PubMed Health⌕ Search

Biomedical subjects

R N Maslova

Publications and source records attributed to R N Maslova.

At least 19 recordsLinked to original sources
PubMed1993

Tritium labeling of antisense oligonucleotides by exchange with tritiated water.

We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides.

Animals↗
PubMed1991

The inhibition of restriction endonucleases due to Z-DNA in negatively supercoiled plasmid.

Plasmid pGC20 containing the (dGC)9 insert in SmaI recognition site has been used to study the inhibition of cleavage by different restriction endonuclease due to Z-DNA formation in (dCG)10 sequence of the negatively supercoiled plasmid. Data obtained indicate the different sensitivity of restriction endonucleases to DNA conformational perturbations resulted from the Z-DNA formation. Therefore, the inhibition of DNA cleavage by a particular restriction endonuclease cannot serve as a criterion for the estimation of the length of B-Z junctions in circular supercoiled DNAs.

Base Sequence↗
PubMed1987

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues.

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.

Base Sequence↗
PubMed1977

A kinetic study of 1H leads to 3H exchange in C(8) H-groups of purinic residues in DNA.

The kinetic study of 1H leads to 3H exchange in C(8) H-groups of purinic residues of DNAs with different G-C content as well as in corresponding dNMP mixtures have been carried out. The present results show that 1H--3H exchange in DNA is retarded (as compared to the exchange in dNMP mixtures) to a lesser extent (Kret =2.4-2.8) than in RNA (Kret=6-8). The degree of retardation in these polymers is practically independent of their nucleotide composition. Assuming the ylide mechanism of exchange reaction it is suggested that the lower rate of 1H leads to 3H exchange in C(8) H-groups of purinic residues in polynucleotides of A-form (RNA and other polyribonucleotides) as compared to those of B-form (DNA and other polydeoxyribonucleotides) might be accounted for by decreased availability of C(8) H-groups for OH-ions of the solvent due to a different microenviroment of these groups in A- and B-type helixes.

DNA↗
PubMed1975

Isotopic hydrogen exchange in reactions catalysed by cysteine lyase and serine sulphhydrase.

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.

Animals↗
PubMedDate not supplied

[Reaction capabilities and structure of poly(rG) and poly(rG)-poly(rC) in solution by the method of the kinetics of hydrogen ion exchange].

Data on the kinetics of 1H greater than 3H exchange between water and C(8)H groups of guanylic residues in the poly(rG) and poly poly(rG)-poly(rC) are presented. Furthermore, optical properties (CD spectra and hyperchromism) of neutral solutions of these polymers from 20 to 100 degrees C are described. It is shown that the exchange in poly(rG) within the temperature range from 20 to 80 degrees C proceeds faster than in rGMP. Within the temperature range from 20 to 40 degrees C such an acceleration of the exchange is observed also in poly(rG)-poly(rC). According to the ylide mechanism of the exchange reaction the observed accleration of the exchanged in in C(8)H groups of guanylic residues is considered as a consequence of an increase of the positive charge at N(7) atoms. This effect is due to formation of additional hydrogen bonds in which N(7) atoms take part. The exchange in poly(rG)-poly(rG) at temperatures hihger than 75 degrees C, when these additional hydrogen bonds are absent, proceeds more slowly than in rGMP. Such picture is usual in other previously studied polynucleotides whose structure in solution is stabilized only by Watson - Crick hydrogen bonds and stacking interactions. The data obtained support a Guschelbauer's model of the four-stranded stranded poly(rG). They also indicate the posibility of associates formation in poly(rG)-poly(rC) solutions at temperature lower than 40 degrees C being stabilized by hydrogen bonds in which N(7) atoms of guanylic residues take part.

Chemical Phenomena↗
PubMedDate not supplied

[Study of conformation characteristics of polydeoxyribonucleotides with non-random base sequence by slow 1H----3H exchange].

