Oligonükleotit ve DNA İle Yeni Nesil Floresans Maddelerin Etkileşimlerinin Kütle Spektrometrisi ve Spektroskopik Yöntemlerle İncelenmesi

Abstract

In the thesis study, the non-covalent interactions of benzimidazole-based platinum (II) and palladium (II) containing metal complexes and perylene diimide derivative structures with single-stranded oligonucleotides, double-stranded short and long DNAs were discussed both through their mechanisms of interaction with DNA and their use for DNA imaging due to the spectroscopic properties of the complexes formed. Various mass spectrometric, spectroscopic and gel electrophoretic methods have been used that provide qualitative and/or quantitative information about binding interactions. N,N-di(n-butyl)-1,6,7,12-tetra(4-hydroxyphenoxy)perylene-3,4,9,10-tetracarboxylic acid diimide (FP-3), Saccharinato-2,6- bis(N-methyl-benzimidazol-2-yl)pyridinepalladium(II) (PT-5), Saccharinato-2,6-bis(N-methyl-benzimidazol-2-yl)pyridineplatin(II) are used in the thesis study as dye structures. Studies on the characterization of the dye structures, single-stranded oligonucleotides, double-stranded DNAs and aptamers used in the thesis, interactions of these dyes and nucleic acids, interaction stoichiometry, stability and determination of the type of non-covalent interaction that is effective in the formation of interaction, DNA binding percentages, the effect of incubation time and incubation concentration on the interaction were examined with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) iv and/or Electrospray Ionization Mass Spectrometry (ESI-MS). Ion Mobility Mass Spectrometry (IM-MS) analyzes were performed in order to examine the conformational differences of oligonucleotides with the same molecular mass in different sequences and the conformational examination of the interactions of the double-stranded DNA (which is formed by the pairing of these oligonucleotides) with PT-5 and PT-6 structures. The absorbance changes of the interactions where binding is appropriate were monitored by Ultraviolet-Visible Region Spectroscopy (UV-vis), and binding constants were calculated, fluorescence efficiencies were monitored by Fluorescence Spectroscopy, and small molecule-DNA interaction mechanisms were investigated. In addition, the use of the structures synthesized for use in the thesis study as a sensor-dye in the Agarose Gel Electrophoresis method and whether they can be used in DNA identification were examined. Thus, alternative imaging agents have been tried to be determined instead of Ethidium Bromide, which is highly carcinogenic, which is based on the observation of DNA, RNA and their fractions, which is frequently used in the literature and applications. The optimum incubation time for all interactions in the specific 1:1 stoichiometry was found to be 24 hours. It was concluded that FP-3 dye, a derivative of perylene dimide, is a good intercalator for long double-stranded DNA structures and can be an alternative to EtBr as an effective imaging agent. It has been determined that PT-6 containing platinum (II) and PT-5 containing palladium (II) dyes, derivatives of benzimidazole, interact with all nucleobases of DNA nucleic acids and platinum creates a stronger and more stable interaction compared to palladium. It has been found that these dyes show weaker fluorescence compared to the EtBr molecule in binding with DNA, but it has been determined that they can be an alternative imaging agent to EtBr, especially for interactions with long double-stranded DNAs. As a result of the thesis studies, it was concluded that these dyes, which were found to interact well with DNA, have a high probability of being used as an alternative to cis-platin, whose side effects and resistance are known, in the field of cancer diagnosis and treatments with further experimental studies.