Nina G. Dolinnaya

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.

Thymidine glycol: the effect on DNA molecular structure and enzymatic processing

Thymine glycol (Tg) in DNA is a biologically active oxidative damage caused by ionizing radiation or oxidative stress. Due to chirality of C5 and C6 atoms, Tg exists as a mixture of two pairs of cis and trans diastereomers: 5R cis-trans pair (5R,6S; 5R,6R) and 5S cis-trans pair (5S,6R; 5S,6S). Of all the modified pyrimidine lesions that have been studied to date, only thymine glycol represents a strong block to high-fidelity DNA polymerases in vitro and is lethal in vivo. Here we describe the preparation of thymine glycol-containing oligonucleotides and the influence of the oxidized residue on the structure of DNA in different sequence contexts, thymine glycol being paired with either adenine or guanine. The effect of thymine glycol on biochemical processing of DNA, such as biosynthesis, transcription and repair in vitro and in vivo, is also reviewed. Special attention is paid to stereochemistry and 5R cis-trans epimerization of Tg, and their relation to the structure of DNA double helix and enzyme-mediated DNA processing. Described here are the comparative structure and properties of other forms of pyrimidine base oxidation, as well as the role of Tg in tandem lesions.

Hairpin-shaped DNA duplexes with disulfide bonds in sugar-phosphate backbone as potential DNA reagents for crosslinking with proteins

Convenient approaches were described to incorporate ‐OP(=O)O−‐SS‐O−(O=)PO‐ bridges in hairpin‐shaped DNA duplexes instead of regular phosphodiester linkages: (i) H2O2‐ or 2,2′‐dipyridyldisulfide‐mediated coupling of 3′‐ and 5′‐thiophosphorylated oligonucleotides on complementary template and (ii) more selective template‐guided autoligation of a preactivated oligonucleotide derivative with an oligomer carrying a terminal thiophosphoryl group. Dithiothreitol was found to cleave completely modified internucleotide linkage releasing starting oligonucleotides. The presence of complementary template as an intrinsic element of the molecule protects the hairpin DNA analog from spontaneous exchange of disulfide‐linked oligomer fragments and makes it a good candidate for auto‐crosslinking with cysteine‐containing proteins.

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding–unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA–RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.

Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.

Specific covalent binding of a NF-κB decoy hairpin oligonucleotide targeted to the p50 subunit and induction of apoptosis

The NF‐κB transcriptional factor regulates various functions such as immune responses, cellular growth and development, and is frequently activated in tumor cells. Thus, inhibition of NF‐κB could suppress tumor cell growth. Using a decoy synthetic hairpin‐shaped oligodeoxyribonucleotide (ODN) containing the κB site with an integrated single diphosphoryldisulfide linkage, we demonstrate its covalent binding to the p50 subunit of NF‐κB. Furthermore, this decoy ODN induces apoptosis in a lymphoblastoma cell line. Thus, such chemically modified decoys could be valuable tools for blocking nuclear factors and tumor cell growth.

Dinucleoside Phosphates Containing Arabinose or Deoxyxylose. Hydrolysis by Exonucleases and Stacking Properties

Abstract Two pairs of isomeric dinucleoside monophosphates containing either 1-β-D-arabinofuranosyl)uracil (arabino-U, aU) or 1-β3-D-2-deoxy-threo-pentofuranosyl) thymine (2′-deoxyxylo-T, dxT) were synthesized. The kinetics of the hydrolysis of these dimers by venom and spleen phosphodiesterases (PDE) were studied. In addition, circular dichroism was used to study their stacking behaviour. A correlation was found to exist between the enzymatic cleavage rate and the conformation of the dimers. It was found that the configuration change at C-2′ or C-3′ in the 5′-terminal nucleoside residue, especially the inversion of 3′-hydroxyl involved into the internucleotide 3′-5′-phosphodiester linkage formation, was critical.

Sequence-dependent structural variations of DNA revealed by chemical ligation

Abstract The comparative study of nick-sealing efficiency under the action of BrCN or water-soluble carbodiimide was carried out for 14 dinucleotide combinations in double helix. The difference between the lowest (17%, GpG) and the highest (94%, CpT) coupling yields was found to be more than five fold, both condensing reagents showing a similar sequence-specific trend. A strong correlation observed between coupling yields at different dinucleotide combinations and 31P NMR parameters supports the idea that variations in chemical ligation efficiency arise from sequence-specific modulations of the helix geometry and confirms close similarity of the intrinsic fine structure of intact and nicked DNAs.

Parallel G-quadruplexes formed by guanine-rich microsatellite repeats inhibit human topoisomerase I

Using UV and CD spectroscopy, we studied the thermodynamic stability and folding topology of G-quadruplexes (G4), formed by G-rich fragments in human microsatellites that differ in the number of guanosines within the repeating unit. The oligonucleotides d(GGGT)4 and d(GGT)4 were shown to form propeller-type parallel-stranded intramolecular G-quadruplexes. The G4 melting temperature is dramatically decreased (by more than 45°C) in the transition from the tri-G-tetrad to the bi-G-tetrad structure. d(GT)n-repeats do not form perfect G-quadruplexes (one-G-tetrad); folded G4-like conformation is not stable at room temperature and is not stabilized by monovalent metal ions. The minimum concentration of K+ that promotes quadruplex folding of d(GGT)4 was found to depend on the supporting Na+ concentration. It was demonstrated for the first time that the complementary regions flanking G4-motifs (as in d(CACTGG-CC-(GGGT)4-TA-CCAGTG)) cannot form a double helix in the case of a parallel G4 due to the steric remoteness, but instead destabilize the structure. Additionally, we investigated the effect of the described oligonucleotides on the activity of topoisomerase I, one of the key cell enzymes, with a focus on the relationship between the stability of the formed quadruplexes and the inhibition degree of the enzyme. The most active inhibitor with IC50 = 0.08 µM was the oligonucleotide d(CACTGG-CC-(GGGT)4-TA-CCAGTG), whose flanking G4-motif sequences reduced the extreme stability of G-quadruplex formed by d(GGGT)4.

Synthesis and properties of modified oligodeoxyribonucleotides containing 9-(2-amino-2-deoxy-beta-D-arabinofuranosyl)adenine

Abstract 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.