The rate constants of 1H----3H exchange between water and C8H-groups of purinic residues of alternating polynucleotides: poly[d(A-T)].poly[d(A-T)] (I), poly[d(G-C)].poly[d(G-C)] (II), poly[d(A-C)].poly[d(G-T)] (III) and homopolynucleotides: poly(dA).poly(dt) (IV), poly(dG).poly(dC) (V), as well as DNA E. coli, was determined in 0.15 M NaCl at 25 degrees C. The retardation of exchange observed at these conditions (compared to that of the B-form DNA) is in agreement with the model of B-alternating structure for the (I) and is attributed to the co-existence of B- and A-conformers for the (V) in solution. Absence of distinguishable differences in exchange rate constants for purinic residues of the (II), (III) and (IV) (compared to that of the B-form DNA) evidences that conformations of these polynucleotides in solution are similar to "canonical" B-form DNA and don't correlate with the model of "heteronomous" DNA which was proposed for (IV).

Base Sequence↗
PubMedDate not supplied

[Study of conformation characteristics of "X-form" of alternating polynucleotides by the method of slow 1H----3H transition].

The rate constants of 1H----3H exchange between water and C8H-groups of purine residues of alternating polynucleotides: poly[d(A-C)].poly[d(G-T)] and poly[d(A-T)].poly[d(A-T)], as well as Escherichia coli DNA, dAMP and dGMP, in solutions with high concentration (4.3 or 6 M) CsF, in water ethanol (60%) solution and (in comparison) in 0.15 M NaCl were determined at 25 degrees C. The 1H----3H exchange rate exchange rate constants for adenylic (kA) and guanylic (kG) residues of polynucleotides were compared with the corresponding constant for DNA and mononucleotides. It was shown that at conditions when poly[d(G-T)] and poly[d(A-T)].poly[d(A-T)] exhibit the "X-form" CD spectrum, alteration of exchange rates in polynucleotides (approximately 2-fold increase in kA in CSF and approximately 1.5-fold decrease in kA and kG in 60% ethanol with 0.15 M NaCl) is due to the effect of solvents on the chemical reactivity of purine residues, but does not reflect a conformational transition. The analysis of these results allows us to conclude, that alternating polynucleotides under the above mentioned conditions retain roughly the conformations inherent in them in 0.15 M NaCl: poly[d(A-C)].poly[d(G-T)] conformation in 4.3 m CsF or 60% ethanol differs only insignificantly from the "canonic" B-DNA, whereas the poly[d(A-T)].poly[d(A-T)] conformation in 6 M CSF corresponds to B-alternating DNA.

Chemical Phenomena↗
PubMedDate not supplied

[Kinetics and mechanism of the 3H to 1H in C(8)H groups of purine derivatives].

The pH-dependence of the 3H to 1H exchange between water and C(8)H groups of purine, adenine, 9-methyladenine, 7-methyladenine, hypoxanthine, guanine, xanthine as well as C(2)H groups of imidazole and benzimidazole was studied. It was shown that within the pH-ranges, where the majority of molecules under study are non-ionized, the values of observed rate constant (kobs) do not depend on pH. Beyond these ranges the values of k(obs) are increased or decreased depending on the type of ionizaiton of the compound under study in appropriate pH range. The observed pH dependence of the 3H to 1H exchange is in a good quantitative agreement with ylide mechanism of the exchange reaction. According to this mechanism the 3H--1H exchange takes place in N(7)-protonated forms of the purine derivatives and in zwitterions with positive charge on N(7). The ylide mechanism of the exchange reaction is also suggested by the fact that the true exchange rate constants (k+) of protonated forms of the studied compounds, calculated from the values of k(ods), rises linearly with the increase of their protonation constant (Ka1)--the tenfold increase of Ka1 leads to about four-fold rise of k+. The knowledge of 1h to 3H exchange mechanism in C(8)H groups of purine derivatives allows to estimate alterations of reactivity of the purine residues in polynucleotides and nucleic acids depending on their conformation.

Binding Sites↗
PubMedDate not supplied

[Study of the intermolecular association of poly(G).poly(c) and poly(dG).poly(dC) in solutions by methods of 1H to 3H exchange and electron microscopy].

