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One of the most popular nonviral delivery systems for gene therapy constructs are carriers based on polyethylenimine (PEI) DNA complexes. A number of disadvantages associated with the lack of targeted delivery and increased cytotoxicity are overcome by adding auxiliary molecules to the complexes. An example of this is chondroitin sulfate (CS). The purpose of this work was to assess the effect of CS on the transfection properties of DNA–PEI complexes under different conditions of their preparation and transfection protocols. All complexes were prepared in solutions with high and low ionic strength. Transfection of C26 cells was performed according to two protocols differing in the presence of serum in the medium. The portion of transfected cells, transgene expression level, and cell viability were the main parameters of assessing the transfection efficiency. In binary DNA–PEI complexes prepared in different salt conditions, using different transfection protocols, the difference in the portion of transfected cells reached ten times. Addition of CS improved this transfection efficiency indicator up to 6.5 times, while the maximum difference in this indicator for the corresponding ternary complexes was reduced to 2.5 times. Changes in the proportion of CS in the composition of the complexes had an insignificant effect on their transfection properties. In the case of complexes prepared in high ionic strength solutions, the order of CS addition was also important. The best results of transfection efficiency were achieved with ternary complexes prepared in low ionic strength solutions, using a serum-free protocol, while these indicators were comparable with the data for Lipofectamine 2000. The addition of chondroitin sulfate improves the transfection properties of DNA–PEI complexes and makes them less dependent on the methods of preparation and transfection.

Naturally occurring positively charged proteins can be promising carriers for nucleic acid transport in gene therapy. The most attractive alternative among them is histones. In this work, we describe expression and purification of recombinant human histones H2A and H2B and of chimeric histone H2A with HIV-1 TAT fragment (TAT-peptide). The proposed method of purification of histone proteins can significantly reduce the content of bacterial endotoxins in the target preparation, which makes it possible to use these proteins in in vivo experiments. The transfection ability of plasmid DNA complexes with core histones H2A and H2B and the chimeric histone was demonstrated. A highly specific and efficient transfection of human HT1080 cell line with the use of histones H2A and H2B was detected, whereas transfection by plasmid DNA complexes with chimeric H2A-TAT protein was observed for many cell lines.

To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus (HSVtk) and mouse OX40L gene (m0X40L) involved in regulation of immune response was constructed. Therapeutic transgene expression was directed by a strong constitutive promoter of human cytomegalovirus (CMV), while translation occurred on an RNA template due to an encephalomyocarditis virus internal ribosome entry site (construct CMV-HSVtk-IRES-mOX40L) or 2A-peptide of Porcine Teschovirus-1 (construct CMV-HSVtk-2AmOX40L) inserted between them. The constructs obtained were functionally validated by transient transfection of C26 cells (mouse colon carcinoma). Both bicystronic vectors demonstrated strong cytotoxic activity and produced cytotoxic protein HSVtk at the similar level. To analyze efficiency of mOX40L expression, C26 transfected cells were immunofluorescence-labelled with anti-OX40L-antibodies conjugated with phycoerythrin following flow cytometric analysis. Production of mOX40L was higher in the case of CMV-HSVtk- 2A-mOX40L. Better performance of this expression vector was correlated with our data. Previously, we have demonstrated that a higher level of transgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence. Thus, transfection with the vector based on the 2A peptide sequence is optimal for achieving high efficiency of expression of both oncotherapeutic genes.

In this study, we evaluated the antitumor activity of a gene therapy complex in which the tumor-specific control of the expression of the effector suicide gene FCU1 was performed using a two-vector system based on the site-specific Cre-LoxP recombinase system. The complex of interest showed a high therapeutic potential in a mouse colon adenocarcinoma model.

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFR&beta, ). In this study, we fused histone H2A with PDGFR&beta, binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFR&beta, positive and PDGFR&beta, negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFR&beta, positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFR&beta, positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFR&beta, positive tumor stromal cells.

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.

The mechanism and kinetics of hydrogen adsorption on Pd/Ni nanostructures on an Al2O3 surface were studied. The dependence of electrical conductivity on the degree of hydrogen adsorption and the temperature effect on the rate of adsorption were determined.

The effect of treatment of Diflon, Makrolon®, and Lexan® polycarbonate films in the cathode fall of a dc discharge was studied. Plasma treatment was shown to result in the hydrophilization of the film surface. The dependence of the contact angle on the discharge current and treatment time was examined. It was found that the discharge induced a negative charge on the polycarbonate surface. The surface-charge density was correlated with the contact angle for various treatment conditions. Space charging processes in glow discharge-treated films were investigated using the thermally stimulated relaxation and depolarization techniques. The role of produced charged entities in an increase in the surface energy of modified polycarbonate films was revealed.

The novel radiation and photo-induced cryochemical solid-state synthesis of metal–polymer materials as well as the structure and physico-chemical properties of synthesized materials were described. The synthesis consists of low-temperature co-condensation of metal and monomer vapors followed by low-temperature solid-state polymerization of the co-condensate under irradiation. Depending on metal nature and monomer structure, this process allows to prepare specific metal–polymer systems: polymers containing organometallic units in polymer chains, metal–polymer complexes or stable metal clusters of various sizes trapped physically inside polymer matrix without coordination bonds between metal and polymer. The thermal treatment of synthesized systems leads to the formation of novel nanocomposite metal–polymer materials characterized by specific electric sensor and magnetic properties, catalytic activity and dielectric anomalies, which are discussed in relation to material structure.

