Nucleobase-Guanidiniocarbonyl-Pyrrole Conjugates as Novel Fluorimetric Sensors for Single Stranded RNA.

We demonstrate here for the first time that a guanidiniocarbonyl-pyrrole (GCP) unit can be applied for the fine recognition of single stranded RNA sequences-an intuitively unexpected result since so far binding of the GCP unit to ds-DNA or ds-RNA relied strongly on minor or major groove interactions, as shown in previous work. Two novel nucleobase-GCP isosteric conjugates differing in the flexibility of GCP unit revealed a fluorimetric recognition of various single stranded RNA, which could be additionally regulated by pH. The more rigid conjugate showed a specific fluorescence increase for poly A only at pH 7, whereby this response could be reversibly switched-off at pH 5. The more flexible derivative revealed selective fluorescence quenching by poly G at pH 7 but no change for poly A, whereas its recognition of poly AH⁺ can be switched-on at pH 5. The computational analysis confirmed the important role of the GCP fragment and its protonation states in the sensing of polynucleotides and revealed that it is affected by the intrinsic dynamical features of conjugates themselves. Both conjugates showed a negligible response to uracil and cytosine ss-RNA as well as ds-RNA at pH 7, and only weak interactions with ds-DNA. Thus, nucleobase-GCP conjugates can be considered as novel lead compounds for the design of ss-RNA or ss-DNA selective fluorimetric probes.

Electrochemistry of double-stranded complexes of synthetic polyribonucleotides having interferonogenic and antiviral activity.

Double-stranded (ds) complexes of poly(C) with poly(G) and poly(G,I) were studied using differential pulse polarography (DPP) and differential pulse voltammetry at a pyrolytic graphite electrode (DPV). The complex formed by copolymer was found to be DPP inactive. On the other hand, poly(G).poly(C) yielded a small DPP peak corresponding to single-stranded (ss) poly(C). It was suggested that ss poly(C) present in the solutions of poly(G).poly(C) appeared due to the existence of segments in poly(G) during the complex-forming process in which guanine residues were unable to be hydrogen-bonded with bases in poly(C). Polynucleotide complexes investigated in this report yielded a DPV peak corresponding to electrooxidation of guanine residues, which was markedly lower than that yielded by ss polymers. Moreover, this DPV peak yielded by the complex prepared from an equimolar mixture of poly(G) and poly(C) was still markedly higher than that yielded by poly(G,I).poly(C), or by poly(G).poly(C) prepared in the excess of poly(C). The lowering of the DPV peak was explained as being particularly due to the presence of the polynucleotide segments with an intact and regular secondary structure. The results of our electrochemical analysis of the complexes investigated were compared with their biological activity reported earlier. This comparison calls attention to the fact that biological effectiveness of these biopolymers may be dependent on details of their secondary structure which may be monitored using the methods of electrochemical analysis.

[Calorimetric study of the polyG-polyC complex]. / Kalorimetricheskoe issledovanie kompleksa poliG-poliTs.

Heat effects of polyG-polyC melting in neutral aqueous solutions have been measured using differential scanning microcalorimeter with an extended temperature range. The limiting value of melting enthalpy is 53 +/- 4 kJ per mole of base pairs and melting temperature dependence on the sodium concentration can be expressed by the empiric relation Tm = 13.2 log(Na+) + 420 K.

[Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex]. / Otsenka razmera nepreryvnogo uchastka poli(G), neobkhodimogo dlia biologicheskoi aktivnosti kompleksa (G).poli(Ts).

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.

[DNA-binding proteins in human serum]. / DNK-sviazyvaiushchie belki syvorotki krovi cheloveka.

Fractions of acid and base blood serum proteins, separated by ion exchange chromatography on QAE-Sephadex (but not the proteins of the whole blood serum, which had the same charge), reacted with DNA preparations. Separate fractions of blood serum proteins were able to react with DNA after electrophoretic separation. Binding of blood serum proteins with DNA did not depend on ion strength within the range of NaCl concentration from 0.1 M to 0.5 M; it was also stable at pH 5.5 = 8.0. Interaction between acid DNA-binding proteins and native DNA was inhibited by denatured DNA, polyguanilic and polyinosinic acids and by other polyanions: dextran sulfate, polyvinyl sulfate, polyanetol sulphonate, polystyrol sulphonate and heparin. Acid DNA-binding proteins showed only a slight affinity to polyadenilic, polyuridilic and polycytidilic acids. The acid and base DNA-binding proteins appear to be contained in blood serum in the form of a loosely bound complex.

Distribution properties of polymononucleotide repeats in molluscan genomes.

A total of 635 DNA sequences from 35 species of mollusks were used as taxonomic support to investigate several distribution features of polymononucleotides in genomic regions of different functionality. We show that all polymononucleotide types in mollusks fit to expectations in exons but not in nonexonic regions, in agreement with a leading role of negative selection on expansions/contractions of transcription-linked poly-(A/T) repeats. The fit of all repeat length types to an exponential decay precludes the existence of a threshold size for replication slippage, a popular but unsatisfactorily explained concept in mutation models for single repeats. The genomic density of poly-(A/T) repeats is not correlated with the DNA content of species, suggesting that the differential density of repeats between species could be better explained by the species-specific performance of its repair mechanisms. This research allows a better understanding of the distribution patterns of single repeats in eukaryotes.

Production of poly-gama-glutamate (PGA) biopolymer by batch and semicontinuous cultures of immobilized Bacilluslicheniformis strain-R

Production of Polyglutamate (PGA) biopolymer by immobilized Bacilluslicheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32- and 2.18-fold increase in comparison with alginate- or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.

Glutathione plus cytosol- and microsome-mediated binding of 1,2-dichloroethane to polynucleotides.

1,2-[1,2-14C]Dichloroethane was metabolized by rat hepatic microsomes to products that irreversibly bound polynucleotides. The polynucleotides were then enzymatically hydrolyzed and the products separated by a high-performance liquid chromatograph (HPLC) equipped with an ODS or a SCX column. The products of microsome-mediated binding were identified in the HPLC eluate as 1,N6-ethenoadenosine to polyadenylic acid, 3,N4-ethenocytidine to polycytidylic acid, and two cyclic derivatives to polyguanylic acid. 1,2-[1,2-14C]Dichloroethane was also metabolized in the presence of a glutathione (GSH)-cytosolic fraction and a polynucleotide. After enzymatic hydrolysis of the polynucleotide, the major peak of radioactivity was eluted from a Sephadex G-25 column in the salt volume which would exclude the presence of a product containing both GSH and a nucleoside. Chromatography by ODS-HPLC of the major peak from Sephadex G-25 indicated the presence of a GSH metabolite of 1,2-dichloroethane that did not contain a nucleoside. A similar hydrophilic peak was obtained for the hydrolysis products of polynucleotides from a glutathione plus cytosol incubation in which the polynucleotide instead of being added prior to the incubation was added after the incubation. The products of the glutathione plus cytosol metabolism of 1,2-[1,2-14]dichloroethane appear to be glutathione metabolites that coisolated with the polynucleotides rather than covalently bound adducts. In conclusion, covalently bound adducts were identified for microsome-mediated binding of 1,2-dichlorethane to polynucleotides, while no evidence was obtained for glutathione plus cytosol-mediated covalent binding to polynucleotides.