Modified Oligonucleotides: New Structures, New Properties, and New Spheres of Application.

Nucleic acids have made a long and arduous journey "from the bench to the bedside." At present, it can be assumed that drugs based on modified oligonucleotides will find a worthy application in personalized medicine of the future.

[10-year results of using segmental pelvic irradiation combined with local irradiation of the prostate in patients with prostate cancer with multiple bone metastases].

The objective was to show the importance of external beam radiotherapy (EBRT) to increase the effectiveness of treatment and improve the quality of life of patients with generalized prostate cancer (PC). From 1992 to 2005 at our institution 205 patients with morphologically verified generalized (stages T1-4N0-1M1b) PC received EBRT. EBRT was conducted against the background of hormone therapy. Irradiation was begun with segmental pelvic irradiation with consistent reduction in the volume of exposure to locoregional and local irradiation of the prostate. The total tumor dose on the prostate was adjusted up to therapeutic level (66-72 Gy). In case of PC generalization with a primary lesion of the pelvis and lumbosacral spine, palliative EBRT according to the proposed method was justified. EBRT not only improved the quality of life of patients but also increased its duration.

Regulation of activity of transcription factor NF-κB by synthetic oligonucleotides.

Eukaryotic dimeric nuclear factor-κB (NF-κB) is one of the main transcription factors that activate expression of genes, products of which play the key role in development of cardiovascular pathologies, carcinogenesis, and inflammatory and viral diseases. In this review, the main attention is given to modulation of the transcription factor NF-κB activity by antisense oligonucleotides and oligonucleotide decoys. Also, current concepts about interactions between NF-κB dimers and DNA and general problems that arise in experimental use of synthetic oligonucleotides in vivo are discussed.

[First experience of using positron emission tomography with 11C-choline in prostate cancer (PC)].

Introduction into clinical practice of combined positron emission technology and computer tomography (PET/CT) allows in one study to identify structural and functional abnormalities. The study involves 32 patients who underwent PET/CT with "C-choline, including 5 patients with prostate cancer (PC), 3--with chronic prostatitis and 24--with biochemical PC recurrence. PET/CT with 11C-choline has a high diagnostic efficacy in detection of local recurrence and PC metastases in patients with biochemical PC recurrence. The results of visual analysis do not permit to distinguish PC from benign prostate diseases.

[Main results of research reported by the Center's Department of Innovation in Radiotherapy].

The efficacy of multimodality conservative treatment for prostate and bladder cancer and cervical carcinoma was improved due to integration of such modem modalities as intra-arterial chemotherapy, local hyperthermia and hyperglycemia and combination of local and systemic radiomodifiers. Our methods use criteria of actual survival and are intended to raise it.

DNA-methyltransferase SsoII as a bifunctional protein: features of the interaction with the promoter region of SsoII restriction-modification genes.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.

Analysis of DNA-protein interactions in complexes of transcription factor NF-kappaB with DNA.

We have applied bioinformatic analysis of X-ray 3D structures of complexes of transcription factor NF-kappaB with DNAs. We determined the number of possible Van der Waals contacts and hydrogen bonds between amino acid residues and nucleotides. Conservative contacts in the NF-kappaB dimer-DNA complex composed of p50 and/or p65 NF-kappaB subunit and DNA sequences like 5 -GGGAMWTTCC-3 were revealed. Based on these results, we propose a novel scheme for interactions between NF-kappaB p50 homodimer and the kappaB region of the immunoglobulin light chain gene enhancer (Ig-kappaB). We applied a chemical cross-linking technique to study the proximity of some Lys and Cys residues of NF-kappaB p50 subunit with certain reactive nucleotides into its recognition site. In all cases, the experimentally determined protein-DNA contacts were in good agreement with the predicted ones.

[Combined radiotherapy and endovascular x-ray therapy for invasive cancer of the urinary bladder].

