Date

2021-2-15

Author

Kara, Nilüfer

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Membrane Protein (MP) targeting remains a challenge because of their amphipathic nature. In the lipid bilayer, both hydrophobic and hydrophilic domains provide MPs their characteristic functions. Therefore in vitrostudies require an environment for mimicking their natural membrane environmentin order for MPs to foldinto their native conformationto function properly. Here, treatment with a detergent is imperative foraMP solubilization to make it a stable individual protein without losing its functionality. In our studies, for aptamer selection we targetedCadaverine/Lysine Antiporter (CadB) as a model protein for MPs, a secondary transporter on the inner membrane of Escherichia Coliwith a critical role in acid stress adaptation.Aptamers are short in vitro-selected RNA or DNA oligonucleotides. The selection of aptamer is engineered by the method known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). This process has iterative rounds to select the best aptamer/s with high affinity and selectivity for a giventarget. Itconsists of three main steps: incubationof the aptamerwith the target, separation of bound oligo-protein complex, and oligo amplification. Interactionswith the target lead to conformational change on the aptamer structure.After aptamer development, theseinteractions may find potential uses indiagnostic or therapeutic applications. To date, many kinds of SELEX techniques have been carried out for aptamer selection. However, some are labor-and time-consuming and may give less specificity against MPs. In this project, before selection, we achieved both solubilization and purification of CadB in a mild detergent,n-dodecyl-β-D-maltopyranoside (DDM). We performed eight rounds of protein-SELEX against CadB in DDM. After completingthe final round, the RNA pool was sequenced, and we finally got 29 distinct sequences. We found three abundant sequences with double copies: CadB30, CadB35, and CadB39. Furthermore, we determined the potential binding motif by multiple sequence alignment analysis. After that, according to the predicted 2D secondary structures (done by RNAfold and KineFold), this motif was found on the loops of CadB35 and CadB39, while it was found on the stem of CadB30. Also, we randomly chose a proto-aptamer (CadB41), which is closed to CadB30 and CadB39, to show their structural similarity with their homology matrixes. Overall, in this study, we successfully generated aptamers against CadB after eight rounds of protein-SELEX. This study shows that CadB, as an MP, can be targeted by protein-SELEX in a suitable mild detergent. In this way, targeting MPs may become more convenient for both diagnosis and therapeutic purposes inthe near future. Protein-SELEX is suitable for all kinds of proteins,andit serves as an efficient, straightforward, time-saving, and cost-effective way for aptamer selection

Subject Keywords

Membrane Protein, CadB, Aptamer, Protein-SELEX, DDM

URI

https://hdl.handle.net/11511/89796

Collections

Graduate School of Natural and Applied Sciences, Thesis