基于外源荧光探针的脱氧核酶切割RNA反应动力学参数测定方法的改进

Improved Assay for Kinetic Constants of Ribonucleic Acid-Cleaving Deoxyribozymes Based on an Extrinsic Fluorescent Probe

对以EB作为外源荧光探针测定脱氧核酶切割RNA反应的动力学参数的方法进行了改进。实验结果表明:对于目前应用最为广泛的1023脱氧核酶和817脱氧核酶,在生理条件下(150mmol/LKCl,2mmol/LMgCl2,pH7.5,37℃)采用改进方法测得的反应动力学参数(kobs1023=0.12min-1,kobs817=0.08min-1)与原方法相比更接近于放射性标记方法的测定结果(k21023=0.13min-1,k2817=0.08min-1)。

An improved method to determine the kinetic constants of small ribonucleic acid (RNA)-cleaving deoxyribozyme was developed, based on the exogenous fluorophore ethidium bromide. Increasing the concentration of ethidium bromide avoids the attenuation of the amplitude of the spectroscopic signal due to increasing of temperature; a good signal-to-noise can be obtained. Thus this method will be more appropriate to the kinetic assay under simulated physiological conditions( 150 mmol/L KCl, 2 mmol/L MgCl2, pH 7.5, 37 degrees C). Using this method to 10-23 deoxyribozyme and 8-17 deoxyribozyme, observed rate constants of cleavage obtained (k(obs10-23) = 0. 12 min(-1), k(obs8-17) = 0. 08 min(-1)) are virtually the same to the rates (k(2) (10-23) = 0. 13 min(-1), k(2 8-17) = 0. 08 min(-1)) based on radioactive substrate. The results demonstrate that this method would be suitable for small deoxyribozymes, which contain little or no secondary structure.