АННОТАЦИЯ
Дегтяр М.В. Взаимодействие ионов двухвалентных металлов с неканоническими структурами нуклеиновых кислот – Рукопись.
Диссертация на соискание ученой степени кандидата физико-математических наук по специальности 03.00.02 – биофизика. – Харьковский национальный университет им. В.Н. Каразина, Харьков, 2002.
Проведено экспериментальное исследование комплексов ионов щелочно-земельных (Ca2+, Mg2+) и переходных (Cu2+, Ni2+, Mn2+) металлов с одно- и мультицепочечными гомополинуклеотидами (polyA, polyU, polyI, A×U, A×2U, 4×polyI) в водных растворах. Измерены дифференциальные УФ-спектры металлокомплексов, что позволило определить влияние ионов на конформацию полинуклеотидов, а также установить места связывания ионов, предложить структуру образующегося комплекса и механизм вызванного ионами разрушения спирали. В широком диапазоне концентраций ионов получены диаграммы конформационных равновесий. Выполнены расчеты констант связывания ионов с одно- и мультицепочечными спиралями. Полученные значения констант связывания позволили в рамках теории лигандов объяснить некоторые особенности фазовых диаграмм. Зарегистрировано образование компактных частиц в присутствии высокой концентрации ионов металлов и определены размеры этих частиц.
Ключевые слова: иони переходних металлов, полинуклеотиды, дифференциальная УФ спектроскопия, термическая денатурация.
SUMMARY
Degtyar M.V. Divalent metal ions interaction with noncanonical nucleic acid structures. – Manuscript.
Thesis for Candidate’s degree of Physical and Mathematical Sciences in Biophysics - Specialty 03.00.02 – V.N. Karazin Kharkov National University, Kharkov, 2002.
Interest in nucleic acid metallocomplexes is caused by the great role which metal ions play in the support of native polynucleotides functioning. All biochemical reactions running with nucleic acids participation require metal ions as coenzymes. The rate and results of reactions depend on the nature and concentration of an ion. In is impossible to understand of metal ions participation in normal functioning of the cell genetic apparatus without studies of metal ions interactions with native and synthetic polynucleotides as well as with noncanonical nucleic acid structures. At the present time a possibility of using triplexes and quadruplexes to treat viral, genetic and carcinogenic diseases is intensively investigated. However under physiological conditions multi stranded helices are usually less stable then corresponding duplexes. Therefore, it is of great interest to study triplexes and quarduplexes stability in the presence of metal ions.
In the present work an experimental study of alcohol – earth (Ca2+, Mg2+) and transition (Cu2+, Ni2+, Mn2+) metal ions complexes with single- (polyA, polyU, polyI), double- (polyA×polyU), three (polyA×2polyU) and four- (4×polyI) stranded homopolynucleotides in aqueous solutions has been performed. Differential UV spectra of metallocomplexes were measured, that gave a possibility to reveal influences of ions on polynucleotides conformations, to establish ions binding sites, to suggest the structure of the complexes formed. Homopolynucleotides conformational transitions induced by heating were observed by the method of thermal denaturation.
It has been shown that Mg2+ and Ni2+ ions interact with phosphate groups of polyA×polyU and polyA×2polyU, ions binding causes the ordering of these polynucleotides. Cu2+, Ni2+, Mn2+ bind to bases of four stranded polyI that results in the destruction of its helical structure. By the ability to destroy the 4×polyI ordered structure these ions follow the sequence Cu2+ > Ni2+ > Mn2+. The dependence of the 4×polyI disordering degree on the Cu2+ content was obtained. Mg2+ and Ca2+ ions do not interact with 4ЧpolyI bases and do not change its conformation.
The formation of particles of single stranded polyA and polyI in the presence of high divalent metal ions concentrations is registered. Effective radia of these particles (about 100A) are by order of magnitude less then the inertial radius of one stranded statistical coil, that evidences compaction of polynucleotides, induced by the high content of metal ions.
Conformational equilibrium diagrams were built in the wide region of temperatures (20-960C) and divalent metal ions concentrations (10-5 – 10-2 M) at different (10-2 – 10-1 M) ionic strengths. The “critical” concentration of Mg2+ and Ni2+ were found at which the conformational transition type changes: in polyAЧ2polyU instead of successive transitions 3→2 and 2→1, 3→1 transition takes place, in polyAЧpolyU, instead of 2→1 transition, 2→3 and 3→1 transitions are observed.
Phase diagrams of polyAЧpolyU and polyAЧ2polyU complexes with Mg2+ are essentially dependent on the Na+ content. Thus, at the Na+ concentration close to the physiological one (0.1М) the increase of the Mg2+ content in polyAЧpolyU solution results in continuous extension of the temperature range of the triple helix stable state, while at 10-2 М Na+ this range does not depend on Mg2+ concentration. Under these conditions the "critical" point at phase diagrams of polyA×2polyU with Mg2+ and Ni2+ ions is absent.
Mg2+ and Ni2+ effects on polyA×polyU and polyA×2polyU conformational transitions are mainly similar. The differences observed are caused by different values of association constants and by the fact that Ni2+ is able to interact with polyA bases while Mg2+ binds to phosphate groups only.
It has been found that 3→2 transition is essentially less cooperative then 2→1 and 3→1 transitions. Cooperativity factors for 3→2 и 3→1 were estimated, that permit us to conclude that the reason of the wide temperature range of the 3→2 transition is the low value of this transition enthalpy.
Ni2+ association constants with phosphate groups of polyA×polyU and polyA×2polyU as well as Mg2+ binding constants with polyA and polyU have been calculated in the framework of the ligand binding theory (Frank-Kamenetskii, 1972). Interaction of metal ions with multi stranded polynucleotides is characterized by negative cooperativity, binding of Cu2+ to single stranded polyI has positive cooperativity. Negative cooperativity of ions binding is due to the electrostatic repulsion between ions absorbed from solution and ions already bound to the polynucleotide. A possible reason of positive cooperativity is the entropy increase which results from polynucleotide dehydration caused by Cu2+ ions induced compaction.
Values of association constants obtained permit us to explain some features of phase diagrams: increase of 3→2, 3→1 and 2→1 transitions temperatures with the rise of divalent metal ions content is due to the low values of metal ions binding constants to single stranded polyU, decrease of the 2→3 transition temperature in the presence of Mg2+ results from the condition KA2U > KAU.
Key words: divalent metal ions, differential UV spectroscopy, thermal denaturation, polynucleotides.
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