Selection of RNA Aptamers that Bind Autophagy-Related BH3 Proteins

Abstract

Background: BH3 interacting domain death agonist (BH3) proteins are key mediators of autophagy. B-cell lymphoma-2 (Bcl-2) family proteins inhibit autophagy by binding to Beclin-1. Studies have shown that blocking the binding of Beclin-1 and Bcl-2 with simulated BH3 can promote autophagy, and this strategy has been applied in the treatment of malignant tumor diseases. In this study, we screened specific RNA aptamers to block the binding of Beclin-1 and Bcl-2 by the systematic evolution of ligands by exponential enrichment (SELEX) method to promote autophagy, which provides a new therapeutic approach for disease research. Methods: Recombinant BH3 protein was obtained and validated using a prokaryotic expression system. Recombinant BH3 protein was immobilized on carboxyl-conjugated magnetic beads, incubated with RNA libraries in SHMCK buffer under different conditions for 16 rounds, and then screened for RNA fittings that bind specifically to BH3 proteins. After screening, the sequence and secondary structure of the adaptors were analyzed. Results: We obtained recombinant BH3 protein of roughly 35KD under the optimized conditions. The main body of RNA aptamers can be divided into two families according to the homology tree analysis after 16 rounds of screening by magnetic bead coupling of target proteins and RNA aptamers. The homology of the two families is 77%, and the secondary structures of the two families are dominated by combinations of multiple stem-loops of different sizes, so it is assumed that aptamer bodies may bind to BH3 proteins through stem-loop topologies. We selected ligands #1 and #7 from both families to further demonstrate the affinities between the ligands and the proteins. According to affinity tests, the No. 1 RNA ligand and the BH3 protein exhibited substantial interaction (p < 0.05). Conclusion: In the study, we developed an innovative RNA aptamers screening method using carboxylated magnetic beads in SELEX technology to screen RNA aptamers specifically binding to BH3 protein. We determined the affinity of RNA aptamers to the BH3 protein using the enzyme-linked immunosorbent assay (ELISA) principle. This study broadens the scope of protein targets that may be screened using SELEX technology and offers a novel technique for determining the binding affinity of RNA aptamers and targets.