The data are presented on treatment of 131 patients with transitional cell carcinoma of the urinary bladder. Radiotherapy was received by 57, regional intraarterial chemotherapy (RIAT)--27. Radiotherapy was combined with RIAT and selective hyperglycemia (HG) in 18 cases. Local super high-frequency (SHF) hyperthermia was given additionally to another 29 patients. Radiotherapy alone was followed by primary clinical cure, a 9.6-months relapse-free period and 3-months survival (36.8%), mean survival time being 26 months. In the RIAT group, these indices were 29.6%, 10.8 mos, 51.9% and 36 mos, respectively. Combination of radiotherapy, RIAT and selective HG yielded significantly improved indices: complete response--44.4%, relapse-free period--13.6 mos, 3-year survival--66.7% and mean survival time--43 mos. After addition of SHF hyperthermia, complete response rose to 69.0%, relapse-free period--18.2 mos, 3-year survival--75.8% and mean survival time--61 mos. Joint use of radiotherapy, RIAT, HG and SHF hyperthermia caused more damage to tumor, stimulated complete response and increased 3-year survival and mean survival time.

[Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group]. / Kovalentnoe sviazyvanie Cys142 metiltransferazy SsoII s DNK-dupleksami, soderzhashchimi fosforildisulfidnuiu mezhnukleotidnuiu grupppu.

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.

[DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins]. / DNK-dupleksy, soderzhashchie 2'-dezoksi-2'-iodatsetamidouridin, kak reagenty dlia affinnoi modifikatsii belkov.

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

[Synthesis and properties of oligodeoxyribonucleotids with mono- and diphosphoryldisulfide internucleotide groupings]. / Sintez i svoistva oligodezoksiribonukleotidov s mono- i difosforildisul'fidnymi mezhnukleotidnymi gruppirovkami.

The synthesis of oligodeoxyribonucleotides bearing mono- and diphosphoryldisulfide internucleotide links was optimized. Oligonucleotide 3'-thiophosphorothioates were modified using the thiophosphoryl-disulfide exchange with preactivated 5'-deoxy-5'-mercaptooligonucleotides or 5'-phosphorothioate derivatives both with and without a complementary template. The lack of template was shown to differently affect the product ratio (homo- and heterodimers) in the reactions of mono- and diphosphoryldisulfide-containing oligonucleotides. A replacement of one natural phosphodiester bond in 15-16-mer duplexes by a mono- or diphosphoryldisulfide group causes a slight thermal destabilization of the corresponding duplex. The disulfide recombination of the resulting compounds was studied.

New chemically reactive dsDNAs containing single internucleotide monophosphoryldithio links: reactivity of 5'-mercapto-oligodeoxyribonucleotides.

Novel modified DNA duplexes with single bridging 5'-SS-monophosphoryldithio links [-OP(=O)-O(-)-SS-CH(2)-] were synthesized by autoligation of an oligonucleotide 3'-phosphorothioate and a 5'-mercapto-oligonucleotide previously converted to a 2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent template-dependent process under the conditions used and does not require strong binding of the reactive oligomers to the complementary strand. The modified internucleotide linkage, resembling the natural phosphodiester bond in size and charge density, is stable in water, easily undergoes thiol-disulfide exchange and can be specifically cleaved by the action of reducing reagents. DNA molecules containing an internal -OP(=O)-O(-)-SS-CH(2)- bridge are stable to spontaneous exchange of disulfide-linked fragments (recombination) even in the single-stranded state and are promising reagents for autocrosslinking with cysteine-containing proteins. The chemical and supramolecular properties of oligonucleotides with 5'-sulfhydryl groups were further characterized. We have shown that under the conditions of chemical ligation the 5'-SH group of the oligonucleotide has a higher reactivity towards N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than does the OH group. This autoligation, unlike disulfide bond formation, proceeds only in the presence of template oligonucleotide, necessary to provide the activated phosphate in close proximity to the SH-, OH- or phosphate function.

Amperometric detection of DNA hybridization on a gold surface depends on the orientation of oligonucleotide chains.

We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.

[Chemical ligation and recombination of DNA fragments by formation (exchange) of disulfide bonds, located in the sugar-phosphate backbone]. / Khimicheskoe ligirovanie i rekombinatsiia fragmentov DNK posredstvom obrazovaniia (obmena) disul'fidnykh sviazei, lokalizovannykh v sakharofosfatnom ostove.

Effective methods of the directed introduction of diphosphoryl disulfide bridges into hairpin DNA duplexes in place of natural phosphodiester groups were developed using the H2O2-effected ligation of 3'- and 5'-thiophosphorylated oligonucleotides or the autoligation of a preactivated oligonucleotide derivative with a phosphorothioate-bearing oligomer. The postsynthetic recombination of the disulfide-linked oligonucleotide fragments was characterized. It was shown that, along with template-directed reactions, out-of-duplex formation and exchange of diphosphoryl disulfide bonds in the DNA sugar-phosphate backbone may occur. In modified hairpin DNA, a spontaneous exchange of disulfide-linked fragments virtually does not take place because of the intramolecular duplex formation.

