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Determination of the binding affinity of RNA aptamer to B‐cell activating factor receptor protein from non‐Hodgkin lymphoma
Author(s) -
Blade Thanh Phan,
Roa Stephanie,
Avrit Tara
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.460.1
Subject(s) - rna , aptamer , b cell activating factor , chemistry , microbiology and biotechnology , electrophoretic mobility shift assay , dissociation constant , rna binding protein , selex aptamer technique , receptor , b cell , biology , biochemistry , gene expression , antibody , gene , systematic evolution of ligands by exponential enrichment , immunology
Non‐Hodgkin lymphoma originates from B‐cells, a type of lymphocytes. Other research labs have shown a high expression of B‐cell activating factor protein (BAFF) binding its protein receptor BAFF‐R on the surface membrane of the malignant B cells. An RNA aptamer was designed not only to bind to BAFF‐R but also to deliver a siRNA into malignant B‐cells to induce cellular apoptosis. The ability of an RNA aptamer to deliver a siRNA in to the cells depends greatly on its high binding affinity to BAFF‐R by yielding a low dissociation constant or K D . We chose an electrophoresis mobility shift assay method to study protein‐RNA interaction using radioactive labeled RNA. After allowing various concentrations of BAFF‐R to incubate with a fixed concentration of RNA to form RNA‐protein complexes, we loaded the samples into wells of a 5% native polyacrylamide gel to separate unbound RNAs and RNA‐protein complexes. Then we exposed the gel to a film which registered the signals from radioactive phosphate RNA. We used Typhoon FLA 7000, a phosphorimager, to convert the film into an image which we analyzed to determine BAFF‐R concentration, which had fifty percent RNA bound to protein. The K D was calculated to be 495nM. In short, the affinity of RNA to BAFF‐R can be determined using EMSA, but we can lower the K D by optimizing the binding‐reaction conditions such as temperature or buffer condition. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .