Biomedical subjects

T S Oretskaya

Publications and source records attributed to T S Oretskaya.

At least 19 recordsLinked to original sources

PubMed2005

Affinity modification of the restriction endonuclease SsoII by 2'-aldehyde-containing double stranded DNAs.

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.

Aldehydes↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Synthetic oligonucleotides: AFM characterisation and electroanalytical studies.

One of the most important steps in designing more sensitive and stable DNA based biosensors is the immobilisation procedure of the nucleic acid probes on the transducer surface, while maintaining their conformational flexibility. MAC Mode AFM images in air demonstrated that the oligonucleotide sequences adsorb spontaneously on the electrode surface, showing the existence of pores in the adsorbed layer that reveal big parts of the electrode surface, which enables non-specific adsorption of other molecules on the uncovered areas. The electrostatic immobilisation onto a glassy carbon electrode followed by hybridisation with a complementary sequence and control with a non-complementary sequence was studied using differential pulse voltammetry and electrochemical impedance spectroscopy. Changes in the oxidation currents of guanosine and adenosine were observed after hybridisation events as well as after control experiments. Modification of the double layer capacitance that took place after hybridisation or control experiments showed that non-specific adsorption of complementary or non-complementary sequences occur allowing the formation of a mixed multilayer.

Biosensing Techniques↗

PubMed2005

Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents.

The review describes key aspects of the synthesis and biological activities of conjugates of oligonucleotides and their analogues with synthetic peptides, in particular aimed towards gene silencing applications. The common methods of synthesis of oligonucleotide-peptide conjugates (OPCs) and PNA-peptide conjugates (PPCs) are described, which include both total solid-phase and fragment coupling approaches. In addition, various applications of conjugates as gene silencing agents are outlined. These include antisense and steric block applications in mammalian cells of OPCs, PPCs and phosphorodiamidate morpholinooligonucleotide (PMO)-peptide conjugates, gene silencing in bacteria, various DNA targeting applications, and recent reports of gene silencing activities of siRNA-peptide conjugates. A table listing all peptides used as oligonucleotide conjugates for gene silencing applications is also included.

Animals↗

PubMed2004

Electrochemical sensing of the behaviour of oligonucleotide lipoplexes at charged interfaces.

Complexes between short oligodeoxynucleotides (ODN) with a variable dG(x)dC(y) base composition and liposomes composed of the cationic lipid DOTAP (ODN lipoplexes) were studied by differential pulse voltammetry at a glassy carbon electrode. Since lipoplexes are spontaneously formed by electrostatic interactions, the objective of the voltammetric study was to investigate their behaviour at the electrode surface/solution interface. It was verified that the peak current in the voltammograms for ODN lipoplexes was due to guanosine oxidation and that it was influenced both by the applied adsorption potential and the lipoplex (+/-) charge ratio used. It was found that for low ODN lipoplexes (+/-) charge ratios the peak current obtained was enhanced when compared to that registered with free ODN for the same concentration. This allowed a higher sensitivity in the determination of ODN by differential pulse voltammetry and a limit of detection of 5.5 ng/mL was achieved. A model that explains the organisation of ODN lipoplexes at the electrode surface/solution interface is proposed. The electrochemical results presented account for a better physicochemical characterisation of lipoplexes at charged interfaces, which can be important for the understanding and development of gene therapy vectors based on ODN lipoplexes.

Biosensing Techniques↗

PubMed2004

AFM and electroanalytical studies of synthetic oligonucleotide hybridization.

The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.

Adsorption↗

PubMed2003

Hybridization of DNA at the surface of phospholipid monolayers. Effect of orientation of oligonucleotide chains.

We studied the properties of lipid monolayers formed at the air-water interface composed of dioleoylphosphatidylcholine (DOPC) with incorporated short (19-mer) oligonucleotides. These oligonucleotides were modified by oleylamine at both (3' and 5') terminals or only at one (3') terminal. Interaction of single-stranded (19-mer) oligonucleotides without oleylamine with DOPC monolayers resulted only in slight increase of surface pressure and the area per phospholipid molecule, while more substantial and significant increase of these values were observed following incorporation of oligonucelotides modified by oleylamine. This influence is similar for both types of oligonucleotide modifications. However, considerable differences in changes of monolayer properties took place after hybridization with complementary oligonucleotides. The hybridization of oligonucleotides with the DNA modified by oleic acid at both 3' and 5' terminals at the surface of lipid monolayer resulted in further increase of surface pressure and in the increase of the area per phospholipid molecule, while decrease of both the surface pressure and the area per phospholipid molecules were observed for hybridization with DNA modified by oleic acid at 3' terminal. It is possible that in latter case, the hybridization caused the loss of hybridized molecules from monolayers. Interaction of noncomplementary chains with DOPC monolayers with incorporated oleyl acid-modified DNA also influenced the properties of monolayers, but the effect was weaker in comparison with that observed for complementary chains.

DNA↗

PubMed2002

A novel method of introducing hydrophobic moieties into oligonucleotides for covalent and non-covalent immobilization on electrode surfaces.

