REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Spiral” research realization in 2005

 All the results obtained do not have scientific analogues in Ukraine and in the world.

1. The spectrophotometric study of the interaction of biologically active drugs: nucleoside 6AZC with thymus DNA in the presence of a derivative of antibiotic actinocin ActII and ethidium bromide (EB) (as competing ligands); actinocin derivative ActII with single-stranded polyadenylic acid (poly(dA)) with different ratios of concentrations of the reactants was carried out in the visible and ultraviolet regions.

Basing on the analysis of titration curves of mixtures 6AZC-DNA-ActII and 6AZC-DNA-EB that were obtained in the visible region of the spectrum, it is concluded that 6AZC interacts with DNA by intercalation, and thus it is a competitor of ActII and EB upon interacting with DNA. It is shown that in the presence of 6AZC the concentration dependences of the absorption spectra ActII and EB with DNA change.

Basing on the analysis of titration curves of mixtures ActII-poly(dA), which were obtained in the visible region of the spectrum at pH = 6.86, it is concluded that Act II interacts with a poly(dA) to form at least two types of complexes.

Using optimization programs DALSMOD various theoretical models of binding were analyzed. Preliminary values ​​of the thermodynamic parameters of the Act II-poly(dA) binding (K₁ ~ 10⁴ M^-1, K₂ ~ 10⁵ M^-1) and 6AZC-DNA binding were obtained (K ~ 10³ M^-1).

Carried out by: -senior researcher Kruglova E.B., junior researcher Gladkovskaya N.A., Ph.D. student Ermak E.L.

2. Using a computer simulation (molecular docking, Monte Carlo methods) the parameters of interaction of antibiotic actinocin derivative ActIII with fragments of double-stranded polyribocytidylic acid (poly(rC)) were calculated.

Basing on the analysis of the results of molecular docking and experimental data on complex formation of actinocin derivative (ActIII) and double-stranded polyribocytidylic acid (poly (rC)) 5 structures of complexes ActIII-poly(rC) were obtained with minimal energy of ligand-target interaction. Complexes are stabilized by intermolecular hydrogen bonds. Cytosine and/or sugar phosphate backbone oxygen atoms of poly(rC), as well as NH₂, C=O, NH-groups of phenazone chromophore and side aminoalkyl radicals of ActIII participate in the formation of hydrogen bonds.

Using a Monte Carlo simulation the effect of solvent (water molecules) on the structure of the complexes obtained by molecular docking was determined. It is shown that the solvent has the ability to additionally stabilize the complexes of ligand and target polynucleotide via water molecules forming “bridges” between the donor-acceptor groups of the target and the ligand. One of the molecular models of the complex ActIII – poly(rC) was suggested as having the most probable structure.

Carried out by: senior researcher Shestopalova A.V., PhD student Miroshnychenko E.V.

3. Using Raman spectroscopy and spectrophotometry in the visible spectrum range, the complexation of actinocin derivative ActIII with double stranded poly(rC) in a wide range of P/D values was investigated. Numerical analysis of spectrophotometric concentration dependences allowed to set the intervals of P/D (P – moles of phosphates, D – moles of ligand), where complexes with different types of ligand binding with double-helical structure of poly (rC) are prevalent (monomer binding and aggregates).

Basing on the Raman spectra of ActIII-poly(C)·poly(C⁺) complexes the groups of atoms of ligand ActIII, involved in the formation of complexes with a polynucleotide matrix via external binding, were determined.

Carried out by: senior researcher Bolbukh T.V., junior researcher Blyzniuk Yu.N., senior researcher Kruglova E.B., senior researcher Semenov M.A.

4. Using EHF dielectrometry, gel electrophoresis and IR spectroscopy the effect of gamma radiation on DNA-caffeine complex was studied. The complexation was shown to be accompanied with the change in dielectric parameters and dehydration of the components – DNA and caffeine molecules. It was found that in the complex radiation does not cause, unlike in the case of DNA, degradation of DNA hydration shell and does not affect the degree of polymerization of native DNA. Analysis of the results shows that the main elements of the structure of DNA in the complex are preserved when exposed to radiation. This finding suggests the protective properties of caffeine.

Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V., senior researcher Krasnitskaya A.A.

5. Сomplex comparative study on characteristics of hydration and conformational transitions of the following collagen structures was carried out: 1) type I collagen from the calf skin from Sigma company; 2) a model collagen-like polytripeptide poly(Pro-Gly-Pro) from Sigma; 3) type I collagen, mineralized in vitro. Mineralization of collagen was carried out using a technique which allows simultaneous formation of nanocrystals of hydroxyapatite and collagen fibrillation. To investigate the hydration IR spectroscopy and piezogravimetry study of films was carried out under a wide range of relative humidity in the atmosphere of light and heavy water. The collagen structures in the range of 0-70% relative humidity were find to typically bind 3-4 water molecules per tripeptide – (Gly-X-Y). At the same time the strengthening of interpeptide hydrogen bonds was observed. These processes are accompanied by low-frequency shift of the amide I absorption band and by the high-frequency shift of the amide II absorption band. On the basis of experimental data the energy of hydration of collagen structures and energies of interpeptide hydrogen bonds were calculated. Mineralization of collagen does not lead to noticeable changes in the spectral parameters of the absorption bands of amide I and amide II.

The frequency shifts of the stretching vibrations of CO – groups that result from the dynamic resonance interactions of carbonyl vibrations of atoms in the structures of collagen (Gly-Pro-Hyp)₂-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)₃ and (Pro-Hyp Gly-)₃-Ile-Thr-Gly-Ala-Arg-Gly-Leu-Ala-Gly-(Pro-Hyp-Gly)₄ were calculated in frames of dipole-dipole approximation based on the theory of molecular excitons. The spectral parameters of the main components of the fine structure of the amide I band of FTIR spectrum of natural collagen were calculated.

Carried out by: junior researcher Melezhik E.P., senior researcher Bolbukh T.V., senior researcher Semenov M.A.

6. Using scanning microcalorimetry the effect of pyridoxine (vitamin B6) on the thermal stability of the secondary structure of DNA was studied. It was shown that the ligand interaction with DNA leads to considerable increase of the melting temperature (Tm) and energy (ΔHm) of structural transition, depending on the concentration of ligand. The reasons for this action of pyridoxine investigated.

Carried out by: senior researcher Gasan A.I., junior researcher Philipskiy A.V.

 Abstract of “Structura” research realization in 2005

A new method of estimating the thermodynamic parameters of ligand binding to DNA, which takes into account the formation of at least two types of complexes and the difference in the temperature dependences of the constants of ligand-DNA binding was developed. Testing of this methodology for the calculation of the thermodynamic binding parameters ΔH, ΔS, ΔG and for the estimation of shifts of melting temperatures (ΔT_m) of  ligand-DNA mixtures has been carried out on ActII-DNA systems with different AT-GC composition and on complexes between ActII and double-stranded and  single-stranded poly(rC). Temperature studies of the interaction of biologically active nucleoside 6-azacytosine (6AZC) with native and irradiated DNA molecules were also conducted.

Carried out by: senior researcher Bereznyak E.G., senior researcher Bolbukh T.V., junior researcher Blyzniuk Yu.N., senior researcher Gasan A.I., junior researcher Gladkovskaya N.A., researcher Dukhopelnikov E.V., senior researcher Kashpur V.A., senior researcher Kruglova E.B., senior researcher Maleev V.Ya., senior researcher Semenov M.A., senior researcher Shestopalova A.V.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Spiral” research realization in 2006

 All the results obtained do not have scientific analogues in Ukraine and in the world.

1. The detailed spectrophotometric study of the binding of ethidium bromide (EB) with DNA was carried out. It was found that it forms with DNA at least two types of complexes. The molar extinction coefficients, binding constants and sizes ​​of binding sites were calculated for both types. The binding of EB with DNA at high degrees of matrix occupancy was shown to lead to the changes in the structure of DNA.

Spectrophotometric studies of competitive binding of cytosine and its analogues with DNA in the presence of EB were conducted. It is shown that unmodified cytidine analogues (cytidine and AraC) do not compete with EB for binding sites on DNA, but lead to change in the titration curves of EB. Upon interaction of cytidine and AraC with DNA the interaction may occur with those groups of DNA bases that are not involved in binding with EB, so that they are not direct competitors of EB, but do create steric barriers to its binding to DNA.

Modified cytidine analogues (6AZC, AZAfur and AZAxyl), that have an active aza-group in the cytosine ring, compete with EB for binding sites on DNA by intercalation. The binding constants of 6AZC, AZAfur and AZAxyl with DNA were determined. The size of the binding site of 6AZC, AZAfur and AZAxyl on DNA is one base per ligand molecule, indicating the absence of AT, GC-specificity of these ligands. The aza-groups of nucleozides and amino groups of DNA bases may be involved in interaction of 6AZC, AZAfur and AZAxyl with DNA.

Carried out by: senior researcher Kruglova E.B., researcher Gladkovskaya N.A., Ph.D. student Ermak E.L., Ph.D. student Minakova E.A.

2. The interaction of DNA fragments and actinocin derivative (ActIІ) at ligand-target ratio of 1:1 and 2:1 in the aqueous environment was studied using Monte Carlo simulations. Low-energy structures of the complexes that make up the most probable molecular models of two types of complexes, namely binding in the minor groove and intercalation in GC-site, were obtained. The energy and structural parameters of complexes were calculated. It is shown that water molecules make additional stabilization due to the formation of water bridges between the donor-acceptor groups of DNA fragments and ligands. The possible sizes of ActIІ binding sites in the minor groove were found to be 3-4 base pairs per ligand molecule. When intercalating in GC-site, ActII molecule occupies binding site that should be more than 4 base pairs per ligand molecule.Carried out by: senior researcher Shestopalova A.V.

3. Using Raman scattering the features of DNA complexation with actinocin derivatives with different side chain length as well as of DNA with the derivative that differs by the structure of its side groups (methyl groups of tertiary amines are replaced by hydrogen atoms) were investigated. All Raman spectra were obtained for ligand-DNA mixtures which have one of possible complex types as predominant: at P/D10 type II complexes (intercalation) are prevalent.

Based on the literature data on the normal vibrations of fenoxazone chromophore and characteristicity of Raman bands, as well as on our previous Raman spectroscopic studies of actinocin derivatives in the free state and in combination with various polynucleotide matrices, the assignment of frequencies of Raman spectra of basic functional groups of the ligands was made. Raman spectroscopy method was also used to define the major atom groups of actinocin derivatives that participate in the formation of various types of complexes (intercalation and external binding) on polynucleotide matrices.
Carried out by: senior researcher Bolbukh T.V., junior researcher Blyzniuk Yu.N., senior researcher Kruglova E.B., senior researcher Semenov M.A.

4. The method of differential EHF dielectrometry was used to obtain the values ​​of the complex dielectric permittivity of DNA-spermine system in native and gamma-irradiated conditions. It is shown that the total hydration extent is the sum of the components’ hydration. The amount of bound water is unchanged after exposure to radiation, indicating the absence of significant changes in the hydration shell of DNA and preservation main elements of its structure. As detected by electrophoresis, exposure to radiation leads to DNA fragmentation both in the complex with spermine and in free of ligand state. In the former case, the range of fragments’ lengths is significantly smaller, indicating the interception of water radiolysis products and a partial protection of DNA structure from damage.Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V., senior researcher Krasnitskaya A.A.

5. The Raman spectra of polytripeptide poly(Gly-Pro-Pro) and type I collagen in deuterium oxide solution were obtained. Carbonyl type A stretching frequency oscillation which lead to the appearance of bands in the Raman spectrum, were calculated according to the model of weakly coupled oscillators in the framework of perturbation theory for the polypeptide structures taking into account dynamic resonance interactions of transition dipoles. The calculated frequencies are in good agreement with the experimental frequencies of the amide I band in the Raman spectra. On the basis of calculations the frequencies of the amide I band components were assigned in experimental Raman spectra of aqueous solutions of poly(Gly-Pro-Pro) polytripeptide and type I collagen.

The piezogravimetry was used to get the hydration isotherms of thin films of poly(Gly-Pro-Pro) and type I collagen. Based on D’Arcy and Watt model of water adsorption the quantitative characteristics of the inner and outer layers of the hydration shell of the molecules with triple helical structure  were obtained,  and the heat of water sorption in Langmuir, Henry layers and multilayers was calculated. Complete hydration enthalpy values for poly(Gly-Pro-Pro) and type I collagen are    -10.9 and -12.2 kJ/mol ща fragment -Gly-XY- respectively. Taking into account the energy characteristics of poly(Gly-Pro-Pro) and type I collagen hydration and direct quantitative estimates of enthalpy of peptide-peptide hydrogen bonds in collagen proteins obtained previously, estimates of the relative energy contributions of different types of interactions into the total energy of stabilization of the triple helical collagen type structures were made.

Carried out by: junior researcher Melezhik E.P., senior researcher Bolbukh T.V., senior researcher Semenov M.A.

Abstract of “Structura” research realization in 2006

1. Differential scanning microcalorimetry and spectrophotometry in the visible and UV regions were used to study the interaction of actinocin derivative ActІІІ with single- and double-stranded polyribocytidylic acid poly(rC). The parameters of ActІІІ complexation with poly (rC) were calculated. It is determined that the interaction of actinocin derivative Act II with poly (rC) leads to a decrease in melting temperature, enthalpy and entropy of poly(rC) in comparison with free polynucleotide. It is shown that the main reason for the decrease of thermodynamic melting parameters of poly(rC) is a conformational change of the matrix caused by the interaction with the ligand ActІІІ.

2. Molecular dynamics simulations of different helical forms of poly(rC) were carried out using AMBER 8 package with explicit ion-water. Molecular dynamics trajectories for single-stranded poly(rC) (with fragment length of 10 cytosines) and two topologies (5’E and 3’E) of 4-helical poly(rC) (two 5bp double-stranded fragments) were obtained. The structure of double-stranded poly(rC) for a relatively short fragments (12, 24, and 48 pairs of cytosines) was unstable and collapsed in the modeling process. The trajectories obtained by the essential dynamics method were analyzed, and the intramolecular entropies in the quasi-harmonic approximation were estimated. It was found that the single-helical form of poly(rC) is the most flexible among all of its helical forms, and 5’E topology of 4-helical poly(rC) is the most rigid.

Carried out by: senior researcher Bereznyk E.G., senior researcher Bolbukh T.V., junior researcher Blyzniuk Yu.N., senior researcher Gasan A.I., researcher Gladkovskaya N.A., researcher Dukhopelnikov E.V., senior researcher Kashpur V.A., senior researcher Kruglova E.B., senior researcher Maleev V.Ya., senior researcher Semenov M.A., senior researcher Shestopalova A.V., Ph.D. student Miroshnichenko E.V., Ph.D. student Khrebtova A.S.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Model” research realization in 2007

 All the results obtained do not have scientific analogues in Ukraine and in the world.

1. Spectrophotometric study of thymus DNA-theophylline binding in the presence of competing ligands was carried out. Compounds absorbing in the visible spectral region and interacting with DNA to form complexes of different types were selected as competing ligands. These were derivative of actinocin ActII and ethidium bromide. It was shown that the constants of theophylline-ActII binding (943 ± 124 M^-1) and theophylline-ethidium bromide binding (177 ± 36 M^-1) are significantly larger than the constants of caffeine binding with the same ligands. Using DALSCOMP and COMPHET, the programs for spectrophotometric concentration dependences optimization, the constant (185 ± 20 M^-1) and the size of the binding sites (n = 1) of theophylline binding with DNA were determined. Basing on these studies it was concluded that theophylline has greater activity than caffeine as acceptor of aromatic molecules in solution. This needs to be considered when simultaneously using these drugs in medicine and biology. Complex of programs for processing experimental data can be used in study of competitive binding of various types of pharmaceutical compounds which interact with DNA. The results obtained can be used for recommendations in combination chemotherapy.

Carried out by: researcher Gladkovskaya N.A., junior researcher Ermak E.L., Ph.D. student Minakova E.A., senior researcher Kruglova E.B.

2. Using DSC the effect of actinocin derivative ActIII on the thermodynamic parameters of the melting of DNA from salmon testes was investigated. The method of calculation of the thermodynamic parameters of ligand binding to DNA from DSC data was suggested. It is shown that by using a model based on the equations of chemical equilibrium, it is necessary to take into account the size of the cooperative unit of DNA melting, the size of binding sites of the ligand and the binding constant dependence on temperature. Corrections that have been added to the traditional model for calculating the thermodynamic parameters of the interaction of ligands with DNA from DSC data allow one to calculate the parameters under multimodal binding more correctly.

Carried out by: senior researcher Kruglova E.B., senior researcher Bereznyak E.G., researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

3. The influence of the sequence of nucleotides on the structure and dynamics of nucleic acids dodecamers was studied using molecular dynamics simulation. A database of molecular-dynamics trajectories of DNA dodecamers that can be used as targets for bioactive compounds was created. Using flexible molecular docking the most probable complexes of anticancer antibiotic derivative actinomycin D with asymmetric side chains (ActIII-II) and DNA fragments with different nucleotides sequences for intercalative and groove binding to double-helix DNA were constructed. The obtained results allow one to simulate molecular complexes of antitumor antibiotics analogues with DNA binding sites of different nucleotide composition.

Carried out by: junior researcher Miroshnychenko E.V., senior researcher Shestopalova A.V.

4. Using the theory of molecular excitons and IR absorption spectra of double-stranded polynucleotides poly(rA)-poly(rU), poly(rG)-poly(rC) and poly(dA)-poly(dT) obtained previously, resonant interactions of valence carbonyl (C=O) and transannular (C= N) vibrations of nitrogenous bases were calculated in the dipole-dipole approximation. On the basis of the calculations the mobility of nitrogenous bases in the process of structural rearrangements of polynucleotides caused by hydration was investigated. It is shown that in the dry state double-stranded polynucleotides have forms slightly different from the double-helical state. The results obtained allow us to describe the molecular mechanisms of the dynamics of nitrogenous bases under structural rearrangements of DNA.

Carried out by:  senior researcher Semenov M.A., researcher Boriskina E.P., senior researcher Maleev V.Ya.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

 Abstract of “Model” research realization in 2008

 All the results obtained do not have scientific analogues in Ukraine and in the world.

1. Differential scanning calorimetry (DSC) and spectrophotometry were used to study the interaction between proflavine (Pf) and thymus DNA. It is shown that Pf forms at least three types of complexes on DNA. At high P/D (Phosphate/Ligand concentrations ratio) values two types of intercalation complexes are formed. Primarily Pf intercalates into GC pairs. At low values ​​of P/D Pf interacts with the DNA via external binding. Spectrophotometric concentration dependences were used to calculate parameters of Pf complexation with DNA. Using DSC thermodynamic parameters of melting of DNA in complexes were obtained.

Carried out by:  senior researcher Kruglova E.B., senior researcher Bereznyak E.G., researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., Ph.D. student Khrebtova A.S.

2. Raman spectroscopy and spectrophotometry in the visible region were used to investigate the complexation of ethidium bromide (EB) with thymus DNA at high and low biopolymer/ligand concentrations ratio. It is shown that EB both intercalates into DNA and interacts with the biopolymer via external binding. From the analysis of Raman spectra of complexes it was found that amino groups of EB form hydrogen bonds with acceptor groups of polynucleotide DNA matrix atoms in both types of complexes. Low degree of DNA occupation by ligand does not change B-form of DNA, and high degree leads to the conformational B-A transition.

Carried out by: junior researcher Blyzniuk Yu.N., senior researcher Bolbukh T.V., senior researcher Gasan A.I., senior researcher Semenov M.A., junior researcher Philipskiy A.V.

3. Using differential EHF dielectrometry the dielectric properties of aqueous solutions of proflavine, myricetin, DNA-proflavin and DNA-myricetin complexes were studied. Basing on the data obtained, the hydration shell of the investigated substances was analyzed and its size was estimated. It was shown that the changes in hydration that accompany DNA-proflavine and DNA-myricetin complexes formation contribute greatly to the stabilization of complexes.

Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V.

4. Three models of building the intercalation sites of the target DNA molecule were considered. These models take into account the peculiarities of insertion of intercalators with different structures: ethidium bromide, daunomycin and 9-amino-DAC. Using molecular docking and molecular mechanics with implicit account for solvent, we show that the construction of intercalation targets requires one to consider only the most significant unwinding of DNA double helix in the intercalation site (for ethidium and 9-amino-DAC) or in an adjacent base pair (pairs) (for daunorubicin). Conformational rearrangements of sugars and asymmetry of their conformations near the site of intercalation were also registered. The influence of DNA bases sequence on the total unwinding angle of DNA-target was found.

Carried out by: senior researcher Shestopalova A.V., junior researcher Miroshnychenko E.V., engineer Sukhanov O.S.

5. The ProtNA-ASA database was created. It has free Internet access (www.protna.bio-page.org). The database contains systematic information about the conformational parameters and surface accessible area of ​​ nucleic acids’ atoms in protein-nucleic acid complexes. Information from the ProtNA-ASA database allows carrying out the statistical comparative analysis of structures of free DNA and DNA in complexes with proteins. Such an analysis significantly expands understanding of the molecular mechanisms of indirect protein-nucleic acid recognition.

Carried our by: Ph.D. student Tkachenko M. Yu., researcher Boriskina E.P., senior researcher Shestopalova A.V.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Model” research realization in 2009

All the results obtained do not have scientific analogues in Ukraine and in the world.

1. Fluorescence spectroscopy and spectrophotometry were used to investigate the interaction of proflavine (Pf) with rolyA-polyU, polyG-polyC matrices and calf thymus DNA. According to experimental spectrophotometric and luminescence titration data Scatchard isotherms for all the systems studied were constructed, binding constants and binding sites were obtained. Pf interaction with natural DNA and synthetic polyribonucleotides PolyA-polyU has the largest binding constant. Combining two optical methods allowed determining that the formation of three types of complexes in Pf-DNA system and only one type of complex in Pf-PolyA-polyU and Pf-polyG-polyC systems are possible. External binding in Pf-polyA-polyU and Pf- polyG-polyC systems is absent due to the fact that the polyribonucleotides have A-form of double helix.

Carried out by: senior researcher Bereznyal E.G., senior researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

2. Molecular docking was used to obtain Pf complexes with fragments of DNA and RNA homopolymers with different nucleotide composition, formed by intercalation and groove binding. It was shown that Pf can form two types of intercalation complexes with nucleic acids, which interaction energies are identical. Only a slight advantage in Pf binding to GC-sequences was established. Upon Pf interaction with B-DNA, complexes are formed in the minor groove of a helix with a maximum energy of binding to AT-sequences. Upon interaction with A-form of RNA and DNA, complexes are formed in the major groove, but such complexes are energetically less favorable than intercalation ones, and for them no dependence of the binding energy on the sequence was found. The possibility of the formation of hydrogen bonds between NH₂-groups of Pf and atoms of sugar-phosphate backbone of nucleic acids upon intercalation of Pf was demonstrated. Upon Pf arrangement in the grooves of helixes, NH₂-groups of the ligand interact with the bases atoms.

Carried out by: senior researcher Shestopalova A.V., junior researcher Miroshnychenko E.V., engineer Sukhanov O.S.

3. Spectrophotometric titration was used to study the interaction between the actinocin derivative and thymus DNA in a wide range of concentrations of monovalent Na⁺ and K⁺. A new model of ligand binding with DNA that takes ions into consideration both as direct competitors of ligand during intercalation and as indirect ligand competitors in case of external binding was suggested. Using this model, we estimated the effect of monovalent Na⁺ and K⁺ ions on complexation constants for the two types of binding and calculated polyelectrolyte contribution to the total free energy of DNA-ligand interaction depending on the type of ions.

Carried out by: senior researcher Kruglova E.B., PhD studen Minakova E.A.

4. IR spectroscopy and piezogravimetry were used to investigate the formation of DNA complexes with vitamin B2 (riboflavin, RBF) at P/D = 8. Using hydration isotherms it was found that DNA-RBF complex formation leads to a decrease of DNA hydration. It indicates that the ligand binds to the hydration active sites of DNA preventing their binding with water. According to IR spectroscopy data, phosphate groups of DNA and the oxygen atom of the ribose ring are involved in the formation of DNA-RBF complexes.

Carried out by: junior researcher Blyzniuk Yu.N., senior researcher Bolbukh T.V., senior researcher Gasan A.I., senior researcher Semenov M.A.

5. The differential EHF dielectrometry in millimeter wavelength was used to get the dielectric characteristics of aqueous solutions of propidium iodide and its complexes with DNA. Basing on the model of hydration, the hydration extent was calculated. It was shown that the stabilization of DNA-propidium complex is caused not only by the interaction between the components, but also by interaction with surrounding bound water. The latter is characterized by appearance of additional water molecules in hydration shell of DNA upon complexation.

Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V.

6. A study of the role of conformational transitions of DNA sugar-phosphate backbone in the mechanisms of indirect recognition of target DNA segments by proteins was carried out. The conformational parameters of the sugar-phosphate backbone and the surface accessible area of ​​the nucleotides in the minor and major grooves of the DNA fragments from 218 protein-nucleic acid complexes were analyzed statistically. It was shown that the frequency of the transition of different nucleotides to alternative conformations varies. We also determined that the transition of DNA sugar-phosphate backbone from classical to alternative conformation leads to characteristic changes in the area of ​​polar and nonpolar accessible surface of DNA in the minor and major grooves for nucleotides with A and B-like conformation of the deoxyribose. This, in particular, makes polar atoms O3′ and O5′ more accessible for interaction with a positively charged amino acid residues. The individual characteristics of the polar-nonpolar profile of different nucleotides in the same conformation were identified. The results obtained indicate an important role of DNA conformational transitions in protein-nucleic acid interactions and enhance the understanding of the molecular mechanisms of indirect recognition.

Carried out by: Ph.D. student Tkachenko M.Yu., researcher Boriskina E.P., senior researcher Shestopalova A.V.

Abstract of “Buksir” research realization in 2009

1. The electromagnetic response of protein macromolecules of integrated biological system on cells and donors’ blood plasma under the combined radiation of mm and submillimeter ranges was analyzed by screening the modulation frequencies of the laser and millimeter radiation. A search of the resonance frequency of macromolecular complexes of sires’ sperm with amplitude-modulated low-intensity electromagnetic radiation in the terahertz range was carried out. With the help of acoustic analysis of data of piezoelectric interaction of sperm suspension it was shown that the frequency of vibration of the bull semen tail can serve as an indicator of ejaculate quality for cryopreservation biotechnology and artificial insemination.

Carried out by: senior researcher Kolesnikov, V.G., junior researcher Dreval N.V.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Model” research realization in 2010

All the results obtained do not have scientific analogues in Ukraine and in the world.

1. Spectrophotometry (SF) and differential scanning calorimetry (DSC) were used to study the complexation of proflavin with DNA from salmon testes. It was shown that depending on the ratio of the concentrations of the reacting components two types of complexes are formed, with the thermodynamic state different from the one of DNA. The parameters of the intercalation of proflavine into DNA were calculated. Binding constants obtained by DSC and UV methods have similar values. By fluorescence spectroscopy, SF and DSC the interaction of flavin mononucleotide (FMN) with DNA from salmon testes was investigated. It was shown that FMN does not almost bind with DNA (K=50 M^-1) and does not affect the DNA melting thermodynamic parameters. It is concluded that in the ternary FMN-ligand-DNA system FMN is not able to regulate the change of biological effect of ligand by protector mechanism.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

2. Using the method of absorption spectrophotometry competitive binding of acridine mutagen ICR-191 and methylxanthines (caffeine and theophylline) with thymus DNA was investigated. The spectra of hetero-associates ICR-191-caffeine and ICR-191-theophylline were obtained. It is shown that ICR-191 interacts with caffeine with higher binding constant (K=1461±95M^-1) than with theophylline (K=404±29M^-1). Spectrophotometric study of ternary systems ICR-191-DNA- methylxanthines have shown that caffeine have greater effect on ICR-191-DNA interaction than theophylline does. Effect of caffeine on acridine mutagene binding to DNA was observed at low concentrations of DNA in solution with a lot of free ligand. The effect of theophylline is observed only at high concentrations of DNA.

Carried out by: researcher Ermak E.L.

3. The dielectric properties of the aqueous solution of ethidium bromide (EB) and DNA-EB complex were studied in EHF range. The hydration extent of EB was estimated basing on the experimental data, its probable hydration active sites were determined. It was found that the complexation process is accompanied by certain changes in the dielectric properties of the solution, which may indicate a tendency towards the increase of the number of bound water molecules during the formation of the complex. Comparison with literature data and analysis using models of DNA-intercalator interaction showed that this increase may be due to the processes of DNA unwinding and lengthening that take place during intercalation.

Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V.

4. Comparative analysis of the dielectric properties of aqueous solutions of DNA complexes with intercalators ActII, proflavine, ethidium bromide and propidium iodide showed that for all studied ligands the formation of their complexes with biopolymer is accompanied with the decrease of the dielectric permittivity, which, as shown by calculations, corresponds to the increase of hydration of DNA, estimated to a few water molecules per one nucleotide.

Carried out by: senior researcher Maleev V.Ya.

5. The method of spectrophotometric titration was used to study ethidium bromide interaction with thymus DNA within a wide concentration range of monovalent Na⁺ ions (from 7.5×10^-4 to 0.2M) using three theoretical models. According to one of the models, ligands form complexes that differ in the type of intercalation (mono-intercalated ligand and ligands on adjacent binding sites); this model was considered as the optimal one. Ions were considered as competitors of ligand in intercalation complex type. Using this model, we estimated the effect of monovalent Na⁺ ions on the parameters of the complexation of ethidium bromide with thymus DNA. We also obtained the dependence of the binding constants logarithms on the logarithm of NaCl concentrations. Using these dependences the contribution of polyelectrolyte component to the total free energy of the interaction of the ligand with DNA was calculated.

Carried out by: senior researcher Kruglova E.B., engineer Minakova E.A.

6. The studies of conformational changes in the sugar phosphate backbone of DNA in nucleosomes were carried out on the basis of statistical analysis of 14 core particles of nucleosome from PDB database. It was found that more than 8% of the nucleotides in the nucleosomal DNA composition have alternative t- and g– conformations of sugar-phosphate backbone angle γ. The dependence of these transitions on the nucleotide composition was revealed. It was also shown that conformation of g angle change leads to changes in geometry and electrostatic potential of the surface of double helix of DNA. Such rearrangements are important for DNA winding on the histone core of nucleosomes and for the implementation of indirect mechanism of proteins recognition of their specific binding sites on DNA.

Carried out by: Ph.D. srydent Tkachenko M.Yu., researcher Boriskina E.P., senior researcher Shestopalova A.V.

7. Working cell for measurement of dielectric parameters of solutions of biological substances in the frequency range from 30 to 40 GHz at different temperatures (0 to 70 ° C) was designed and manufactured. Measurements of the dielectric parameters of sucrose solutions at different temperatures and concentrations were carried out. The energy of activation of the dipole relaxation at various temperatures was calculated.

Carried out by: researcher Glibitskiy G.M.

Abstract of “Buksir” research realization in 2010

 A study of the electromagnetic response of biological objects under the combined effect of electromagnetic waves in the millimeter (35.5¸37.7 GHz) and terahertz (f = 0,89 THz) ranges was carried out. The objects of study were the suspension of red blood cells, unicellular algae (samples from reservoirs of Kharkiv region and pure cultures).

1. The effect of electromagnetic waves of THz range on lipid peroxidation and osmotic resistance of erythrocytes with different morphological and functional disorders was studied. The positive effect of the THz radiation on the osmotic resistance of erythrocytes which were exposed to osmotic hemolysis in the presence of NaCl salt solution (0.45%) for 30 minutes was discovered. The resistance of the membrane of red blood cells increased almost two times, and the level of peroxidation products remained almost unchanged.

Practical value: Selected and investigated exposure modes may be suggested for practical medicine in the treatment of chronic allergic dermatitis. The pathogenesis of the majority of the dermatitis is associated with the disruption of the normal arrangement of the membrane bilayer and with the damage of the membrane-associated proteins that are responsible for the inactivation of membrane receptor complexes and of systems of water and ions transport through the membrane of erythrocytes.

2. We investigated the electromagnetic response of unicellar microalgae in terms of the parameters of solar activity. The response was registered in the millimeter wavelength range with the activation of the cell suspension by terahertz radiation. A correlation between the real part of the complex permittivity e¢ and the sunspots number was found. The sunspots number characterizes the level of solar activity, and the correlation coefficient (r) was -0.71.

Practical value: We showed the possibility to predict the productivity of the upper layer of the water area depending on solar activity.

Carried out by: senior researcher Kolesnikov V.G., junior researcher Dreval N.V.

REPORT OF DEPARTMENT OF BIOLOGICAL PHYSICS

Abstract of “Model” research realization in 2011

 All the results obtained do not have scientific analogues in Ukraine and in the world.

1. Using spectrophotometry and differential scanning calorimetry the complexation of the actinocin derivative ActII with DNA from salmon testes was studied at different ratios of concentrations of DNA and ligand. The sizes of the binding sites, binding constants and energy parameters of ActII- salmon testes DNA binding were obtained.

Carried out by: senior researcher Bereznyak E.G., researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

2. To analyze the data of spectrophotometric titration we built a new system of multi-threaded parallel optimization of the absorption spectra of BAS using genetic algorithms. Testing of new optimization program was carried out on ligand1-ligand1, ligand1-ligand2 systems. The constants of self- and hetero-association of proflavine, ethidium bromide, actinocin derivative, flavin mononucleotide were calculated.

Carried out by: senior researcher Dukhopelnikov E.V., junior researcher Kulishenko S.F.

3. The dielectric properties of aqueous solutions of chlorophyllin and its complex with DNA were studied using differential EHF dielectrometry. It was found that the complexation process is accompanied by a change in the dielectric properties of the solution due to changes in hydration shell of the nucleic acid. Basing on the data obtained and using model conceptions, we calculated the extent of hydration of the ligand and its complex with DNA. It was discovered that the process of complex formation is accompanied by a decrease of amount of bound water in the DNA-chlorophyllin system.

Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V., Ph.D. student Pesina D.A.