The kinetic of 1H leads to 3H exchange between water and C(8)H-groups of the guanylic residues in poly(G) . poly(C) and poly(dG) . poly(dC) was investigated within the temperature range from 30 to 90 degrees in 0.5 M NaCl (pH 7.2). It was shown that the exchange in freshly dissolved preparations at temperatures lower than 50 degrees proceeds faster than that in the case of GMP. According to the ylide mechanism of the exchange reaction the observed acceleration of the exchange is considered as a consequence of associates formation in poly(G) . poly(c) and poly(dG) . poly(dC) solutions at temperatures lower than 50 degrees. Associates are stabilized by intermolecular hydrogen bonds in which N(7) atoms of guanylic residues take part. The increase of the temperature is accompanied by gradual disappearance of the exchange acceleration. The retardation of exchange, which is characteristic of most non-associated double-stranded polynucleotides and nucleic acids is observed at the temperatures above 60 degrees. The retardation points to thermal destruction of the associates at temperatures higher than 50 degrees. The associates which are characterized by ordered structure including several "side by side" arranged double-stranded molecules were observed by electron microscopy. The addition of EDTA to solutions as well as the increase of temperature leads to destruction of the associates whereas the addition of Mg2+ makes the associates more stable.

Chemical Phenomena↗
PubMedDate not supplied

[Study of conformational differences between DNA composing chromatin and compact particles by the slow 1H--3H exchange method].

The rate of 1H leads to 3H exchange between water and C(8)H groups of purinic residues in chromatin as well as in optically active and optically inactive compact DNA particles, formed in PEG-containing water-salt solutions was measured. In chromatin the rate of exchange is shown to be about 15% lower as compared to that in the open form of DNA. Assuming the ylid mechanism of exchange the retardation observed may be interpreted as a consequence of screecing of C(8)H groups by proteins of chromatin which hampers the contacts of these groups with OH- ions of the solvent. In optically active compact particles the rate of exchange is shown to be as high as 20% but in optically inactive particles up to 60% as compared to the open form of DNA. This fact indicates the difference of the secondary structure of DNA in these two compact forms. The ratio between the rate constants in guanylic and adenylic residues (kG : kA) in the open form of DNA as well as in chromatin and in optically active compact particles is equal 3.0, whereas the ratios in mononucleotides and in optically inactive particles are equal 1.6 and 2.2, accordingly. Comparison of the 1H leads to 3H exchange kinetic data with literature data on the small-angle X-ray scattering reflexes for optically active and optically inactive compact particles of DNA, suggests that the secondary structure of DNA in chromatin and in optically active compact particles is a highly-ordered one, whereas in optically inactive compact particles of DNA the bases are to a great extent disordered.

Animals↗
PubMedDate not supplied

[Comparative study of slow 1H to 3H exchange in synthetic polynucleotides of A- and B-type conformations].

The rate of 1H leads to 3H exchange between water and C(8)H-groups of purinic residues in synthetic polynucleotides in wide temperature range measured. At temperatures below their Tm the rate of the exchange is shown to be lower as compared with that in corresponding mononucleotides. In the case of polynucleotides of A-conformation (poly(A).poly(U), poly(A).2poly(U) and poly(dA).2poly(dT), and poly(G).poly(C) the exchange is retarded by a factor of 5.7--7.5, whereas in the case of those of B-conformation (poly(dA).poly(dT), poly(dA--dT).poly(dA--dT) and poly(dG).poly(dC)) the exchange is retarded only by a factor of 2.3--2.5. Assuming the ylide mechanism of exchange the retardation is interpreted as a consequence of sterical hidrance in polynucleotides helical structure, which hampers contacts between purinic C(8)H-groups and OH-ions of solvent. Analysis of atomic arrangement around C(8)H-group and interatomic distances calculated on the basis of published atomic coordinates support our general conclusion that the sterical hindrance is more significant in the A-form as compared with that in the B-form. Elucidated correlation between the degree of the retardation in purine-containing polynucleotides and their conformation in solution allows to estimatf the type of conformation of polynucleotide with unknown structure on account of the slow 1H leads to 3H exchange data.

Hydrogen↗