Selection Of RNA Aptamers That Bind Autophagy-Related BH3 Protein

Objective: In this study,RNA aptamers was selected to block the binding of Beclin-1 and Bcl-2 to promoting autophagy,which provides a new treatment for disease research.BH3 domain protein is an important domain protein in Bcl-2/Bcl-XL protein,which participates in the regulation of apoptosis,autophagy and other important cellular physiological activities of Bcl-2/Bcl-XL,and has important biological significance.As one of the key targets of autophagy,Bcl-2 family proteins inhibit its autophagy promotion 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,becoming a new research hotspot.RNA aptamers are obtained by SELEX screening,and SELEX(systematic evolution of ligands through exponential enrichment)is a very emerging and powerful method for determining the binding sites of proteins on RNA.It depends on the ability of RNA binding proteins to select high-affinity RNA aptamers from random RNA library.Methods: The recombinant BH3 protein was obtained by prokaryotic expression system,and 6×His and Flag tags were added at the C-terminus of the recombinant BH3 protein for verification.After purification by nickel column,the purified product was verified by 12% SDS-PAGE electrophoresis and western blot.ds DNA libraries with fixed random sequences at both ends and appropriate length were designed as templates for initial random RNA libraries.The initial RNA library was obtained by reverse transcription after PCR amplification,and the recombinant BH3 protein was fixed on the carboxy-conjugated magnetic beads.The recombinant BH3 protein was incubated with the RNA library in the SHMCK buffer for 14 rounds under different conditions,and then RNA apperors binding the specific BH3 protein were selected.After screening,the aptamers’ sequence and secondary structure were analyzed.Results: Escherichia coli containing recombinant protein was cultured under optimized conditions,and 12% SDS-PAGE analysis was performed after cryogenic ultrasonography.After analysis,the protein bands obtained after the treatment of the broken bacteria were concentrated at about 35 KD,and after purification,the protein bands were concentrated at 35 KD.Western blot further verified that the 35 KD bands were indeed the recombinant BH3 protein required by our experiment.Magnetic beads method coupling target protein and RNA aptamers after14 rounds screening,cloning sequencing by RNA aptamers level sequence,after removal of mutant,according to the same tree analysis of RNA aptamers body can be divided into two families,two homologous degree of 76%,and the secondary structure of the two families with multiple stem ring size combination is given priority to,speculation in the screening of RNA aptamers body may be through stem ring structure and restructuring BH3 protein in combination.The ligands No.1 and No.7 of the two families were selected to further verify the affinity between the ligands and proteins.Affinity tests showed that there was a strong binding force between BH3 protein and No.1 RNA ligands(p<0.05).The results of this RNA aptamer screening indicated the success of this screening process and provided a new idea for the further study of autophagy in the treatment of diseases.