4. The textures were recorded in films obtained from the solutions of DNA and alkali and/or alkaline earth metal salts. The textures have structure that is inherent to the forms of liquid crystals. The ratio between the plane of the texture on the film and the plane of the whole film depends on the properties of the ions which were added to the solution, and on the ratio of metal salts and DNA concentrations in solution. Most of the textures which are formed in the films containing DNA and various metal salts are the fractal type textures. The fractal dimension of the textures can be used for a preliminary assessment of mutagenic level of biologically active substances in vitro.

Carried out by: researcher Glibitskiy G.M., junior researcher Glibitskiy D.M., senior researcher Maleev V.Ya.

5. By means of IR spectroscopy it was shown that the presence of silver nanoparticles in the DNA films causes the denaturation of DNA. The changes observed in the IR spectra indicate breakage of the hydrogen bonds which are formed with sugar-phosphate backbone groups of native DNA. These results confirm the hypothesis that the formation of textures on the surface of the films is determined by the degree of intactness of DNA.

Carried out by: researcher Glibitskiy G.M., junior researcher Glibitskiy D.M., senior researcher Semenov M.A.

6. A study of the mechanisms of competitive binding in the ternary system proflavin-DNA-caffeine in a wide range of relative concentrations of DNA/ligand1, ligand2 was carried out. It was shown that in the ternary system caffeine demonstrates both interceptive and protective mechanisms.

Carried out by: senior researcher Bereznyak E.G., researcher Gladkovskaya N.A., engineer Minakova E.A.

7. The research of the distribution of nucleotides with alternative conformations of nucleosomal DNA was performed according to the data of statistical analysis of 14 nucleosome core particles from the PDB database.

Carried out by: junior researcher Zhitnikova M.Yu., senior researcher Shestopalova A.V.

Abstracts of “Buksir” research in 2011

1. A measurement of the response of native cells to synchronized action of radiation from the HCN-laser and the source of mm-range radio waves was carried out to detect and study the resonant interaction. Measurement and control of complex action of radio waves in the range of extremely high and terahertz frequencies were carried out by means of EHF complex with a power of 20 MW (frequencies 35 GHz – 37.7 GHz) and Terahertz complex with a power of 10 MW (frequencies of 1.0 THz – 2.0 THz). The measurements were performed by research device “Device for the study of biological objects by waveguide methods in the range of EHF”.

2. In the modeling of osmotic hemolysis of the erythrocyte suspension the antioxidant effect of terahertz radiation was shown; a significant increase in resistance of erythrocytes to the membrane hemolysing agent (0.45% NaCl solution) was registered at terahertz laser exposure (f = 0,89 THz, P = 1,5 mW / cm², irradiation time 60 min).

3. The possibility of simulation of the solar activity by terahertz laser radiation under laboratory conditions was demonstrated. Correlation between the number of sunspots and hydration of suspension of unicellular algae was registered. It was shown that the terahertz radiation (f = 0,89 THz, P = 1,5 mW/cm²) refers to the “algophilic” frequencies of the electromagnetic spectrum.

Carried out by: senior researcher Kolesnikov V.G., junior researcher Dreval N.V.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Model-2” research realization in 2012

Testing of a new model of DNA complexation with biologically active compounds that have different type of binding has been designed and carried out to interpret microcalorimetry data. Thermodynamic equilibrium constants and binding parameters of the systems containing two classes of biologically active compounds (double systems) and DNA with two different classes of biologically active compounds (ternary systems) were established. For double systems intermolecular hydrogen bonds were found experimentally (by vibrational spectroscopy). These bonds additionally stabilize the formation of heteroassociates of biologically active compounds in aqueous solution. The results obtained are necessary to build models of the interaction of DNA with biologically active compounds that act like drugs and have antitumor effect.

Improvement of measuring and recording complex for studying the combined effect of electromagnetic waves of submillimeter and mm ranges on biological objects allowed us to conduct research under the agreement of scientific and practical cooperation between O.Ya. Usikov Institute for Radiophysics and Electronics NASU and Institute of Dermatology and Venereology of Academy of Medical Sciences of Ukraine.

All the results obtained do not have scientific analogues in Ukraine and in the world.

1. A model of DNA-BAS complexation which allows calculating the melting and binding parameters for the systems from differential scanning calorimetry data was developed. Testing of the model was carried out on DNA-proflavin system.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov EV, researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

2. To establish the equilibrium parameters of self- and hetero-association of BAS and of their complexation with DNA the next problems were studied using spectrophotometry:

– Hetero-association of double ligand1-ligand2 systems: flavin mononucleotide-ethidium bromide, ethidium bromide-chlorophyllin and proflavin-chlorophyllin;

– Complex formation in ternary systems DNA-ligand1-ligand2: DNA-ethidium bromide-flavin mononucleotide, DNA-ethidium bromide-chlorophyllin, DNA-proflavin-chlorophyllin.

Self- and hetero-association constants obtained independently under the same experimental conditions are necessary for calculating the parameters of complex formation in the DNA-ligand1-ligand2 system.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

3. Methods of IR and Raman spectroscopy were used to study the formation of hetero-complexes of biologically active substances: flavin mononucleotide (FMN), ethidium bromide (EB), proflavine (PRF), theophylline (TPH) and caffeine (CAF) in neutral aqueous solutions. Analysis of IR and Raman spectra showed the formation of intermolecular hydrogen bonds between the functional atomic groups (C = O and NH₂) in FMN-EB, FMN-PRF and TPH-EB hetero-complexes. Detected shifts of vibrational frequencies of the carbonyl groups in the IR spectra allowed us to estimate the value of the enthalpy of hydrogen bonds. We show that intermolecular hydrogen bonds additionally stabilize the stacking structures of the investigated hetero-complexes.

Carried out by: senior researcher Semenov M.A., junior researcher Blyzniuk Yu.N.

4. The differential EHF dielectrometry in mm range (λ = 7,6 mm) was used to study the dielectric parameters of aqueous solutions of chlorophyllin, human serum albumin and its complex with chlorophyllin. The experimental results obtained by dielectrometry for serum albumin-chlorophyllin solutions were analyzed. On the basis of model concepts the extent of hydration of chlorophyllin, human serum albumin and their complex was calculated.

Carried out by: senior researcher Kashpur V.A., researcher Khorunzhaya O.V., Ph.D. student Pesina D.A.

5. The self-organization of DNA under the dehydration was studied in the presence of Na⁺, Ag⁺ ions and silver nanoparticles. It is shown that nature of the formation of dendritic textures, which is determined by the size of the area of such structures on the surface of the dehydrated DNA film, depends on the conformational state of DNA and correlates with the mutagenic properties of the added compounds.

Carried out by: researcher Glibitskiy G.M., junior researcher Glibitskiy D.M., senior researcher Semenov M.A.

6. The development and upgrading of software for recording the interaction of blood elements with millimeter radiation was completed. The discretization of the spectrum of the measuring track in the range from 0 to 96000 Hz is not worse than 0.17 Hz. Software upgrades allowed us to carry out the calculations of the imaginary part of the dielectric constant taking into account the significant absorption of formed elements of the blood in the 8-mm wavelength range..

