Diastereoselectivity of 5-Methyluridine Osmylation Is Inverted inside an RNA Chain.
Diastereoselectivity of 5-Methyluridine Osmylation Is Inverted inside an RNA Chain.
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DOI：
10.1021/acs.bioconjchem.6b00403
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发表时间：
2016-09
影响因子：
4.7
通讯作者：
L. Tserovski;M. Helm
中科院分区：
文献类型：
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作者：
L. Tserovski;M. Helm
在本研究中，我们研究了四氧化锇-联吡啶配合物与嘧啶在RNA中的反应。该试剂与5-6非对映异构体双键反应，从而形成两个非对映异构体，过去用于标记DNA中的胸苷和5-甲基胞嘧啶。鉴于对转录后RNA修饰的兴趣日益增长，我们解决了该试剂是否可用于标记自然发生的RNA修饰5-甲基胞嘧啶和5-甲基尿嘧啶的问题。在核苷水平上，5-甲基胞嘧啶和5-甲基尿嘧啶分别比它们的非甲基化等价物显示出5倍和12倍的偏好。在RNA水平上进行反应，我们可以表明，五核苷酸的空间环境对渗透化反应速率有主要的有害影响。有趣的是，这种反应性的下降是由于非对映选择性的巨大变化，而非对映选择性又由于si侧的首选攻击受阻而导致的。因此，在核苷水平上，5-甲基尿嘧啶酰化反应的主要产物的绝对构型为(5R,6S)-5-甲基尿嘧啶乙二醇-二氧基-联吡啶，与RNA五核苷酸反应产生相应的(5S,6R)-非对映体作为主要产物。非对映选择性的改变导致在五核苷酸环境中对5-甲基胞嘧啶的选择性几乎完全丧失，而5-甲基尿嘧啶的反应性仍然是典型嘧啶的8倍。基于这些发现，我们评估了四氧化锇-联吡啶在全转录组研究中作为5-甲基尿嘧啶修饰的潜在标记的有效性。
In this study, we investigated the reaction of the osmium tetroxide-bipyridine complex with pyrimidines in RNA. This reagent, which reacts with the diastereotopic 5-6 double bond, thus leading to the formation of two diastereomers, was used in the past to label thymidine and 5-methylcytosine in DNA. In light of the growing interest in post-transcriptional RNA modifications, we addressed the question of whether this reagent could be used for labeling of the naturally occurring RNA modifications 5-methylcytosine and 5-methyluridine. On nucleoside level, 5-methylcytosine and 5-methyluridine revealed a 5- and 12-fold preference, respectively, over their nonmethylated equivalents. Performing the reaction on an RNA level, we could show that the steric environment of a pentanucleotide has a major detrimental impact on the reaction rate of osmylation. Interestingly, this drop in reactivity was due to a dramatic change in diastereoselectivity, which in turn resulted from impediment of the preferred attack via the si side. Thus, while on the nucleoside level, the absolute configuration of the major product of osmylation of 5-methyluridine was (5R,6S)-5-methyluridine glycol-dioxoosmium-bipyridine, reaction with an RNA pentanucleotide afforded the corresponding (5S,6R)-diastereomer as the major product. The change in diastereoselectivity lead to an almost complete loss of selectivity toward 5-methylcytosine in a pentanucleotide context, while 5-methyluridine remained about 8 times more reactive than the canonical pyrimidines. On the basis of these findings, we evaluate the usefulness of osmium tetroxide-bipyridine as a potential label for the 5-methyluridine modification in transcriptome-wide studies.