The alteration of properties of both sides of the laminated polyimide-fluoropolymer film PMF-351 by treatment in a low-frequency glow discharge, depending on their position with respect to the electrode, was investigated. By the dynamic capacitance method, it was found that both positive and negative charges can be induced on both sides of the film modified in the cathode fall, depending on the arrangement of its polyimide and fluoropolymer sides with respect to the electrode. It was shown that the formation of a negative charge in PMF-351 is in all cases due to trapping of plasma electrons injected into the polymer surface layers.

New phosphoramidite reagent 7 suitable for incorporation of dC analogue was synthesized. ODNs containing diazaphenoxaziione residues in defined positions were prepared. The stability of duplexes formed was increased up to 3-5°C per modified base. Preliminary results of molecular biological testing were reported. This work was partly supported by RFBR grant 96-04-49209, by grant HHMI#75195-544201, LLNL P99U, and grant DOE agreement No. B307902 mod. 04. The authors would like to thank Dr. Tatyana L. Azhikina for valuable help in DNA sequencing and Dr. Natalya P. Potapova for NMR spectra recording.

Data on the synthesis of aluminum oxide nanostructures on the silicon surface by the oxidation of atomic aluminum layers with a high-frequency inductively coupled oxygen discharge plasma are reported. The conditions for the formation and the properties of aluminum oxide nanolayers were determined. It was found that this method affords oxide films with uniform structure and composition.

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine and 5-propynyl-2'-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments from mixtures.

Oligonucleotides containing the modified bases 5-methylcytosine and 2-aminoadenine in place of cytosine and adenine, respectively, have higher than normal affinity for complementary sequences The strong binding oligonucleotides (SBO) are much better than their normal counterparts in PCR amplification: they yield significantly more product per cycle, allow amplification at annealing temperatures as high as 72 degrees C and, unlike their normal counterparts, allow efficient priming from within a palindromic sequence. We propose that such strong binding oligonucleotides will be valuable in numerous PCR applications, including: (i) minimization of the frequency of mutants among PCR products; (ii) when only short specific primers can be designed based on available sequence information; (iii) when the material available for the analysis is limited in quantity; and (iv) when primer binding is blocked by DNA secondary structure involving a primer binding site, or chain extension is impeded by secondary structure in downstream sequences.

New phosphoramidite reagent 7 suitable for incorporation of dC analogue was synthesized. ODNs containing diazaphenoxaziione residues in defined positions were prepared. The stability of duplexes formed was increased up to 3-5°C per modified base. Preliminary results of molecular biological testing were reported. This work was partly supported by RFBR grant 96-04-49209, by grant HHMI#75195-544201, LLNL P99U, and grant DOE agreement No. B307902 mod. 04. The authors would like to thank Dr. Tatyana L. Azhikina for valuable help in DNA sequencing and Dr. Natalya P. Potapova for NMR spectra recording.

Laser spectroscopy and kinetic mass spectrometry of polyatomic isolated molecules have been carried out by using stepwise laser ionization of molecules in a mass spectrometer. The optical absorption spectrum of NO(2) has been recorded, for example, by using pulsed dye-laser excitation and H(2) vacuum-ultraviolet laser ionization of molecules. The kinetics of excited electron states and of the production of molecular and fragmented ions of some polyatomic molecules have been investigated with high temporal resolution. The sensitivity achieved is 10(-10) cm(-1) of the absorption coefficient and 10(9) (10(5)) molecules in the ground (electronically excited) state in the irradiated volume.

Background Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system. Methods We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte–macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models. Results We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan. Conclusions We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier – a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0433-0) contains supplementary material, which is available to authorized users.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerse directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.

We use modified oligonucleotides with enhanced strength of complementary DNA binding for primer walking DNA sequencing with strings of short contiguous oligonucleotides as primers. Such an approach allows us to reduce the probability of primer failures due to unstable binding of oligos with templates. In this paper the factors affecting the priming efficiency of segmented primers (strings composed of several short oligonucleotides contiguously juxtaposed on the template) used for DNA sequencing were investigated. Modified oligonucleotides were used to discriminate the effects caused by intrinsic properties of the oligonucleotides and by template features. It was shown that the most crucial factor is the stability of the duplex formed by the template with the 3'-outermost oligonucleotide of a string. The data were obtained with a model M13 template and in the process of sequencing the region flanking a long terminal repeat of human endogenous retrovirus HERVK mapped on chromosome 19. The sequencing was done by primer walking with strings of contiguous modified hexanucleotides. The effects revealed should be taken into consideration when choosing oligonucleotide units of segmented primers and for construction of minimised libraries composed of short unit oligonucleotides.

