Use of dimethyl sulfate to probe RNA structure in vivo.
Use of dimethyl sulfate to probe RNA structure in vivo.
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DOI：
10.1016/s0076-6879(00)18071-1
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发表时间：
2000
影响因子：
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通讯作者：
Sandra E. Wells;John M. X. Hughes;A. Igel;M. Ares
中科院分区：
文献类型：
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作者：
Sandra E. Wells;John M. X. Hughes;A. Igel;M. Ares
理解RNA的工作原理需要协调使用不同的实验方法。了解RNA的结构及其与功能的关系是解释RNA作用的生物学机制的重要组成部分。利用遗传、系统发育、生物物理和计算方法来预测RNA结构和结构动力学，通过在溶液中应用RNA结构的化学和酶探针得到了极大的增强。用于研究RNA和核糖核蛋白结构的最通用的化学探针是硫酸二甲基（DMS），它可以直接给RNA中a、C和G残基上的特定氢键接受环氮提供甲基。甲基化的效率报告了每个碱基中敏感环氮的化学环境：氢键或较差的溶剂可及性导致甲基化保护，而溶剂暴露或不寻常的化学环境可能会增强甲基化1。甲基化的效率可以通过评估逆转录酶对引物延伸的甲基化依赖终止来估计RNA链上许多位置的甲基化效率2,3。这些信息提供了关于单个核苷酸环境的线索，可以与RNA结构模型和关于RNA功能的假设进行比较。DMS的一个主要的实验便利之处在于它能迅速渗透到细胞的所有区室。这一特性使得探测各种细胞中的RNA结构成为可能，包括革兰氏阴性细菌、革兰氏阳性细菌、酵母、原生动物和植物，包括细胞核、核仁和叶绿体。在生理温度下较短的孵育时间允许对体内RNA结构进行快速快照，并将干扰或次要效应导致观察到的结构的担忧降至最低。该方法可同时应用于多种培养，便于直接测定不同突变或处理对靶RNA折叠的影响。使用特定于许多rna的引物，可以在同一样品中确定许多rna的结构。本文介绍了利用DMS检测酵母细胞RNA结构的方法。
Understanding how RNA works requires the coordinated use of diverse experimental approaches. Knowledge of RNA structure and its relationship to function is an essential ingredient for interpreting the biological mechanisms of RNA action. Use of genetic, phylogenetic, biophysical, and computational approaches to divining RNA structure and structural dynamics is greatly enhanced by the application of chemical and enzymatic probes of RNA structure in solution. Among the most versatile chemical probes available for studying RNA and ribonucleoprotein structure is dimethylsulfate (DMS), which can directly donate a methyl group to specific hydrogenbond accepting ring nitrogens on A, C, and G residues in RNA. The efficiency of methylation reports the chemical environment of the sensitive ring nitrogens in each base: hydrogen bonding or poor solvent accessibilty results in protection from methylation, whereas solvent exposure or an unusual chemical environment may enhance methylation1. The efficiency of methylation can be estimated at many positions along the RNA chain by evaluating methylation-dependent stops to primer extension by reverse transcriptase2, 3. This information provides clues about the environment of individual nucleotides that can be compared to RNA structural models and hypotheses about RNA function.A major experimental convenience of DMS is its rapid penetration into all compartments of the cell. This feature has allowed probing of RNA structure in a wide variety of cells including gram negative4, 5 and gram positive6 bacteria, yeast7, protozoa8, 9 and plant10, including the nucleus7, nucleolus11, and chloroplasts12. Short incubation times at physiological temperatures allow for a quick snapshot of RNA structure in vivo with a minimum of perturbation or concern that secondary effects lead to the observed structure. The method can be applied to many cultures simultaneously, facilitating direct determination of the effect of different mutations or treatments on folding of the target RNA. Using primers specific for a number of RNAs, the structure of many RNAs can be determined in the same sample. In this paper we present methods for the probing of RNA structure in yeast cells using DMS.