Theoretical Study of Antagonists and Inhibitors of Mammalian Adenosine Deaminase: II. Isomeric Aza-deazaanalogues of Adenosine

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of electrostatic potential around their van der Waals molecular surface were calculated using the ab initioSTO-3G method. The conformational studies were carried out by the MM⁺ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.     

Theoretical study of antagonists and inhibitors of mammalian adenosine deaminase. I. Adenosine and its aza- and deaza-analogs

Aza- and deazaanalogues of adenosine, including their 1-protonated forms (except for that of 1-deazaadenosine), were studied by computer computation to find a relationship between their molecular structures and substrate properties for the mammalian adenosine deaminase. The atomic charge distribution and maps of the electrostatic potential around their van der Waals molecular surface were calculated for these compounds using the ab initio STO-3G method. The conformational studies were carried out by the MM+ method of molecular mechanics. The mechanism that determines the substrate selectivity of mammalian adenosine deaminase is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Binding of unnatural alpha,beta-oligocytidylates with DNA duplexes

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain unnatural alpha-anomers along with natural beta-nucleotides; the nucleotide composition is selected according to putative pattern of unconventional triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study complexation of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg2+. Binding is observed at neutral pH values, while more basic pH (8.0) prevents complexation of the AT duplex and oligocytidylate. Contrary to oligonucleotides of irregular composition, a regular dA30:dT30 duplex does not bind with the dC strand. It is also shown that alternating self-complementary duplex d(AT)16 and oligocytidylate d(CbetaCalpha)15 do not form complexes, and poly-dC self-associates are formed instead. The effect of 2'-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.     

Theoretical Study of the Structure of Adenosine Deaminase Complexes with Adenosine Analogues: I. Aza-, Deaza-, and Isomeric Azadeazaanalogues of Adenosine

Conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13-isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme [the structure of ADA complex with 1-deazaadenosine (1ADD) was used] within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1ADD known from X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.     

Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.     

Parallel double DNA spiral. A conformational analysis of regular spirals of poly(dA).poly(dT) with different variations of joining bases

We have performed a conformational analysis of DNA double helices poly(dA).poly(dT) with parallel directed backbone strands in heteronomic model frames. All possible models of base pairs and various mutual orientation of base pair and sugarphosphate backbones were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of parallel double polynucleotides were calculated. The dependences of conformational energy on the base pair structure were studied.     

A triple-stranded "clip" from the oligonucleotide 3'-(dA)10-pO(Ch2Ch2O)3p-(dT)10-pO(CH2CH2O)3-p-(dT)10(-5)]

The temperature dependence of the UV- and CD-spectra of the oligonucleotides 3'-d(A)10-L-(T)10-5' [anti(AT)], 3'-d(A)10-L-d(T)10-3' [par(AT)] and 3'-d(A)10-L-(dT)10-L-(dT)10-5' [tripl(ATT)] (L = -PO(CH2CH2O) 3p-) in the phosphate buffer at pH 7 under different concentrations of NaCL and in the presence or absence of 0.01 M MgCl2 was studied. All registered structural changes are the result of intramolecular processes if the concentrations of the oligonucleotides is low (about 2.2.10(-5) M). Par(AT) and anti(AT) exist in the only two forms, transforming into each other: under low temperatures they exist as hairpins with the parallel or antiparallel orientation of chains accordingly which transform into unfolded chains when the temperature increased. In contrast trip(ATT) exists in the three different forms depending on the temperature and ion conditions. They are: the three- stranded clip, the two-stranded hairpin with a single stranded "tail" and completely unfolded chain. For the first time this work presents thermodynamic parameters of the triplex formation from deoxyoligonucleotides depending on NaCl concentration. We have registered the CD spectra to one-, two-, and three-stranded forms. Ethidium bromide binding to three-stranded "clip" was investigated, and it was established that molecules of the dye may intercalate into the "clip" with formation of stable complexes (the constant of association 10(6) M-1). It is maximum three molecules of ethidium bromid which may bound to one molecule of the three-stranded clip. It has been shown that the suggested synthetic model (three oligonucleotide blocks combined by hydroxyalkyl chains) is the most convenient for physico-chemical investigations of triplexes today.     

Structure of d(GT)n repeating sequences with parallel and antiparallel chains]

The ability of oligonucleotides 3'-d(GT)5pO(CH2)5Opd(GT)5-5' (anti[d(GT)]) and 3'-d(GT)5pO(CH2)6Opd(GT)5-3' (par[d(GT)]) to form hairpins and higher associates is studied. Optical methods of thermal denaturation and circular dichroism as well as the fluorescence of ethidium bromide and acridine orange bound to oligonucleotides were used. At room temperatures the formation of hairpin structure with parallel and antiparallel strands is possible. Thermodynamic parameters of par[d(GT)] and anti[d(GT)] are similar and equal to delta H = -15 kcal/mol, delta S = -50 cal/mol. deg. In the temperature range 3-10 degrees C par[d(GT)] and anti[d(GT)] form four-stranded structures with parallel chains, in which layers of four G-residues alternate with unpaired T-residues being bulged out easily. On comparison of occurrence of alternating (GT)n, (GC)n and (G)n sequences in genome it can be stated that (GT)n biological functions could be connected with conformational possibilities of the four-stranded parallel structures with unpaired T-residues.