An effective method for the introduction of oleylamine-modified cytidine units into predetermined position(s) of the oligodeoxyribonucleotide (ON) chain during automated ON synthesis has been developed. The high yields of the condensation products upon the introduction of the modified units allow the methods suggested to be used for the synthesis of ONs with two hydrophobic substituents. We also suggest a simple method for obtaining ONs with 5'-terminal hydrophobic linker with free thiol group. The functionality of synthesized ON modified by thiol group and that with hydrophobic spacer for the detection DNA hybridization has been approved in conductometric experiments.

Base Sequence↗

PubMed2001

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.

Chromatography, High Pressure Liquid↗

PubMed2000

Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking.

A target sequence-specific DNA binding region of the restriction endonuclease Sso II was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-IdU) at the central position of the Sso II recognition site (CCNGG). For this purpose the Sso II-DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was approximately 50%. In the presence of excess unmodified oligodeoxynucleotide or with oligode-oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked Sso II-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxy-nucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of Sso II to its DNA substrate.

Base Pairing↗

PubMed2000

Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E. coli RNA polymerase using regioselective cross-linking.

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E. coli RNA polymerase in the open complex model were studied. A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified. It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1). Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1). Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.

Base Sequence↗

PubMed1999

Oligonucleotide-peptide conjugates as potential antisense agents.

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.

Adenosine↗

PubMed1998

Role of DNA definite structural elements in interaction with repair enzyme uracil-DNA glycosylase.

Interaction of different oligodeoxyribonucleotides (oligos), their analogs and oligonucleopeptide with uracil-DNA glycosylase (UDG) from human placenta was investigated. It is shown that there is no considerable contribution of heterocyclic bases of DNA to UDG-substrate binding but the UDG interaction with some DNA phosphate groups is necessary for enzyme-substrate recognition. However the phosphate group adjacent to single dU from the 3'-end in oligo is not involved into the electrostatic contact with UDG. It is found that UDG has the high affinity to its reaction product. An oligonucleotide containing a single 2'-deoxy-2'-aminouridine is a non-hydrolyzable substrate analog for UDG.

DNA↗

PubMed1997

A new technique for the characterization of long-range tertiary contacts in large RNA molecules: insertion of a photolabel at a selected position in 16S rRNA within the Escherichia coli ribosome.

A new approach for inserting a photo-label at a selected position within the long ribosomal RNA molecules has been developed. The Escherichia coli 16S rRNA was cleaved at a single internucleotide bond, 1141-1142, with RNase H in the presence of a complementary chimeric oligonucleotide. 4-Thiouridine 5', 3'-diphosphate was ligated to the 3'-end of the 5'fragment at the cleavage site with T4 RNA ligase. The 16S rRNA fragments containing this added photo-reactive nucleotide were assembled together with total 30S ribosomal proteins into small ribosomal subunits. The ability of such 30S particles containing fragmented rRNA to form 70S ribosomes has been demonstrated previously. Crosslinks were induced within the 30S subunits by mild UV irradiation. The sites of crosslinking within the 16S rRNA were then analyzed using RNase H digestion and reverse transcription. Two crosslinks from the thio-nucleotide attached to nt C1141 of 16S rRNA were observed, namely to nt U1295 and G1272. These results are in agreement with the established proximity of helix 39 and 41 in the 3D structure of the 30S ribosomal subunit, as shown by other intra RNA crosslinking data. These data furthermore allow us to refine the structural arrangement of helices 41 and 39 relative to one another.

Bacteriophage T4↗

PubMed1997

Chemical cross-linking of the human immunodeficiency virus type 1 Tat protein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues.

A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.

Amino Acid Sequence↗

PubMed1997

[Optimization of conditions for ion-pair HPLC of oligonucleotides].

Regularities of ion-pair HPLC of oligonucleotides were studied. An algorithm was suggested for the calculation of the acetonitrile gradient providing proportionality between the number of nucleotide residues and the retention time of the oligonucleotide (tR) (equidistant separation of oligonucleotides). The algorithm is based on extrapolation of the results of separation of a test mixture of five oligonucleotides varying in length from 10 to 30 units. It was shown that the logarithmic gradient of acetonitrile C0 + m ln(t) leads to the equidistant separation of oligonucleotides. The separation is characterized by two measured parameters: the retention time step and the extent of equidistantness. A functional dependence of the measured parameters on two varying parameters of the logarithmic function (C0 and m) was established experimentally. A method for calculating the expected retention time of an oligonucleotide from its nucleoside composition was suggested.

Acetonitriles↗

PubMed1997

[Synthesis of modified oligodeoxyribonucleotides containing 9-(2'-amino-2'-deoxy-beta-D-arabinofuranosyl)adenine].

A method for preparing a modified derivative of 2'-amino-2'-deoxy-arabino-adenosine ready for directed insertion into an oligonucleotide chain during solid-phase synthesis was elaborated. A series of the title oligonucleotides (6-25-mers) containing a 2'-amino-2'-deoxy-arabino-adenosine fragment were prepared. A high reactivity of the 2'-amino group during the acylation with carboxylic acid anhydrides was demonstrated. It was shown that the insertion of the modified fragments into oligonucleotides did not inhibit the formation of the DNA duplex.

Acylation↗

PubMed1996

Synthesis and properties of cross-linked DNA duplexes.

A method has been devised to synthesize DNA duplexes with covalently connected strands. The structure of cross-linked duplexes was confirmed by a reaction with the restriction endonuclease AluI. The thermal stability of the resulting compounds was investigated.

Base Sequence↗