Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.
Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.
复制标题
通过含有新型碱基类似物的三链体形成寡核苷酸靶向哺乳动物细胞中中断的多嘌呤：多嘧啶序列。
DOI：
10.1021/bi100797z
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Seidman,MM
中科院分区：
文献类型：
--
作者：
Semenyuk,A;Darian,E;Liu,J;Majumdar,A;Cuenoud,B;Miller,PS;MackerellJr,AD;Seidman,MM
DNA三螺旋由位于完整DNA双链体的大沟中的第三条核酸链组成。最稳定的三链体在多嘌呤上形成：多嘧啶序列，嘌呤链中的嘧啶中断是不稳定的。序列严格性由双链体中第三链碱基和嘌呤碱基之间的特异性Hoogsteen氢键赋予。三螺旋形成寡核苷酸（TFO）的适当碱基和糖修饰赋予活细胞中的染色体靶向活性。然而，TF 0作为哺乳动物细胞中的基因靶向试剂的广泛利用受到对高嘌呤靶序列的要求的限制。尽管已经描述了许多碱基类似物，它们似乎有希望作为三链体靶向扩增的候选物，但没有一个在生物系统中进行过研究。我们采用了合成后的策略来制备一系列的TFO，在一个确定的位置与碱基类似物。在评估对具有单个C：G倒位的三链体靶标的亲和力之后，合成具有第二代类似物的TFO。其中之一，TFO-5a，在对应于靶序列中C：G中断的位置处具有2′-OMe-胍基乙基-5-甲基胞嘧啶，被进一步修饰以赋予生物活性。在哺乳动物细胞系中测量了与peptide连接的该TFO的活性，所述哺乳动物细胞系通过定向序列转化工程化以携带具有单个C：G中断的三链体靶标。TFO-5a对该靶标有活性，对具有不间断多嘌呤：多嘧啶序列的相应靶标无活性。
The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2′-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence.