SITE-SPECIFIC ENZYMATIC INCORPORATION OF AN UNNATURAL BASE, N(6)-(6-AMINOHEXYL)ISOGUANOSINE, INTO RNA
SITE-SPECIFIC ENZYMATIC INCORPORATION OF AN UNNATURAL BASE, N(6)-(6-AMINOHEXYL)ISOGUANOSINE, INTO RNA
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DOI：
10.1021/ja00064a007
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发表时间：
1993-06-02
影响因子：
15
通讯作者：
DERVAN, PB
中科院分区：
文献类型：
--
作者：
TOR, Y;DERVAN, PB
描述了一种有效的酶促方法，用于将可官能化的修饰碱基序列特异性掺入RNA分子中。DNA模板中的脱氧-5-甲基异胞苷（d（Me）isoC）指导T7 RNA聚合酶将N6-（6-氨基己基）异鸟苷（6-AH-isoG）掺入转录的RNA产物中。在ATP存在下，与T相对的异GTP衍生物的错误掺入被消除，并且在异GTP衍生物存在下，与d（Me）isoC相对的A的错误掺入可以忽略不计。使用修饰的模板的RNA产物的分离产率是使用天然模板的反应的约50%。用N-羟基琥珀酰亚胺活化的生物素或乙二胺四乙酸的二酐对反应性伯氨基进行转录后修饰，提供适合于进一步研究的位点特异性修饰的RNA序列。这种用于产生含有适合于转录后修饰的伯胺的RNA分子的方法应该可用于通过亲和切割和亲和标记来绘制折叠的RNA聚合物和RNA-蛋白质复合物的结构。
An efficient enzymatic method is described for the sequence-specific incorporation of a functionalizable modified base into RNA molecules. A deoxy-5-methylisocytidine (d(Me)isoC) in the DNA template directs the T7 RNA polymerase incorporation of N6-(6-aminohexyl)isoguanosine (6-AH-isoG) into the transcribed RNA product. The misincorporation of isoGTP derivatives opposite T is eliminated in the presence of ATP, and the misincorporation of A opposite d(Me)isoC is negligible in the presence of isoGTP derivatives. The isolated yield of RNA products using modified templates is approximately 50% that for reactions using natural templates. A post-transcriptional modification of the reactive primary amino group with N-hydroxysuccinimide-activated biotin or the dianhydride of ethylenediaminetetraacetic acid affords site-specifically modified RNA sequences suitable for further studies. This method for the generation of RNA molecules containing a primary amine suitable for post-transcriptional modification should be useful for mapping the structure of folded RNA polymers and RNA-protein complexes by affinity cleavage and affinity labeling.