Carried out by: senior researcher Kolesnikov V.G, researcher Khmel N.V.

7. A waveguide improvement of the measuring and recording complex with the additional insertion of circulators was carried out in order to find the resonance frequencies of erythrocytes, blood plasma, microalgae and other biological objects.

Carried out by: senior researcher Kolesnikov V.G, researcher Khmel N.V.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Model-2” research realization in 2013

A new model describing the complexation of DNA with biologically active compounds that interact with DNA in different ways under the conditions of competitive binding was developed for the interpretation of microcalorimetry data. For spectrophotometric data processing a program module of calculating the parameters of the ligand1-ligand2 hetero-association was created taking into account the self-association of each ligand. To optimize the search of hetero-association parameters various modifications of the genetic algorithm were used. The original method of assessing the impact of biologically active compounds (BAS) on biopolymers was developed. The method is based (1) on the analysis of the area occupied by textures on the films grown from the solutions of biopolymer-BAS mixtures; (2) on the presence of scale-invariant fractal textures on the surface of the films and (3) on the estimates of fractal dimensions of textures of biopolymers films. For the first time the results on the antioxidant effect of the laser terahertz radiation in the modeling of the induced hemolysis caused by hypotonic sodium chloride solution were obtained. The possibility of applying EHF dielectrometry in rapid diagnosis of organism sensitization to anesthetic drugs was shown. It was tested in the study of the molecular action of local anesthetic artifrin on cellular structure of red blood cells.

All the results obtained do not have scientific analogues in Ukraine and in the world.

1. A system of equations describing the interaction of two ligands with DNA in a competitive binding was obtained. In the wide range of ratio of ligands concentrations and of binding parameters the theoretical dependencies of the change of the specific heat capacity of the DNA-ligand1-ligand2 complex on temperature were constructed and thermodynamic parameters of melting were calculated.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., junior researcher Khrebtova A.S., Ph.D. student Fomin A.V.

2. For the processing of the spectrophotometric data, a program module for calculating the parameters of ligand1-ligand2 hetero-association was created. The program takes into account self-association of each ligand. Testing of various genetic algorithm modifications for the optimization of search of the parameters of hetero-association of ligands was carried out.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., junior researcher Kulishenko S.F., Ph.D. student Fomin A.V.

3. Differential scanning calorimetry was used to study ethidium bromide-DNA and ethidium bromide-flavin mononucleotide-DNA systems. It was shown that in solutions with low ionic strength the addition of flavin mononucleotide leads to an increase in temperature and enthalpy of DNA melting in the ternary system as compared with the double system.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., junior researcher Khrebtova A.S.

4. Using IR spectroscopy in the range of absorption of carbonyl vibrations the self-association of flavin mononucleotide was carried out in solutions and films grown from neutral (pH=7.0) and slightly acidic (pH=6.2) solutions. In neutral solutions the spectral parameters of carbonyl vibrations (absolute intensities, dipole moments and other parameters) were determined. In the IR absorption spectra of films grown from slightly acid solutions of flavin mononucleotide for the first time new band of high-frequency carbonyl vibrations C4=O at 1729 cm^-1 was detected. Within the framework of the molecular theory of excitons in dipole-dipole approximation the origin of this band was explained, and an n-dimensional model of self-association of flavin mononucleotide was built.

Carried out by: senior researcher Semenov M.A., junior researcher Blyznyuk Yu.N., Ph.D. student Glibitskiy D.M., engineer Minakova E.A.

5. The differential EHF dielectrometry in mm range (λ = 7,6 mm) was used to study the dielectric parameters of aqueous solutions of bovine serum albumin (BSA) and its complex with chlorophyllin. Basing on the data and model conceptions, the extent of hydration of the protein and its complex with the ligand was calculated. It was found that the formation of the complex is accompanied by a decrease in the number of bound water molecules in BSA-chlorophyllin complex.

Carried out by: senior researcher Kashpur V.A., senior researcher Maleev V.Ya., researcher Khorunzhaya O.V., Ph.D. student Pesina D.A.

6. The electrostatic potential of free DNA fragments from structural databases was calculated. The dependence of the distribution of the electrostatic potential of minor groove of the double helix on the nucleotide sequence and the type of adjacent nucleotides in the sequence was detected.

Carried out by: junior jesearcher Zhitnikova M.Yu., senior researcher Shestopalova A.V.

7. The original method of assessing the impact of biologically active compounds (BAS) on the biopolymers was developed. The method is based on the analysis of the area occupied by textures on the films grown from the solutions of biopolymer-BAS mixtures; on the presence of scale-invariant fractal textures (SIFT) on the surface of the films, and on the estimates of fractal dimensions of textures of biopolymers films. It was shown that the textures of the films obtained from solutions containing ions of sodium, potassium and rubidium form SIFT on the surface while textures of the films obtained from the solutions containing zinc ions, mutagens, copper and silver at certain concentrations do not form SIFT. An application for a patent “Method for determining the effect of a substance on biopolymers” has been submitted.

Carried out by:  senior researcher Glibitskiy G.M., junior researcher Glibitskiy D.M., senior researcher Semenov M.A., senior researcher Gasan A.I.

8. The data on antioxidant effect of the laser terahertz radiation in modeling of the induced hemolysis caused by hypotonic 0.45% sodium chloride solution were obtained.

Carried out by:  senior researcher Kolesnikov V.G., researcher Khmel N.V.

9. The results of the study of the molecular action of local anesthetic artifrin on cellular structure of red blood cells obtained by EHF dielectrometry showed the possibility of using this ligand in the express diagnosis of the organism sensitization to anesthetic drugs.

Carried out by:  senior researcher Kolesnikov V.G., researcher Khmel N.V.

REPORT OF THE DEPARTMENT OF BIOLOGICAL PHYSICS (№25)

Abstract of “Model-2” research realization in 2014

Spectrophotometric temperature study of the effect of caffeine, chlorophyllin, flavin mononucleotide on interactions in DNA-ethidium bromide and DNA-proflavine systems was carried out. It was shown that caffeine exhibits both protective and interceptive properties at high relative concentrations (the concentration of caffeine must be 10⁴ times greater than the concentration of biologically active ligands). Chlorophyllin is a powerful interceptor, it reduces the concentration of proflavine or ethidium bromide by forming stable heterocomplexes with them. Flavin mononucleotide shows no competitive effect.

Basing on the results of analysis of formation of fractal textures on the surface of films prepared from solutions of the biopolymers in the presence of various ions, a new method for analyzing the influence of biologically active substances on the biopolymer was proposed.

To determine the influence of conformational changes in the sugar-phosphate backbone of DNA on the electrostatic potential of the minor groove calculations were made basing on the solution of the nonlinear Poisson-Boltzmann equation (using software package DelPhi). It was shown that the electrostatic properties of free DNA depend on the type of base (are sequence-specific) and the conformation of the sugar-phosphate backbone. These structural differences and the related changes in the physical properties of the double helix can explain the mechanisms of indirect recognition of DNA by changing the affinity of proteins in specific DNA sequences.

