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

作　　者：刘彬[1] 王雪丽[1] 张华利[1] 程瑛琨[1] 李正强[1] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,长春130023

出　　处：《分析化学》2005年第5期595-598,共4页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金资助项目(No.20273022)

摘　　要：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.

关 键 词：外源荧光探针 脱氧核酶 RNA 反应动力学 测定方法 技术改进 

分 类 号：O629.74[理学—有机化学]