Template-mediated synthesis of lariat RNA and DNA
Template-mediated synthesis of lariat RNA and DNA
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DOI：
10.1021/jo035002r
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发表时间：
2003-10-31
影响因子：
3.6
通讯作者：
Damha, MJ
中科院分区：
文献类型：
--
作者：
Carriero, S;Damha, MJ
核酸“幼虫”作为信使RNA（幼虫RNA内含子）生物合成中的剪接中间体，自20年前被发现以来，一直引起生物界的极大兴趣。我们在这里报道了通过模板介导的y型寡核苷酸化学连接首次合成的幼虫DNA和RNA。该方法允许合成任何碱基组成的幼虫DNA以及更具生物学相关性的幼虫RNA。通常，支链前体和互补线性模板（“夹板”）以等摩尔比溶解，总浓度为10(-4)M，在pH 7.6的缓冲液中加入溴化氰促进结扎。模板导向环化是非常有效的，因为在所有情况下观察到的环化的lariproduct的量在40-60%的范围内。经聚丙烯酰胺凝胶电泳纯化，MALDI-TOF质谱鉴定其结构和核苷酸组成。lariat:RNA和lariat:DNA双链的热变性和圆二色性研究完全支持分离的“套索”结构。通过脾脏磷酸二酯酶（一种3'-核酸外切酶）和RNA分枝脱支酶（一种特异性的2',5'-磷酸二酯酶）的酶降解进一步进行了表征。
Nucleic acid "lariats" have been of great interest to the biological community since their discovery two decades ago as splicing intermediates in the biosynthesis of messenger RNA (lariat RNA introns). We report here the first synthesis of lariat DNA and RNA via template-mediated chemical ligation of Y-shaped oligonucleotides. The method allows for the synthesis of lariat DNA of any base composition as well as the more biologically relevant lariat RNA. Typically, branched precursors and complementary linear templates ("splints") were dissolved in an equimolar ratio at a total concentration of 10(-4) M, and ligation was promoted by addition of cyanogen bromide in a pH 7.6 buffer. The template-directed cyclization was very efficient, since the amount of circularized lariat product observed in all cases was in the 40-60% range. The lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition confirmed by MALDI-TOF mass spectrometry. Thermal denaturation and circular dichroism studies of lariat:RNA and lariat:DNA duplexes were fully supportive of the isolated "lasso" structures. Further characterization was conducted by enzymatic degradation with spleen phosphodiesterase (a 3'-exonuclease) and the RNA lariat debranching enzyme, a specific 2',5'-phosphodiesterase.