Mechanism of protein priming DNA replication of B.subtilis phage M2.

B.subtilis phage M2 uses a protein, instead of RNA, as the primer of its DNA replication. Hence this protein encoded in the phage genome is called as the primer protein (PP). At the initiation of DNA replication, a hetero dimer complex with its own DNA polymerase and the PP supposed to interact with the terminal protein (TP), which is covalently bound to the template DNA (TP-DNA). PP contained an important adhesive amino acid sequence, Arg-Gly-Asp (RGD), near the carboxyl terminal. We have recently showed that the synthetic RGD peptide inhibited the transfection of phage M2. By site-directed mutagenesis, we introduced different amino acid into the RGD site of PP. These altered PP decreased obviously the priming activity in vitro.     

Enhancement of star vector-based gene delivery to endothelial cells by addition of RGD-peptide

This study aimed to investigate the feasibility of using a cationic nonviral gene carrier in endothelial cells for enhancing gene expression by the addition of an integrin-binding RGD peptide. A 4-branched cationic polymer of poly( N,N-dimethylaminopropylacrylamide) (star vector), developed as a gene carrier, could complex with the luciferase-encoding plasmid DNA under a charge ratio of 5 (vector/pDNA) to form polymer/DNA complexes (polyplexes). The addition of the RGD-containing peptide (GRGDNP) to the polyplex solution led to a decrease in the zeta-potential from ca. +30 to +20 mV along with the reduction in the particle size from ca. 300 to 200 nm. Additionally, a marked inhibition of polyplex aggregation was observed, indicating the coating of the polyplex surface with RGD peptides. A transfection study on endothelial cells showed that the luciferase activity increased with the amount of RGD peptides added to the polyplexes and exhibited minimal cellular cytotoxicity. The transfection activity further increased when cyclic RGD peptides (RGDFV) were used; the activity with RGD peptide addition was approximately 8-fold compared to that without RGD peptide addition. Gene delivery to endothelial cells was significantly enhanced by only the addition of RGD peptides to star vector-based polyplexes.     

An inhibitory effect of RGD peptide on protein, priming reaction of bacteriophages ?29 and M2

Summary The amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages 29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of proteinprimed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of 29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.     

Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG5K-succinyl-ACDCRGDCFCG-COOH (DSPE-PEG5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of alphavbeta3 and alphavbeta5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.     

Targeting of Lipid-Protamine-DNA (LPD) Lipopolyplexes Using RGD Motifs

Abstract

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG_5K-succinyl-ACDCRGDCFCG-_COOH (DSPE-PEG_5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG_5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of αvβ3 and αvβ5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.     

DNA transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis

Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.     

Transfecting activity of Pseudomonas aeruginosa bacteriophage SM

Factors affecting the efficiency of transfection of Ps. aeruginosa PAO1 cells by the temperate SM bacteriophage DNA have been determined. The efficiency of transfection by DNA preparations isolated from the wild type bacteriophage SMc+ or its thermoinducible mutant SM cts6 is practically the same. The frequency of transfection is (7-9) X 10(4) of infectious centers per mkg of transfecting DNA. Variability in the frequencies of transfection has been registered depending of the infection conditions or on the transfer of the Ps. aeruginosa PAO1 recipient strain population into the competence phase. The efficiency of transfection is increased by the addition of Ca2+ or Mg2+ ions affecting the adsorption and absorbtion of phage DNA by the recipient cells. Optimal concentrations of the bivalent metal ions are 0.15M CaCl2 and 0.2M MgCl2. The results obtained have been used for optimizing the conditions of Ps. aeruginosa PAO1 transfection by SM bacteriophage DNA.     

The role of the cell cycle on the efficiency of photochemical gene transfection

The efficiency of gene transfection mediated by nonviral vectors is limited because of nonoptimal intracellular trafficking of transfecting DNA. Most nonviral vectors deliver transfecting DNA into a cell through endocytosis. However, poor escape from endocytic vesicles and inefficient transport of DNA into the nucleus often limits a success of gene transfection. Photochemical transfection is a new method, based on light-induced permeabilisation of endocytic vesicles, liberating transfecting DNA into the cytosol, concurrently increasing the chances for DNA to enter the nucleus.The aim of this study was to investigate the role of the cell cycle for the efficiency of photochemical transfection. It was demonstrated that in asynchronous human colon carcinoma HCT 116 cells photochemical treatment increased the transfection mediated by the nonviral vectors, the cationic polypeptide polylysine and the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (beta AE-DMRIE/DOPE), by 30- and 2.5-fold, respectively. In aphidicolin-synchronised cells, photochemical transfection mediated by polylysine was dependent on the cell cycle: transfection level was 4-fold higher when illumination, inducing photochemical reactions, was performed during the G2/M phase as compared to the G1/early-S phase. The cell cycle influence on photochemical transfection mediated by beta AE-DMRIE/DOPE was very low: only 20% difference between G2/M and the G1/S phase was observed. We suggest that transgenes, photochemically liberated close/during mitosis, perhaps have the highest opportunity to enter the nucleus and be expressed. However, the dependence of photochemical transfection on the cell cycle might be partially disguised by various factors induced by photochemical treatment.     

