Shabarova Za

Chemical reactions within DNA duplexes Cyanogen bromide as an effective oligodeoxyribonucleotide coupling agent

Cyanogen bromide was found to condense oligodeoxyribonucleotides on a complementary template in aqueous solution. Optimum conditions for this vigorous and effective reaction were developed. CNBr proved to be useful for incorporation of phosphoramidate or pyrophosphate internucleotide bonds in DNA duplexes.

Synthetic nucleotide-peptides.

Publisher Summary This chapter deals with synthetic nucleotide-peptides, with phosphoamide and phosphoester bonds. It reviews that synthetic nucleotide-peptides proves to be very useful in elucidating the mode of action of enzymes of nucleotide exchange from the point of view of the activation of the phosphate residue of the mononucleotide, as well as from that of noncovalent interactions between the nucleotide and the active center of enzymes. Application of physical methods for the study of noncovalent interactions in the molecules of model nucleotide-peptides with closely arranged amino acid radicals and heterocyclic bases, open new perspectives for investigation of the structural arrangement of nucleoproteins in ribosomes and viruses. Besides, synthetic nucleotide-(P-N)-peptides are also useful for elaborating a model for the enzymatic synthesis of internucleotide linkages, as these compounds have some features of enzyme–substrate complexes. It concludes that the steric closeness of such synthetic “enzyme-substrate complexes” may give rise to formation of new internucleotide linkages. One may hope that this principle may become a basic one for the self-assembly of oligonucleotides to form double-stranded polynucleotides.

Modification of Oligo (Poly) Nucleotide Phosphomonoester Groups in Aqueous Solutions

Abstract Selective modification of oligo (poly) nucleotide phosphomonoester groups in an aqueous medium by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide in the presence of various nucleophilic agents has been investigated. Optimal conditions of the modification by amino- and hydroxycompounds have been found. Based on these studies a general efficient method for preparation of oligo (poly) nucleotide phosphoamidates and phosphodiesters in an aqueous solution has been developed. The method allows to prepare both oligodeoxyribonucleotide derivatives at 3′- and 5′-terminal phosphate groups and oligoribonucleotide derivatives at 5′-terminal phosphate groups with 80–100% yields.

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.

The behaviour of 2'-deoxy-2'-fluorouridine incorporated into oligonucleotides by the phosphoramidite approach

Abstract 2′-Deoxy-2′-fluorouridine has been chemically incorporated into an oligodeoxynucleotide of the structure 5′ACGGAX 3′ (X=U(2′-F)) using the phosphoramidite method and the behaviour of the product has been studied. 5′-O-Monomethoxytrityl-2′-deoxy-2′-fluorouridine was fixed on silica gel at the 3′-end and the chain elongated on a DNA-synthesizer using nucleoside methoxyphosphoramidites. After alkaline work-up two products were observed. One was found to be the desired fluoro containing hexamer, whereas the other corresponds to an araU-hexamer (X=arabino-furanosyluridine). The latter compound is supposed to be a product of alkaline hydrolysis of the C-2′-F-bond. The oligomers containing 2′-fluoro- and ara-U at their 3′-end were chemically sequenced by a solid phase method on CCS-paper which confirmed the right primary structure.

Addressed fragmentation of RNA molecules

Many problems of molecular biology require specific fragmentation of nucleic acids. For DNAs this can be done with the help of specific endonucleases (restriction enzymes). However, no reliable and efflcient methods are known for strictly specific cleavage of RNA molecules. This paper describes an experimental approach to addressed fragmentation of polyribonucleotides. It is known that RNase H splits RNA in RNA-DNA heteroduplexes [l] or in the heteroduplexes made of monotonous synthetic polyribonucleotides and

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non‐specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N‐terminal His residues was shown to be cross‐linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non‐cognate DNAs in the absence of cofactor, Mg2+ ions.

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments: EcoRII endonuclease cleavage of substrates with repeated natural and modified recognition sites

Interaction of EcoRII restriction endonuclease with a set of synthetic concatemer DNA duplexes with natural and modified sites for this enzyme has been studied. DNA duplexes with repeated natural sites are cleaved by EcoRII. Substitution of central AT‐pair in the recognition site for a non‐complementary TT‐ or AA‐pair reduces the rate of cleavage, this effect being much more pronounced in the last case. Absence of site flanking in one strand from the 5′‐terminus also results in very slow cleavage. The results obtained testify to the interaction of EcoRII with both strands of the substrate.

Cyanogen Bromide-Induced Chemical Ligation: Mechanism and Optimization of the Reaction Conditions

Abstract Chemical ligation induced by BrCN in the presence of N-substituted morpholine has been studied in details. It has also been shown that the phosphate activation by BrCN can be used for the synthesis of nucleotide derivatives in aqueous solutions.