Aptamer Bağlı Yüzeylerde Vegf ve İnterferon-y Proteinlerinin Zenginleştirilmesi ve Maldı-Ms ile Tayinleri

Abstract

Aptamers are DNA, RNA oligonucleotides that consist of various numbers of nucleotides. Non-covalent interactions of nucleotides such as hydrogen bond and van der Waals bond enable to consist three dimensional structure of aptamers. Thus, these special three dimensional structure of aptamers interact with target molecules with high specific selectivity. Aptamers are similar to antibodies in terms of their ability to interact high selectivity with the target molecules so aptamers are called “chemical antibodies”. When working conditions and features are compared, aptamers are much prefer than antibodies. The most important superior of aptamers can be synthesis in the laboratory as “in vitro”. Bonding their target molecules with high specifity in respect to suitable working conditions, aptamers have many applications fields such as purifications, diagnosis, treatment, biotechnology, and biosensor. The most common method of application is that aptamers are immobilized on a surface for intreaction with target molecule. Aptamers show specifity not only to small molecules such as drugs, peptides but also to large molecules such as proteins, virus, vitamin, and bacteria spores. Although iv aptamer interact with many molecules, aptamer and protein interactions are very noteworthy in these interactions. In the scope of this thesis, enrichment of various concentration of IFN-γ and VEGF were studied owing to aptamer structures. Firstly, aptamers and proteins in various matrix were analyzed by MALDI-MS, because suitable matrix choice is most important factor in MALDI-MS for aptamers and proteins. To examine whether aptamer bind on the surface or not, it was checked and then control studies carried out for aptamer-protein interactions. Before enrichment and after enrichment, analyses of proteins in the solutions were done. Analysis results show that lowest concentration of IFN- was succeed to be enriched and detected for 0.007 pmol/μL and this was found to be 0.002 pmol/μL for VEGF. It was noticed that study of enrichment are sucessful in this study for IFN- and VEGF. Optimization studies such as effect of polymeric surface area, effect of buffer solution using in the experiments and peptide presence on the surface were tested for the purpose of the best enrichment. Then specifity of aptamers to target protein was checked in the presence of other proteins and it was noticed that aptamers show high specificty to target protein that aptamers only interacted with target protein even if other proteins were exist in the mixture solutuions. Specifity of aptamers was investigated in the real World sample that have large or small molecule together such as commerical human blood plasma. In addition to these studies, ziptip procedure (this procedure enable to get rid of the chemicals used in protein digestion such as DTT, IAA or salt) was applied to very diluted VEGF solution and the procedure effect on peptide signal intensity was examined. According to MALDI-MS analysis results, using the method proposed in this thesis, enrichments of low concentration of IFN- and VEGF proteins were successfully done. Also undetectable peptide signals in rutin procedure could be observed very clearly compared to the signal of those peptide at the same concentration in the solution without preconcentration using MALDI-MS.