[The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA].

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

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.

SsoII-like DNA-methyltransferase Ecl18kI: interaction between regulatory and methylating functions.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Delta(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

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.

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.

Synthesis of 2'-modified oligonucleotides containing aldehyde or ethylenediamine groups.

Oligonucleotides carrying 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2'-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2'-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.

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.

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.

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.

[Synthesis of modified oligodeoxyribonucleotides containing 9-(2'-amino-2'-deoxy-beta-D-arabinofuranosyl)adenine]. / Sintez modifitsirovannykh oligodezoksiribonukleotidov, soderzhashchikh 9-(2'-amino-2'-dezoksi-beta-D-arabinofuranozil)adenin.

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.

The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding.

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.