Riboswitch analysis technology based on high resolution capillary electrophoresis

Abstract

Riboswitch is the cis-regulatory element in 5’-UTR and composes of an aptamer domain where the metabolites can be bound and an expression platform domain which regulates the downstream coding sequence by changing the structure. When riboswitch bound with the small molecules, conformational change of riboswitch occurs and alters the expression in transcription or translation level. To understand the mechanism and characteristic of riboswitch, technology that sensitively analyzes the conformational difference of riboswitch is needed. So, this research establishes new riboswitch analysis technology based on high resolution Capillary Electrophoresis (CE). This technology was performed by ABI PRISM® 310 Genetic Analyzer and riboswitch detection was done by PEO-PPO-PEO tri-block copolymer, 0.5x TB buffer condition (90mM Tris base, 89mM Boric acid, 1mM MgCl2, pH 8.5) showed supreme resolution compared to other conformation sensitive analysis methods. The riboswitch analysis using high-resolution CE is able to rapidly analyze the target with small amounts of sample, and has advantages of high reproducibility and repeatability. In this research, dynamics of riboswitch was revealed with the mobility shift in high resolution CE system. SAM-Ⅰand coenzyme B12 riboswitch that are isolated from Bacillus subtilis and lysine riboswitch that is originated from Escherichia coli were used as target riboswitches. In order that mobility of riboswitch can be detected by capillary electrophoresis, riboswitch was labeled by fluorescence. Three kinds of riboswitch were analyzed by high resolution CE system. Mobility difference between the free state and bound state was observed and equilibrium dissociation constant (KD) could be also obtained from the mobility difference. Equilibrium dissociation constant is very important parameter to understand affinity between riboswitch and ligand. This parameter was calculated by peak area of electropherogram from high resolution CE system. So, this technology could be used as an identification method for the new riboswitch-ligand pair screening.