E. coli Hücrelerinden Polimerik Mikrokriyojeller ile RNA Saflaştırılması

Abstract

RNA purification is a critical first step in many life science studies, particularly in the diagnosis of viral, bacterial and parasitic diseases, in the diagnosis of hereditary disorders, tumors and in a number of preparatory and analytical methods. In this context, pure and pristine RNA molecules are required in order to obtain sensitive and reliable results with the applications of molecular biology techniques. Affinity chromatography is a method for separation, removal and purification of a particular molecule or group of molecules in a mixture using molecular interactions such as ionic/hydrophobic interactions, hydrogen bonding, disulfide bridge. Affinity chromatography; It is frequently used in biochemical and biotechnological processes such as the separation and purification of many biomolecules, including antibodies, enzymes, hormones, DNA, receptors and vitamins. Boronate affinity chromatography is a common purification method used for the isolation and purification of cis-diol containing biomolecules. Recently, chromatographic strategies utilizing specific affinity interactions have been increasingly developed. Leveraging effective techniques developed for RNA separation, boronate affinity chromatography is a promising approach for bioselective separation and enrichment of a wide variety of biomolecules containing cis-diol groups. Conventional particle-based adsorbents are frequently used in chromatography because they can be added to columns at different scales. However, they usually have relatively small pore sizes and cause some disadvantages such as slow diffusion mass transfer, low flow rates and high pressure drop. Therefore, different adsorbents have been developed to overcome these limitations. One of the best-known alternatives is cryogel-based adsorbents. Cryogels are macroporous polymeric materials produced by gelation at subzero temperatures. In addition, cryogels are prepared in monolithic form in the original mold dimensions and are usually only used in a particular column that conforms to its shape. Within the scope of the presented thesis, micron-sized cryogels that can be synthesized in the desired scale and shape were named as microcryogels, and were manufactured as boronate affinity adsorbent. PHEMA and poly(HEMA-VPBA) microcryogels synthesized in two different types were characterized by various methods such as SEM, FT-IR, µCT, and optimum adsorption conditions were determined in the batch system. In the thesis study, optimum conditions were obtained by using poly(HEMA-VPBA) microcryogels due to its suitable characterization, pH 8.5 ethyleneamine buffer 0.1 M CaCl2 salt presence at 37oC. As a result of RNA adsorption experiments from aqueous solution, it was found that adsorption saturation was reached after 50 minutes, and the maximum adsorption capacity was 303 mg/g polymer at 0.5 mg/ml RNA concentration. In the adsorption isotherm studies, it was determined that it conforms to the Langmuir isotherm and overlaps with the second-order isotherm kinetics. The reusability examinations of the microcryogels were performed 8 times and the adsorption capacity value of 223.4 mg/g was reached. In the thesis, Escherichia coli cells (E. coli), which are frequently preferred in biochemical processes due to their ease of experimentation and control, were used and RNA extract was obtained from E. coli cells as a result of incubation and centrifugation. The RNA extract was washed with 0.1 M borate buffer 0.5 M CaCl2 as desorption agent. As a result of washing and agarose gel electrophoresis analysis, it was proved that RNA was purified with a high degree of success.