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1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.

Oligonucleotides with 1,2-diol group were prepared from 2′-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3′-phosphoramidite. The thermal stability of modified DNA duplexes and their ability to form complexes with the p50 subunit of the NF-ϰB transcription factor and (cytosine-5)-DNA methyltransferase M.SsoII were studied. The periodate oxidation of the 1,2-diol group of the oligonucleotides resulted in reactive 2′-aldehyde derivatives. The opportunity of their use for the affinity modification of DNA-recognizing proteins was studied.

This review outlines the design and synthesis of oligonucleotides bearing 2'-modified residues at predeter- mined positions within the strand. The relative merits of incorporation of reactive carboxyl, carbonyl, iodoacetamide and disulfide-containing groups into oligonucleotides were considered along with solid-phase synthesis of DNA 2'- conjugates. The specific cross-linking of 2'-substituted oligonucleotides to nucleic acid-binding proteins (transcription factor NF- B, restriction-modification enzymes) was shown to be helpful in scanning the protein-DNA interface and studying the conformational dynamics of biopolymer ensembles. The future perspectives of chemically reactive DNA constructs as specific protein decoys are discussed.

We describe the preparation of two batches of a polymer support for the incorporation of folic acid into oligonucleotides. The method permits the regioselective attachment of a target nucleic acid sequence through its 3'-end to either the alpha-or gamma-carboxyl group of L-glutamic acid, respectively. The supports have been tested in solid-phase synthesis of oligonucleotide-folate conjugates for cell delivery studies.

Oligo-2′-O-methylribonucleotides containing residues of phenylalanine, histidine, and lysine amides were synthesized with the use of new phosphoramidites of 2′-aminoacid derivatives of uridine.

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore, viral integrase is of interest as a target for new antiviral drugs. Conjugates of 11-mer single-stranded oligonucleotides with hydrophobic molecules were shown to be efficient integrase inhibitors, inducing dissociation of the integrase-viral DNA complex. The dependence of the conjugate inhibitory activity on the oligonucleotide length and structure as well as on the structure of hydrophobic molecules was studied. Conjugates with eosin and oleic acid proved to be the most active. Conjugates of these molecules with 2′-O-methyl-oligonucleotide inhibited integrase at concentrations 50–100 nM but did not influence some other DNA-binding enzymes.

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

An efficient method for synthesis of oligonucleotide 2′-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2′-O-carboxymethyl group were obtained by use of a novel uridine 3′-phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2′-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide–oligonucleotide conjugates containing an amide linkage between the 2′-position of an oligonucleotide and the N-terminus of a peptide.

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 thymidine 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-κB.

The published data on the methods of chemical synthesis of oligonucleotides containing 5'- and 3'-terminal or internal carboxy or aldehyde functional groups are considered. The known methods are systematised and their advantages and disadvantages are discussed. The bibliography includes 65 references.

The published data on the methods of chemical solution and solid-phase synthesis of peptide–oligonucleotide conjugates are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to the solution synthesis of peptide–oligonucleotide conjugates are systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates,viz., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 references.

The synthesis of modified oligodeoxyribonucleotides# containing 2′-amino-2′-deoxyarabinoadenosine residues (aAn) was carried out by means of the standard phosphoramidite chemistry. A high reactivity of such compounds to electrophilic reagents was shown. The cross-link formation between 2′-amino group of aAn and carboxyl function introduced into complementary strands occurs with 55% yield. The aAn residues was shown to induce the increased resistance of modified oligomers towards the enzymatic cleavage and provide the insignificant destabilization of DNA duplexes. This publication is dedicated to Professor Tsujiaki Hata, who made a valuable contribution to the chemistry of nucleosides and nucleotides.

An efficient method for the synthesis of DNA or RNA oligonucleotide 2'-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2'-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH(3)CN. The resulting oligonucleotide 2'-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH(3)CN reduction to afford novel 2'-conjugates.

The published data on the methods of chemical solution and solid-phase synthesis of peptide–oligonucleotide conjugates are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to the solution synthesis of peptide–oligonucleotide conjugates are systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates,viz., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 references.

Synthesis of new non-nucleosidic building blocks that incorporate 2,2'-biquinoline is described. Starting from readily available bicinchoninic acid, the corresponding bifunctional monomeric reagents for solid-phase oligonucleotide synthesis, phosphoramidite and H-phosphonate, have been prepared. The compounds developed may be useful for the design of sequence-specific artificial nucleases.

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

2'-O-[2-(2,3-Diacetoxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite was prepared and used in solid-phase synthesis to obtain oligonucleotides containing a 1,2-diol group, which may then be converted into a 2'-aldehyde group. The oligonucleotides were conjugated efficiently to various molecules by chemoselective ligation that involves an addition-elimination reaction between the 2'-aldehyde group and a suitable nucleophile, such as a hydrazine, a O-alkylhydroxylamine or an 1,2-aminothiol. The method was applied successfully to the conjugation of peptides to oligonucleotides at the 2'-position.

The preparation of two novel 2'-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2'-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO4 to generate 2'-aldehyde oligonucleotides. These were successfully conjugated to acceptor molecules. The latter amidite also showed good coupling yields, but the hydrazide function was demonstrated to be labile under basic deprotection conditions.

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.

Certain amino acids and short peptides are known to act as enhancers of ribozyme-mediated RNA cleavage at low concentration of magnesium ion. Thus, covalent conjugates of oligonucleotides and some amino acids may have a potential for development as sequence-specific artificial ribonucleases. Here we would like to report an incorporation of basic amino acids, lysine and histidine, into oligonucleotides at the 2'-position of a uridine residue. The approach involves the 2'-O-alkylation and attachment of the corresponding amino acid by the alpha-amino group through a urethane-type linker.