[A potentiometric sensor designed on the basis of urease immobilized in polyelectrolyte microcapsules].

A potentiometric biosensor has been designed on the basis of glass pH-electrode with a sensing device of the microcellular polyelectrolytic coating containing urease. The polymeric walls of the coating are readily permeable for low-molecular weight compounds, including urea, but are impermeable for macromolecules. The main characteristics of the biosensor in various experimental solutions containing urea, low-molecular-weight salt, and buffer have been obtained. The sensor has been shown to be stable for at least three weeks. The standard curves of the sensor are linear in the range of urea concentrations from 0.2 to 20 mM.

[The effect of low intensity electromagnetic irradiation on hydration of DNA film]. / Ussledovanie vliianiia EMI maloi intensivnosti na gidratatsiiu plenok DNK.

By using the IR-spectroscopy it has been shown that electromagnetic radiation (frequency 8.15-10.0 GHz, energy flux density 5 microWt/cm2) reduces the rate of water desorption from DNA films. It was found that the irradiation of samples with high humidity did not change spectral characteristics of DNA molecules in the range of 900-4000 cm-1, that means their molecular structure remains intact. At the same time the irradiation changed conformation liability of these biopolymeric molecules, that is their ability of conformational transformations under the influence of outer factors. Drying of non-irradiated humid films induced rapid (for a few minutes) transition of DNA from B to A conformational state, whereas in the irradiated films this transition took several hours after humidity reducing.

Hydrogen-ion binding of polycytidylic acid immobilized between Langmuir layers of dimethyldioctadecylammonium (DODA) in thin multilayer films.

The report describes the study of hydrogen-ion binding of Langmuir-Blodgett films contained with polycytidylic acid. A variety of multilayer films are analyzed and their UV absorption spectra are recorded. Poly (C) molecules established between dimethyldioctadecylammonium (DODA) layers are shown to exist in double stranded and semiprotonated form, independent of the pH value of the solution from which the films were made. A large hysteresis was found between forward and back proton titration of poly(C) immobilized in the LB films. This hysteresis points to a marked transference of both types of molecules during the film titration. This behavior also depends upon the types of molecules from which the films were made.

[Conformation and molecular and ionic transformations of polycytidylic acid immobilized in multilayer Langmuir and polyelectrolyte films]. / Konformatsionnoe sostoianie i osobennosti molekuliarno-ionnogo prevrashcheniia politsitidilovoi kisloty, immobilizovannoi v mul'tisloinykh lengmiurovskikh i poliélektrolitnoi plenkakh.

Multilayer films of complexes of polycytidylic acid with dioctadecyldimetylammonium were obtained by the Langmuir-Blodgett method (LB films), and complexes of poly(C) with polycations (poly-L-lysisne, polyethyleneimine, polyallylamine) were obtained by the method of alternate adsorption (polyionic assembly) from solutions of oppositely charged polyelectrolytes on the solid carrier (SA films). It was shown that poly(C) exists in SA films in a single-stranded state irrespective of whether in the starting solution it occurred in the single-stranded nonprotonated or double-stranded protonated conformation. Conversely, in the LB film poly(C) preferred to be in a double protonated conformation. UV-spectra of water-insoluble LB and SA films at different pH values of surrounding water medium were investigated. Proton titration curves of poly(C) immobilized in LB films were obtained. The analysis of the shape of titration curves showed that the molecular-ionic transformation of poly(C) in LB films is accompanied by both the conformational transition of the polynucleotide and the molecular rearrangement in the whole film. Poly(C) was found to transform from the double- to single-stranded state and vice versa in the "deprotonation-protonation" cycle of LB film due to cooperative release/binding of hydrogen ions by cytosine bases. In contrast, poly(C) "protonation-deprotonation" in SA films occurred without conformational transitions of the polynucleotide. As opposed to poly(C) in solution a rather big hysteresis of forward and back titration curves was found for both types of multilayer films, indicating molecular rearrangements in films. The reason for the structural transformations of poly(C) upon fabrication of LB or SA films and the mechanism of molecular ionic transformations of poly(C) in films are discussed in terms of a simple model of ion exchange. An assumption about the nature of structural transformations of LB and SA films during their protonation-deprotonation is put forward.

Sequence-dependent binding of metal ion to DNA oligomeres. A comparison of molecular electrostatic potentials with NMR data.

Experimentally observed sequence-selective binding of metal ion to DNA oligonucleotides have been compared with variations of electrostatic potential (EP) along the helix. Calculations of EP have been performed for three atomic models of the oligonucleotide duplex [d(CGCGAATTCGCG)2] using several variants of EP calculations, including a solution of non-linear Poisson-Boltzmann equation (NPBE). N7 atom of guanine adjacent to adenine base was identified as a region with the most negative electrostatic potential in the major groove. The EP value for the Me ion binding site surpasses the value for N7 of other guanines by 10-26% depending on particular duplex conformation. Qualitatively, the sequence dependent variations of EP near guanine N7 atoms are in agreement with the sequence-selective behavior of Mn(II) and Zn(II) ions as revealed by NMR experiments. But the difference in EP between the two most negative regions near guanine N7 atoms does not exceed 1.25 kT/e. Simple model suggests that metal ions are capable to form ion-hydrate complexes with G-Pu steps of DNA duplex. These complexes are formed via one Me...G and five Me...water coordination bonds with water molecules hydrogen bonded to two adjacent purine bases in the same chain. We suppose that such a stereospecific structural possibility is the main factor which control the sequence-selectivity in the metal ion binding. A combination of both mechanisms allows to explain sequence specific Mn(II) and Zn(II) binding to a set of oligonucleotides.

