Trinucleotide repeat system for sequence specificity analysis of RNA structure probing reagents
Trinucleotide repeat system for sequence specificity analysis of RNA structure probing reagents
复制标题
DOI：
10.1016/j.ab.2010.03.021
复制
发表时间：
2010-07-01
影响因子：
2.9
通讯作者：
Krzyzosiak, Wlodzimierz J.
中科院分区：
文献类型：
--
作者：
Sobczak, Krzysztof;Michlewski, Gracjan;Krzyzosiak, Wlodzimierz J.
化学和酶结构探针已经使用了几十年，以获得关于各种rna分子结构的快速和全面的信息。尽管它们被广泛使用，但这些RNA结构探测试剂的序列特异性尚未得到彻底的表征。在这项研究中，我们重新审视了常用的结构探针的性质，如Pb（II）离子，核糖核酸酶V1，核糖核酸酶T2，以及S1和绿豆核酸酶，通过在具有3‘和5’相邻核苷酸的每种可能组合的高度规则的三联体重复序列上进行测试。我们发现，Pb（II）离子优先在嘧啶后裂解，而Si核酸酶对G残基后的磷酸二酯键具有先前被忽视的特异性。我们还观察到绿豆核酸酶更倾向于切割ApN键，RNase V1主要识别单链和双链rna中的U残基。这些数据对于结构探测实验结果的准确解释和对单个RNA分子正确结构的分配是重要的。这里描述的三联体重复转录系统可以被认为是确定用于探测RNA结构的其他试剂的序列特异性的可靠平台。(C) 2010爱思唯尔公司版权所有。
Chemical and enzymatic structural probes have been used for decades to obtain rapid and comprehensive information regarding the molecular architecture of various RNAs. Despite their widespread use, the sequence specificity of these RNA structural probing reagents has not yet been thoroughly characterized. In this study, we revisited the properties of commonly used structural probes such as Pb(II) ions, ribonuclease V1, ribonuclease T2, and the S1 and mung bean nucleases by testing them on highly regular triplet repeat sequences representing phosphodiester bonds with every possible combination of 3' and 5' adjacent nucleotides. We show that Pb(II) ions preferentially cleave after pyrimidines and that Si nuclease possesses a previously overlooked specificity toward phosphodiester bonds following G residues. We also observed that mung bean nuclease shows a preference for cleaving ApN bonds and that RNase V1 mainly recognizes U residues in both single- and double-stranded RNAs. These data are important for accurate interpretation of the results of structure probing experiments and for assignment of the correct structure to individual RNA molecules. The triplet repeat transcript system described here may be considered as a reliable platform for determining the sequence specificity of other reagents used to probe RNA structure. (C) 2010 Elsevier Inc. All rights reserved.