Hairpin-shaped DNA duplexes with disulfide bonds in sugar-phosphate backbone as potential DNA reagents for crosslinking with proteins.

Convenient approaches were described to incorporate -OP(=O)O(-)-SS-O(-)(O=)PO- bridges in hairpin-shaped DNA duplexes instead of regular phosphodiester linkages: (i) H2O2- or 2,2'-dipyridyldisulfide-mediated coupling of 3'- and 5'-thiophosphorylated oligonucleotides on complementary template and (ii) more selective template-guided autoligation of a preactivated oligonucleotide derivative with an oligomer carrying a terminal thiophosphoryl group. Dithiothreitol was found to cleave completely modified internucleotide linkage releasing starting oligonucleotides. The presence of complementary template as an intrinsic element of the molecule protects the hairpin DNA analog from spontaneous exchange of disulfide-linked oligomer fragments and makes it a good candidate for auto-crosslinking with cysteine-containing proteins.

Plasmodium falciparum: effect of chemical structure on efficacy and specificity of antisense oligonucleotides against malaria in vitro.

Antisense oligodeoxynucleotides (AS ODNs) have shown promise both as potential anti-malarial chemotherapeutic agents and as a means for identifying genes critical for parasite survival. Because conventional ODNs containing phosphodiester (PO) groups are subject to rapid nuclease degradation, ODNs with phosphorothioate (PS) groups are commonly used. However, at high concentration, these lose target specificity, and in some animal models, they become toxic. We compared a variety of chemical modifications (PO, PS, PO-PS hybrids, 2'-O-methyl-2'-deoxy chimeras) and structural modifications (sequence alterations favoring self-stabilizing loop formation) for their ability to inhibit Plasmodium falciparum malaria cultured in vitro. All modifications were done using an AS ODN sequence targeted against dihydrofolate reductase thymidylate synthase (DHFR). Inhibition by PO-PS hybrids containing as few as three PS groups at the 3'- and 5'-ends did not differ significantly from that obtained using compounds containing all-PS groups. Similarly, inhibition by PS chimeric compounds containing 2'-O-methyl modifications did not differ significantly from that of conventional PS compounds. In contrast, while inhibition by PO-PS hybrid chimeras did not differ significantly from that of all-PS compounds at low concentrations, at 1 microM they inhibited parasite growth 25% less (P < 0.001) than all-compounds or PS 2'-O-methyl-2'-deoxy chimeras. Extension of the nucleotide sequence to increase stem-loop formation yielded two compounds which inhibited parasite growth about 20% more than unmodified compounds, though this difference was not significant. Furthermore, most of this increase appears to correlate with the greater number of PS groups associated with the increased ODN length. We conclude that limiting the number of PS groups and inclusion of PO 2'-O-methyl groups may yield compounds with high antisense activity but low non-sequence-dependent effects. Such compounds are currently being tested in vivo.

[Current methods of radiation therapy in patients with prostatic cancer: subtotal body radiation, local hyperthermia]. / Sovremennye metodiki luchevoi terapii bol'nykh rakom predstatel'noi zhelezy: subtotal'noe obluchenie tela, lokal'naia gipertermiia.

The paper deals with the preliminary data of treatment of patients with prostatic cancer by using unconventional methods of radiation therapy (RT), such as subtotal radiation of the body (STRB) and thermoradiation treatment (TRT). Out of 72 patients receiving RT, 16 and 8 had STRB and TRT, respectively. Systemic and local drug therapies were made to prevent radiation reactions and injuries. In all cases, STRB and TRT showed a significant objective and subjective effect. To evaluate the long-term results of treatment needs further studies.

[HPLC of oligodeoxyribonucleoside phosphorothioates ]. / V'EAZKH oligodesokciribonukleozidtiofosfatov.

Conditions are described for the separation of synthetic oligodeoxyribonucleoside phosphorothioates by HPLC using a SCOUT WP PEI weak anion exchanger and for the separation of oligodeoxyribonucleotides with mixed phosphate and phosphorothioate internucleotide bonds on a Partisphere C18 support in ion-pair conditions. The influence of the nucleoside composition and the number of phosphorothioate bonds on the retention time was studied.