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