Site-specific modification of the 5"-terminal fragment of PGY1/MDR1 gene mRNA by reactive conjugates of antisense oligonucleotides

The site-specific modification of the 5"-terminal fragment of PGY1/MDR1 mRNA by oligodeoxyribonucleotide conjugates bearing residues of bleomycin A₅ (Blm), cobalt(ii) tetracarboxyphthalocyanine (Phcn), 4-[N-(2-chloroethyl)-N-methylamino]benzylamine (RCl), or perfluoroarylazide (Az) was studied. Conjugates of oligonucleotides complementary to the RNA sequences 123—138 and 155—166 selectively modify RNA in the vicinity of these regions. The highest efficacy (up to 50%) was achieved in reactions with alkylating and perfluoroarylazide conjugates of oligonucleotides. Conjugates of perfluoroarylazide with 2"-O-modified oligonucleotides are much more efficient than analogous conjugates with oligodeoxyribonucleotides (extents of RNA modification are 40—50% and 20%, respectively).     

Catalytic DNA-and RNA-hydrolyzing antibodies from milk of healthy human mothers

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab′)₂ and Fab fragments. Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of cat alytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infec tions until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.     

Design and synthesis of artificial ribonucleases based on 1,4-diazabicyclo[2.2.2]octane and imidazole

The review surveys the results of our studies devoted to the design of highly efficient catalysts of hydrolysis of the phosphodiester bonds in RNA. These catalysts contain the imidazole residue in the catalytic domain, one or several bis-quaternized rings of 1,4-diazabicyclo[2.2.2]octane as a polycationic RNA-binding domain, and a lipophilic radical. A versatile approach to artificial ribonucleases of this type was proposed, which allows one to vary not only the number of positive charges in the RNA-binding domain, the structure of the catalytic site, and their mutual arrangement but also the domain structure of the molecule as a whole. Analysis of the catalytic properties of the synthesized constructs makes it possible to optimize the domain structure and the geometry of the molecule ensuring its maximum ribonuclease activity.     

Proton source size measurements in the eA-->e'ppX reaction

Two-proton correlations at small relative momentum q were studied in the eA(3He,4He,C,Fe)-->e(')ppX reaction at E(0)=4.46 GeV using the CLAS detector at Jefferson Lab. The enhancement of the correlation function at small q was found to be in accordance with theoretical expectations. Sizes of the emission region were extracted, and proved to be dependent on A and on the proton momentum. The size of the two-proton emission region for He was measured in eA reactions for the first time.     

Secretory immunoglobulin a from health y human mothers' milk catalyzes nucleic acid hydrolysis

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.     

Suppression of MDR1 gene expression by chemically modified siRNAs

The ability of chemically modified siRNAs targeted to MDR1 mRNA to inhibit P-glycoprotein expression and to restore sensitivity of cancer cells to antibiotic vinblastine was investigated. The effects of chemical modifications on RNA stability in cell culture medium and inhibition of MDR1 gene expression were tested. We found that siRNAs containing 2′-O-methyl ribonucleotides within either sense or/and antisense strands display high stability in serum but exhibit a significant reduction in the biological activity. The protection of 3′-ends of siRNA by introduction of 3′-3′-inverted phosphodiester bonds and two 2′-O-methyl ribonucleotides in protruding 3′-ends considerably increase their biological activity, which allows a 30-fold decrease in the cytostatic agent concentration required for cancer cell death. The data obtained demonstrate that the chemically modified siRNAs can be considered as potential therapeutics, which enhances the efficiency of cancer chemotherapy. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1227–1235, July, 2006.     

Artificial ribonucleases. 5. Synthesis and ribonuclease activity of tripeptides composed of amino acids involved in catalytic centers of natural ribonucleases

The characteristic features of the spatial arrangement of the main functional groups involved in catalytic centers of ribonucleases and nucleases were revealed by computer analysis of the catalytic centers of these enzymes. Based on the results of computer simulation, tripeptides containing Lys, Arg, His or Hia, Thr, and Asn in different combinations were synthesized. In these tripeptides, the distances between the corresponding functional groups are equal to those observed in natural enzymes. The efficacy of RNA cleavage with Arg- and His-containing tripeptides depends on their structure and correlates with the overall positive charge of these compounds. Of all the tripeptides under consideration, compounds bearing the overall charge of +4 exhibit the highest ribonuclease activity.     

Sensitized photomodification of oligonucleotide-binding proteins displayed on the eucaryotic cell surface

Interactions of double-stranded nucleic acids with cell surface proteins, which are involved in binding and transport of extracellular nucleic acids, were studied by the photoaffinity modification with a binary system of oligonucleotide conjugates. The photoreactive double-stranded complex involved an oligonucleotide template and two complementary to adjacent sequences oligonucleotide conjugates. One conjugate contained a photoreagent, viz., 4-azido-2,3,5,6-tetrafluorobenzaldehyde N-(3-aminopropionyl)hydrazone, at the terminus located in proximity to the terminus of another conjugate containing the sensitizer, viz., 9-aminomethylanthracene. Binding of photoreagent and the sensitizer to a single-stranded template yields the photoreactive center. Upon irradiation with visible light (400—580 nm), this photoreactive double-stranded complex forms covalent cross-linkages with oligonucleotide-binding surface proteins of eucaryotic SPEV cells.     

Investigation of surface oligonucleotide-binding proteins of eucaryotic cells by affinity modification with reactive oligonucleotide derivatives

Interactions between oligodeoxyribonucleotides (ODN) with different sequences and cell proteins were examined using the affinity modification by [³²P]-labeled reactive oligonucleotide derivatives. 3"-Terminal ribouridine oxidized with sodium periodate, 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine, and the maleimide residue were used as reactive groups. All the compounds used are specific reagents. The set of the discovered nucleic acid-binding (NA-binding) proteins depends on the chemical properties of the affinity reagent. The presence of the hydrophobic group at the 5"-terminus of the ODN molecule is the key factor determining the variety of the discovered NA-binding proteins. The cells of different origin (A431, HeLa, KB, MCF-7, Hap-2, K562, Cos-7, NIH/3T3, human-lung primary epithelial cells, and porcine kidney primary cells) are characterized by the same set of NA-binding proteins whose affinity modifications depends on the conditions of incubation of oligonucleotides with the cells. Treatments of cells disturbing the integrity of the cellular membrane (scrapping, treatment with trypsin, or cell permeabilization with streptolysin O or saponin), disrupt interactions between NA-binding proteins from native cells and ODN.