Effect of Synthetic Hydroxy Isothiocyanates on a Bacterial Virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on φK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Effect of synthetic hydroxy isothiocyanates on a bacterial virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on psiK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.     

Preferential transfection with M13mp2 RF DNA synthesized in vitro

Single-strand DNA binding protein (SSB) from Escherichia coli abolishes transfection of E.coli by viral M13mp2 DNA at levels that inhibit transfection by M13mp2 replicative form (RF) DNA by approx. 25%. Synthesis of M13mp2 RF DNA (SS leads to DS) has been carried out using DNA polymerase I (Klenow fragment) and a unique 15-nucleotide primer. A time course for in vitro synthesis showed that the increase in transfection in the presence of SSB paralleled DNA synthesis after an initial lag period for transfection. Digestion of replication products with restriction endonucleases and S1 endonuclease indicates that only those molecules that are fully or almost fully duplex transfect competent cells in the presence of SSB.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.     

An archaeal histone-like protein as an efficient DNA carrier in gene transfer

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.     

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7.

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.     

Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell.

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the efficiency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the linear DNA than for the closed circular DNA. Transfection of Escherichia coli bacteria by several plasmids indicates that the transfection efficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (In-) DNA (Xie, T. D., L. Sun, H. G. Zhao, J. A. Fuchs, and T. Y. Tsong. 1992. Biophys. J. 63:1026-1031). To resolve these conflicting observations, we have systematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSVcat, pRSVneo, and pRSVgpt. Mg(2+)-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after treatment with PEF, were monitored by 3H-labeled plasmids. Transfection efficiency was evaluated by both the transient expression of chloramphenicol acetyltransferase (cat) activity 2-3 days after, and the permanent expression of neomycin phosphotransferase (neo) and xanthine-guanine phosphoribosyltransferase (gpt) genes in the transformants 2 weeks after the PEF treatment. Our results indicate that cell surface binding and PEF-induced cell uptake of DNA did not depend on the topology of DNA. However, both the transient and the permanent expression of the plasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the in-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, in vitro transfection activity and physicochemical characterization of novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) amphiphilic derivatives

A novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives was synthesized and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and cholesterol. Physicochemical properties of lipid-DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance technique at 23 degrees C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a +/-charge ratio of 2. In agreement with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A2 and 38.7 mN/m, respectively. This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved transfection activity.     

Structure of Bacillus subtilis bacteriophage SPP1 DNA in relation to its transfection activity.

The availability of a detailed restriction map of SPP1 DNA allowed defined manipulations of such molecules. These were performed to investigate structural requirements for SPP1 transfection. (i) The transfection activity of SPP1 DNA was destroyed by degradation with restriction enzymes. Biological activity could be regenerated when transfection was performed with a combination of two different restriction endonuclease digests, provided that such digests generated widely overlapping DNA fragments. (ii) Unique DNA molecules were constructed from the natural population of circularly permuted SPP1 DNA molecules by using genetic engineering techniques. Such molecules had the same specific transfection activity as did the circularly permuted SPP1 DNA. These results are discussed in the context of current models of DNA processing in transfection.     

Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins

A cyclic RGD peptide-conjugated block copolymer, cyclo[RGDfK(CX-)]-poly(ethylene glycol)-polylysine (c(RGDfK)-PEG-PLys), was synthesized from acetal-PEG-PLys under mild acidic conditions and spontaneously associated with plasmid DNA (pDNA) to form a polyplex micelle in aqueous solution. The cyclic RGD peptide recognizes alphavbeta3 and alphavbeta5 integrin receptors, which play a pivotal role in angiogenesis, vascular intima thickening, and the proliferation of malignant tumors. The c(RGDfK)-PEG-PLys/pDNA polyplex micelle showed a remarkably increased transfection efficiency (TE) compared to the PEG-PLys/pDNA polyplex micelle for the cultured HeLa cells possessing alphavbeta3 and alphavbeta5 integrins. On the other hand, in the transfection against the 293T cells possessing no alphavbeta3 and a few alphavbeta5 integrins, the TE of the c(RGDfK)-PEG-PLys/pDNA micelle showed no increase compared to the TE of the PEG-PLys/pDNA micelle. Flow cytometric analysis revealed a higher uptake of the c(RGDfK)-PEG-PLys/pDNA micelle than the PEG-PLys/pDNA micelle against HeLa cells, consistent with the transfection results. Furthermore, a confocal laser scanning microscopic observation revealed that the pDNA in the c(RGDfK)-PEG-PLys micelle preferentially accumulated in the perinuclear region of the HeLa cells within 3 h of incubation. No such fast and directed accumulation of pDNA to the perinuclear region was observed for the micelles without c(RGDfK) ligands. These results indicate that the increase in the TE induced by the introduction of the c(RGDfK) peptide ligand was due to an increase in cellular uptake as well as facilitated intracellular trafficking of micelles toward the perinuclear region via alphavbeta3 and alphavbeta5 integrin receptor-mediated endocytosis, suggesting that the cyclic RGD peptide-conjugated polyplex micelle has promising feasibility as a site-specifically targetable gene delivery system.