Multilayer films containing immobilized nucleic acids. Their structure and possibilities in biosensor applications.

Langmuir-Blodgett (LB) and film technologies based on electrostatic attraction self-assembly (SA) are shown to be useful for immobilization of nucleic acids (DNA, polynucleotides) onto solid supports in sensor devices. The nucleic acids were immobilized in complexes with cationic surfactants (for LB) and polycations (for SA). Infrared spectral studies showed that DNA unfolds in multilayer LB films with octadecylamine and conserves its double helical structure in the LB films with dioctadecyldimethylammonium and in the SA films with polyallylamine, polyethylenimine and poly-L-lysine. Atomic groups and the types of interactions determining the complex formation of these films have been identified. The hydration of LB and SA films was studied to find out binding sites of water molecules and to evaluate the flexibility of nucleic acid compounds in the multilayer films. The possibilities of biosensor applications of these LB and SA films were monitored on binding of specific reagents for DNA by DNA-containing films and mononucleotides by a complementary single-stranded polynucleotide immobilized on a positively charged solid support.

[IR-spectroscopic study of the interaction of chromium salts with native DNA]. / IK-spektroskopicheskoe izuchenie vzaimodeistviia solei khroma s prirodnoi DNK.

Infrared spectra of some unoriented films of DNA contained CrCl3, K2Cr2O7 and Cr2(SO4)3 have been obtained at relative humidities (r.h.) 0 to 93%. Chromium cations were shown to interact with phosphoric groups of DNA molecules. Cr2(SO4)3 or K2Cr2O7 content exceeds 0.1 Cr/P, the sugar-phosphate backbone of the double helix becomes irregular and nucleic bases unstacked, i.e. denaturation of DNA occurs. A very small amount of the salt, less than 0.01 Cr/P contained in DNA films prevents the conformational transition of the double helix from the B to A form induced by r.h. decreasing. The efficiency of the chromium salts in preventing the transition falls in the row: Cr2(SO4)3 > K2Cr2O7-CrCl3.

Infrared and X-ray diffraction study of the effect of protonation of DNA on its B-to-A transition.

The influence of H+ on the secondary structure of DNA and on its B-to-A transition has been studied by employing X-ray diffraction and infrared spectroscopy. Helical parameters for DNA molecules with different degrees of protonation were determined. It was shown that H+ binding stabilizes the B-form of DNA in fibers over a wide range of water and inorganic salt content. Only 0.03 H+ bound per nucleotide is sufficient to prevent the B-to-A transition caused by decreasing relative humidity in DNA fibers containing 4% NaCl. The effectiveness of B-form stabilization by H+ is explained by changes in DNA-solvent molecule interactions, especially in the major groove of double helices.

[The effect of hydrogen ions on the B-A transition in DNA]. / Vliianie vodorodnykh ionov na B-A perekhod v DNA.

The effects of hydrogen ions binding to DNA on its secondary structure and B to A transition were studied by methods of X-ray diffraction and infrared spectroscopy. Helical parameters of DNA molecules with different degrees of protonation were determined. It was shown that H+-ions binding stabilize the B form of DNA in fibers in the wide range of water and inorganic salt content. Only 0.03 H+-ions bound to each nucleotide are sufficient to prevent B to A transition caused by a relative humidity decrease in DNA fibers, containing 4% of NaCl. The effective stabilization of the DNA B form by H+-ions binding is explained by modifications in DNA - solvent molecules interactions, especially in the major groove of double helices.

[Study of DNA-interaction with protons of the medium by means of buffer capacity method]. / Izuchenie vzaimodeistviia DNK s protonami sredy metodom bufernoi emkosti.

pH-dependences of buffer capacity of native and denaturated DNA have been obtained. These dependences were expanded into basic functions each corresponding to ionised groups of equal pK. The pH dependence of buffer capacity of native DNA have two narrow g- and s-peaks and are shifted (relatively to nucleosides mixture) to acidic pH values, whereas those of heat denaturated DNA have only one wide assymmetric peak shifted to neutral pH. It was shown that s-and g-peaks of pH dependences of buffer capacity correspond to polymorphic transformation of the double helix (h leads to s transition) and to its disordering (s leads to g transition) respectively. From our data it follows that h leads to s transition at mu = 0.01 M NaCl is observed at temperatures T less than 50 degrees C. The independence of DNA protonation degrees, at which h leads to s leads to g transitions occur, on ionic strength of the solution is, apparently, related to linear dependence of the positions of g- and s-peaks maxima on (lg mu). This points to an important role of protonated bases in double helix destabilisation upon salt elimination. High sensitivity of the buffer capacity method allows us to reveal some differences between states of heat and acid denaturated DNA. Analysing pH dependences of buffer capacity of denaturated DNA it was shown that at neutral pH it contained some double or triple helix regions formed by H-linked bases holding unshared proton. Simms' method was shown to be inapplicable to determine base ionization constants both in native and denaturated DNA.

[X-ray diffraction study of polymorphism of the DNA double helix at protonation]. / Rentgenostrukturnoe issledovanie polimorfizma dvoinoi spirali DNK pri protonirovanii.

X-ray diffractograms of DNA fibers have been obtained at various degrees of protonation (alpha). At the protonation degrees preceding acid denaturation, DNA double helix undergoes polymorphic transformation. At 75% relative humidity DNA helix of 11 : 1 type protonated up to alpha = 0.3 changes to 10 : 1 type.