The polymerase chain reaction with degenerate primers corresponding to the most conservative amino acids 16-21 (ELEKEF) and 49-54 (WFQNRR) of the Antennapedia class homeodomain was used for the amplification of cDNA from regenerating planarians (asexual race of Dugesia tigrina). A total of six new Antennapedia-like homeobox sequences, designated Dutarh-1-Dutarh-6 (Dugesia tigrina asexual race homeobox gene), were obtained. Their comparison with other homeobox genes using a "Genebee" software (the EMBL Data Library) showed that all sequences except Dutarh-6 belong to the Antennapedia class. Dutarh-6 is closely related to a recently described novel homeobox gene subfamily that includes mouse mesodermal homeobox genes Mox-1 and Mox-2 and rat homeobox gene Gax.

Modified oligonucleotides containing 5-methylcytidine and/or 2-aminoadenosine form tighter hybrids with DNA and are, therefore, more efficient primers for DNA sequencing as compared to their natural counterparts. Strings of contiguous modified pentanucleotides can be used for DNA sequencing by primer walking.

We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver). Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin. After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer. The method has been applied to purification of fragments from a 2.9 kb plasmid added to E. coli DNA at equimolar quantity. Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR.

The chemically induced dynamic neclear polarization (CIDNP) method showed that the photodissociation of 4-benzoyl-4-phenyl-1,3-dioxolane takes place according to a Norrish type I mechanism from the triplet excited electron state. Reactions involving benzoyl and 4-phenyl-1,3-dioxolyl radicals were unambiguously detected in the analysis of the polarization of the signals in the CIDNP spectra of the products of photolysis of benzoylphenyldioxolane.

1. We have detected and investigated chemical polarization of protons and carbon nuclei in photochemical reactions ofα-hydroxyketones. 2. On the basis of CPN, we have formulated a mechanism for the radical photoconversions of ketones.

Methods of obtaining oligodeoxyribonucleotide 3′-phosphates based on the triester synthesis of oligodeoxyribonucleotides are discussed. These methods can be divided into two classes: I) phosphorylation of the 3′-OH group in a protected oligo-deoxyribonucleotide; and II) modification of a terminal nucleotide 3′-phosphate. It has been shown that a universal method is the preparation of an oligonucleotide with a 3′-terminal uridine residue followed by its oxidative elimination. The methods discussed in the paper have been used for obtaining a set of oligodeoxyribonucleotide 3′-phosphates required for the chemical “cross-linking” of oligonucleotides in DNA duplexes.

Two methods for synthesizing oligodeoxyribonucleotides containing 3'-terminal phosphosphate groups have been developed. The first consists in the synthesis and the introduction into the oligonucleotides of a block containing a 3'-terminal bis(2-cyanoethyl) phosphate group and the second in the use of the transesterification reaction of oligonucleotides with 2-cyanoethanol in the presence of cesium fluoride. Both methods permit the fairly effective synthesis of oligodeoxyribonucleotide 3'-phosphates.

It has been established by kinetic and spectral methods that the cyclohexadiene carbene generated in the photolysis of 2,6-di-tert-butyl-1,4-benzoquinone diazide in benzene predominantly in the singlet state reacts with benzene to form a norcaradiene derivative, which isomerizes thermally to 4-phenylcyclohexadiene-2,5-one. The latter is converted thermally to 2,6-di-tert-butyl-4-phenylphenol.

1. 1H and13C NMR spectroscopy was used to study the photoconversion of methyl n-propyl ketone, methyl benzyl ketone, and methyl adamantyl ketone. The13C and1H CIDNP signs indicated that the polarization arises upon the cleavage of excited ketones in the triplet state. 2. The competition of the radical photodecomposition (Norrish type-I) and “biradical” photodecomposition was studied in the photolysis of methyl n-propyl ketone. The ratio of the probabilities of the two radical pathways (recombination and disproportionation) is 3∶1, while the ratio of the two biradical pathways (photocyclization and Norrish type-II decomposition) is 3∶7. 3. A mechanism was established for the steps in the conversions of the radicals and radical pairs arising in the photolysis of the ketones studied.

A simple method is proposed for obtaining two kinds of polymeric supports of the polystyrene type for the solid-phase synthesis of oligonucleotides containing 5′-terminal phosphate groups. For both kinds of supports the optimum conditions of adding the first nucleotide and of splitting out the nucleotide material from the polymer have been worked out and the synthesis of a number of di- and trinucleotides of the deoxy series has been performed.

This review deals with studies on primary photoionisation and molecular decomposition processes and ion–molecule reactions in the gas phase. Problems concerning the nature of the electronic and vibrational states arising on photoionisation and their role in unimolecular and bimolecular reactions of organic compounds are discussed. Various pathways for the formation of secondary products in ion–molecule reactions have been determined. Data are presented on the energy relations of ion–molecule reactions and the proton affinities of molecules. The bibliography includes 69 references.

The relation between the ionisation potentials of aromatic and heterocyclic compounds and their electronic structures is considered. The nature of the orbitals in the ground states of molecules of different structures is examined on the basis of an analysis of the ionisation potentials. It has been shown how substituents affect the positions of the electronic levels of aromatic compounds. The bibliography includes 57 references.