The modernization of hardware and recording complex, which includes the EHF dieleсtrometer, waveguide and generating circuits and software, was carried out. The software allows one to use the computer to record the electromagnetic response of biological objects using a fast Fourier transform. Using the upgraded complex, dielectric permittivity of the erythrocytes was measured in parallel with the measurements of erythrocyte sedimentation velocity in the presence of anesthetics (artifrin, septonest and ultrakain). A high correlation coefficient (r = 0,917) of the results obtained using two techniques indicates the possibility of applying the EHF dielectrometry to diagnose the limited scleroderma in a clinical setting. It also means that this method is informative in assessing the sensitization of erythrocytes to anesthetic of new generation. Measurements on blood cells and plasma were carried out in a licensed Institute of Dermatology and Venereology AMS of Ukraine.

All the results obtained do not have scientific analogues in Ukraine and in the world.

1. Spectrophotometry was used to carry out the thermal study of binding in DNA systems with ethidium bromide and proflavine in the presence of competing ligands (flavin mononucleotide, chlorophyllin, caffeine). It was found that caffeine has both protective and interceptive properties at high relative concentrations (the concentration of caffeine must be 10⁴ times greater than the concentration of biologically active ligands). Chlorophyllin exhibits high interceptive properties by forming stable heterocomplexes with ligands and reducing their concentration in the complexes with DNA. Flavin mononucleotide shows no competitive action. Spectrophotometric study of proflavin-chlorophyllin system showed that the hetero-association constant does not almost dependent on the temperature, i.e. the enthalpy of the process is equal to zero.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., researcher Gladkovskaya N.A., Ph.D. student Fomin A.V.

2. A model for the construction of the theoretical curves of heat absorption of DNA-ligand systems was developed for three cases of multimodal binding. The curves of heat absorption, binding isotherms and change of concentrations of various complexes formed depending on the type of multimodal binding were analyzed.

Carried out by: senior researcher Bereznyak E.G., senior researcher Dukhopelnikov E.V., junior researcher Khrebtova A.S., junior researcher Kulishenko S.F.

3. The hydration isotherms of films of flavin mononucleotide (FMN) grown from the neutral (pH=7.0) and slightly acidic (pH=6.2) solutions were obtained using piezogravimetry. It was shown that the level of sorption of water on the film from the neutral solution is higher (~ 30%) than sorption level on the film from the slightly acidic solution. It is suggested that self-association of isoalloxazine rings of FMN reduces the access of water molecules to the centers of hydration of FMN. IR spectroscopic study of FMN films revealed that the absorption bands of the transannular vibrations of N5 and N1 atoms have frequency shifts under the changing of pH of solution, the films were grown from, from neutral (pH = 7.0) to slightly acidic (pH = 6.2 ) values. However, in a series of experiments carried out in solutions with D₂O these effects are not revealed.

Carried out by: senior researcher Semenov M.A., junior researcher Blyzniuk Yu.N., Ph.D. student Glibitskiy D.M., engineer Minakova E.A.

4. The results of the measurements of the dielectric permittivity of solutions of chlorophyllin complexes with DNA, bovine serum albumin and human serum albumin, conducted using differential EHF dielectrometry, were analyzed. The dependence of hydration changes on the presence of fatty acids bound to the protein upon complex formation in systems protein-chlorophyllin was discovered.

Carried out by: senior researcher Kashpur V.A., senior researcher Maleev V.Ya., researcher Khorunzhaya O.V., junior researcher Pesina D.A.

5. To establish the influence of conformational changes in the sugar-phosphate backbone of DNA on the minor groove electrostatic potential, the calculations of its values were made on the basis of the solution of the nonlinear Poisson-Boltzmann equation (using software package DelPhi). It was shown that the electrostatic properties of free DNA depend both on the type of base (are sequence-specific) and on the conformation of the sugar phosphate backbone (namely on the angle γ and the conformation of the deoxyribose). It was also discovered that the same DNA sequences can have different electrostatic potential, and, conversely, the various DNA sequences have similar conformations with the same electrostatic potential distribution in the minor groove. These structural differences and the related changes in the physical properties of the double helix can explain the mechanisms of indirect recognition of DNA by changing the affinity of proteins in specific DNA sequences.

Carried out by: junior researcher Zhitnikova M.Yu., senior researcher Shestopalova A.V.

6. The influence of Fe ions on the formation of the textures on the surface of the films obtained from solutions of bovine serum albumin (BSA) was investigated. It was shown that the surfaces of BSA films form fractal textures of the “zigzag” type. Reducing the specific length of the fractal textures correlate with increasing concentration of Fe ions in the solution with BSA. The dependence of the changes in the UV and photoluminescence spectra of BSA solutions on concentration of iron, fluorine, bromine and chlorine ions was analyzed. It was shown that the differences in the textures of the films result from the interaction of different types of anions of fluorine, chlorine, bromine and BSA. Basing on these results, a new method for analyzing the effect of biologically active substances on the biopolymer was suggested.

Carried out by: senior researcher Glibitskiy G.M., junior researcher Glibitskiy D.M., senior researcher Semenov M.A., senior researcher Gasan A.I.

7. The modernization of hardware and recording complex, which includes the EHF dielectrometer, waveguide and generating circuits and the PC software, was carried out. The software allows one to record the electromagnetic response of biological objects using a fast Fourier transform. The modernization of hardware and recording complex was conducted by a specially designed and constructed waveguide filter for the terahertz radiation input into the measuring cell in experiments on biological objects. Elements of the measuring cells for using in strong magnetic fields were designed and manufactured. The software for processing and analyzing the amplitude of the primary data array that have various extensions of registering files was upgraded.

Carried out by: senior researcher Kolesnikov V.G., researcher Khmel N.V.

8. The hydration and surface tension of the blood plasma of patients with limited scleroderma were studied in the EHF range of radio waves (f = 35,6¸37,7 GHz) where the dispersion of the dielectric permittivity of free water takes place. Reaction index is calculated in the relation to the presence of DNA autoantibodies using the immuno-enzymatic test system “DNA antibodies-IEA”. Analysis of the real part of the complex permittivity e¢ showed a decrease in hydration in blood plasma samples with a more expressed reaction index of DNA_native. Measurements of the dielectric constant of the erythrocytes in parallel with the measurements of their sedimentation velocity in the presence of anesthetics (artifrin, septonest and ultrakain) were carried out. A high correlation coefficient of results obtained using two techniques (r = 0,917) indicates the possibility of adapting the EHF dielectrometry to diagnose the limited scleroderma using blood plasma in a clinical setting and that EHF dielectrometry is informative for assessment of the sensitization of erythrocytes to anesthetics of new generation. Measurements on blood cells and plasma were carried out in a licensed Institute of Dermatology and Venereology AMS of Ukraine.

Carried out by: senior researcher Kolesnikov V.G., researcher Khmel N.V.