鉴定一种选择性聚合酶可用于检测 RNA 中的 N(6)-甲基腺苷。
Identification of a selective polymerase enables detection of N(6)-methyladenosine in RNA.
机构信息
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
出版信息
J Am Chem Soc. 2013 Dec 26;135(51):19079-82. doi: 10.1021/ja4105792. Epub 2013 Dec 11.
Abstract
N(6)-methyladenosine (m(6)A) is the most abundant mRNA modification and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m(6)A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m(6)A. We show that the enzyme can be used to locate and quantify m(6)A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m(6)A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m(6)A in mammalian 28S rRNA is nucleotide 4190.
摘要
N(6)-甲基腺苷（m(6)A）是最丰富的 mRNA 修饰，与人类健康有重要联系。虽然最近的研究已经成功地鉴定出数千种含有修饰的哺乳动物 RNA 转录本，但要确定任何特定 m(6)A 的的确切位置却极其困难。在这里，我们鉴定出一种具有逆转录酶活性的聚合酶（来自 Thermus thermophilus），它在将胸腺嘧啶掺入未修饰的 A 对 m(6)A 时的选择性高达 18 倍。我们表明，该酶可通过分析暂停带用于定位和定量合成 RNA 中的 m(6)A，并且已将该酶与非选择性聚合酶串联使用，以定位高丰度细胞 RNA 中 m(6)A 的存在和位置。通过这种方法，我们证明了 m(6)A 在哺乳动物 28S rRNA 中的长期未确定位置是核苷酸 4190。
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