B-cell activating factor (BAFF) is a member of the TNF ligand superfamily. Tumor necrosis factor (TNF) is a multifunctional cytokine with a wide range of functions, one being inducing necrosis of tumor cells. It has been shown that upon binding to BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), the ligand-receptor interaction plays a role in the maturation of B and T-cells. The interaction also increases cell proliferation of normal and malignant B-cells, resulting in an increase of cancer cells over normal cells. Overexpression of BAFF bound to BAFF-R has been identified in Non Hodgkin’s Lymphoma, NHL. NHL stems from the lymphocytes, white blood cells or B and T-cells, and spreads throughout tissues. This cancer can be either aggressive, fast growing, or non-aggressive, slow. RNA interference among the BAFF - BAFF-R interaction lowers the expression of these genes. This leads to the hypothesis that aptamers have therapeutic capabilities. An aptamer is a single stranded DNA or RNA that can bind to select target areas with high specificity. Binding of the BAFF-R aptamer to the BAFF-R protein blocks and limits the binding of the BAFF protein to the receptor. This hinders and slows the productions of the malignant cells. Since the malignant cell growth is usually much faster than normal B-cells, this approach allows normal B-cells to catch up and aide in preventing these malignant cells from taking over. The objective of this research project is to successfully synthesize and purify the RNA aptamer that interacts with the BAFF-R protein. Upon obtaining two DNA oligonucleotides, a DNA template was created and amplified by DNA PCR method. Once the template was created, a 10% agarose gel purification was performed using gel electrophoresis. After the gel confirmed the expected band size of 100 base pairs, a gel extraction procedure using an extraction kit followed. The extracted and purified templates were then transcribed using EPICENTER transcription kit. The newly transcribed RNA strands were run on a denaturing 2.5% polyacrylamide gel using electrophoresis once again to obtain the expected band size. Agarose gel electrophoresis and purification obtained an expected band size of 100 base pairs indicating the correct DNA template. Following, the polyacrylamide gel confirmed the expected RNA aptamer size of 81 bases. The above results confirmed the success of using PCR and transcription methods.

Synthesis and purification of BAFF-R RNA aptamer

251st National Meeting (2016)

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