report that circular plasmid DNA is auto-packaged into a uniform dual-toroidal-spool conformation in response to mechanical stress stemming from sharp bending and un-winding by atomic force microscopic analysis. The mechanism underlying this phenomenon was deduced with basic physical principles to explain the auto-packaging behaviour of circular DNA. Based on our observations and previous studies, we propose a dynamic model of how chromosomal DNA in E. coli may be organized during a cell division cycle. Next, we test the model by monitoring the development of HNS clusters in live E. coli during a cell cycle. The results were in close agreement with the model. Furthermore, the model accommodates a majority of the thus-far-discovered remarkable features of nucleoids in vivo.

A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI.

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5'-TCGA-3' sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5'-GCGC-3' sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA.

Iron(II) supramolecular helicates condense plasmid DNA and inhibit vital DNA-related enzymatic activities.

The dinuclear iron(II) supramolecular helicates [Fe2 L3 ]Cl4 (L=C25 H20 N4 ) bind to DNA through noncovalent (i.e., hydrogen-bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA-condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe2 L3 ]Cl4 do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA-processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site-specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe2 L3 ]Cl4 helicates on DNA-related enzymatic activities have been proposed.

The influence of novel gemini surfactants containing cycloalkyl side-chains on the structural phases of DNA in solution.

Very important to gene therapy is the delivery system of the nucleic acids (called a vector), which will enhance the efficiency of the transport of new DNA into cells whilst protecting against damage. A promising alternative to the currently used viral vectors are the systems based on amphiphilic compounds - lipoplexes. Among them, gemini surfactants, which consist of two hydrophobic chains and two cationic heads connected by a linker - spacer group, appear to be promising candidates. The subject of this study involves two gemini surfactants, alkoxy derivatives of bis-imidazolium quaternary salts, differing in the length of their spacer groups and how they interact with two types of salmon sperm DNA (low and high molecular weight (MW)) or plasmid DNA (pDNA). The mixtures of gemini surfactants with nucleic acids of differing p/n ratios (positive-to-negative charge ratio) were characterised by small angle X-ray scattering (SAXS) of synchrotron radiation, dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), transmission electron microscopy (TEM) and gel electrophoresis techniques. This analysis allows for the selection of the most suitable and promising candidates for non-viral vectors in gene therapy, determination of the conditions needed to form stable complexes, identification of conformational changes in the DNA molecules upon interactions with gemini surfactants and in some cases, determination of the structures formed in these lipoplexes.

Intracellular microenvironment responsive polymers: a multiple-stage transport platform for high-performance gene delivery.

A new strategy for promoting endoplasmic gene delivery and nucleus uptake is proposed by developing intracellular microenvironment responsive biocompatible polymers. This delivery system can efficiently load and self-assemble nucleic acids into nano-structured polyplexes at a neutral pH, release smaller imidazole-gene complexes from the polymer backbones at intracellular endosomal pH, transport nucleic acids into nucleus through intracellular environment responsive multiple-stage gene delivery, thus leading to a high cell transfection efficiency.

Combined bidimensional electrophoresis and electron microscopy to study specific plasmid DNA replication intermediates in human cells.

Replication interference by specific chromosomal sequences-such as trinucleotide repeats-plays a causative, though undefined role in the aetiology of human disease, especially neurodegenerative syndromes. However, studies on these mechanisms in human cells have been hampered by poorly defined replication origins on genomic DNA. Simian Virus 40 (SV40)-based plasmids were useful in the past to overcome these experimental limits, but have been rarely amenable for the most complex and revealing molecular biology approaches to study in vivo DNA replication interference. This chapter describes a new, safe, SV40-based episomal system that replicates with very high efficiency in human cells and allows isolation of in vivo replication intermediates with high yield and purity. We describe how to use this experimental system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy.

[Putative temperature sensitive riboswitchers in Salmonella genome].

Currently, a number of structurally and functionally different temperature-sensitive elements like as RNA thermometers which control a variety of biological processes of bacteria, including virulence, are known. Well-known RNA thermometers correspond to one long step-loop structure or few hairpins which can be matched or mismatched. Based on the computer and thermodynamical analysis of 25 isolates of Salmonella enterica with complete genome, algorithm and the criteria of search for putative RNA thermometers were developed. It will permit to perform the search of potential riboswitchers in genome of socially significant pathogens in the future. For S. enterica, in addition to well-known 4U RNA thermometer, four step-loop structures that may be new RNA thermometers were identified and two of them are localized in 5'-UTR of virulence regulators gltB and yaeQ. They correspond to necessary and sufficient conditions of RNA thermometer formation as far as these highly conservative structures are found in genome of all 25 isolates of S. enterica. Matched hairpins forming cruciform structure in supercoiled pUC8 plasmid were visualized by atomic force microscopy.

DNA cloning: a personal view after 40 years.

In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer's laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning.

Chain relaxation dynamics of DNA adsorbing at a solid-liquid interface.

We have used scanning force microscopy (SFM) to elucidate the dynamic behavior of open (torsionally unconstrained) circular and long linear DNA molecules during the relaxation process following adsorption onto mica. We find that bending stress and excluded volume effects drive the conformational equilibration via segmental out-of-plane dynamics.

Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy.

Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.

Superstructure of the centromeric complex of TubZRC plasmid partitioning systems.

Bacterial plasmid partitioning systems segregate plasmids into each daughter cell. In the well-understood ParMRC plasmid partitioning system, adapter protein ParR binds to centromere parC, forming a helix around which the DNA is externally wrapped. This complex stabilizes the growth of a filament of actin-like ParM protein, which pushes the plasmids to the poles. The TubZRC plasmid partitioning system consists of two proteins, tubulin-like TubZ and TubR, and a DNA centromere, tubC, which perform analogous roles to those in ParMRC, despite being unrelated in sequence and structure. We have dissected in detail the binding sites that comprise Bacillus thuringiensis tubC, visualized the TubRC complex by electron microscopy, and determined a crystal structure of TubR bound to the tubC repeat. We show that the TubRC complex takes the form of a flexible DNA-protein filament, formed by lateral coating along the plasmid from tubC, the full length of which is required for the successful in vitro stabilization of TubZ filaments. We also show that TubR from Bacillus megaterium forms a helical superstructure resembling that of ParR. We suggest that the TubRC DNA-protein filament may bind to, and stabilize, the TubZ filament by forming such a ring-like structure around it. The helical superstructure of this TubRC may indicate convergent evolution between the actin-containing ParMRC and tubulin-containing TubZRC systems.

Interference of intrinsic curvature of DNA by DNA-intercalating agents.

It has been demonstrated in our studies that the intrinsic curvature of DNA can be easily interrupted by low concentrations of chloroquine and ethidium bromide. In addition, the changes of DNA curvature caused by varying the concentration of these two DNA intercalators can be readily verified through using an atomic force microscope.

Long-term structural changes of plasmid DNA studied by atomic force microscopy.

Long-term stability of plasmid DNA (pDNA) conformations is critical in many research areas, especially those concerning future gene therapy. Despite its importance, the time-evolution of pDNA structures has rarely been studied at a molecular resolution. Here, the time-evolution of pDNA solutions spanning four years was observed with atomic force microscopy (AFM). The AFM data show that the pDNA molecules changed over time from isolated supercoiled structures, to aggregated supercoiled structures, to thin, branched network structures, and finally to wider, branched network structures. Additional topographical analysis of the AFM data suggests that the actions of residual proteins could be the main mechanism for the structural changes in our laboratory-prepared pDNA.

Orientation-defined alignment and immobilization of DNA between specific surfaces.

DNA-based single-molecule studies, nanoelectronics and nanocargos require a precise placement of DNA in an orientation-defined manner. Until now, there is a lack of orientation-defined alignment and immobilization of DNA over distances smaller than several micrometers. However, this can be realized by designing bifunctionalized DNA with thiol at one end and (3-aminopropyl) tri-ethoxy silane at the other end, which specifically binds to a gold and SiO2 layer after and during alignment, respectively. The electrode assembly consists of platinum as the electrode material for applying the AC voltage and islands of gold and silicon dioxide fabricated at a distance of about 500-800 nm by electron-beam lithography. The orientation-defined alignment and covalent binding of pUC19 DNA to specific surfaces are carried out in frequency ranges of 50 Hz-1 kHz and 100 kHz-1 MHz and observed after metallization of DNA by palladium ions by field emission scanning electron microscopy (FESEM). The bifunctionalized 890 nm long DNA was effectively aligned and immobilized between a gap of 500 to 600 nm width.