Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Chembiochem. 2010 Oct 18;11(15):2132-7. doi: 10.1002/cbic.201000329.
We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple-helix formation at adjacent positions on a specific purine-rich target sequence of duplex DNA. One fluorescein-labeled probe contains an α-azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double-stranded DNA, at least for homopurine-homopyrimidine target sites, under native and nondenaturing conditions.
我们描述了一种用于检测溶液中特定双链 DNA 序列的模板荧光化学的发展。在这种方法中，两个经过修饰的同聚嘧啶寡脱氧核苷酸探针被设计为在双链 DNA 的特定嘌呤丰富靶序列的相邻位置通过三螺旋形成结合。一个荧光素标记的探针含有一个α-叠氮醚连接子到荧光猝灭剂;第二个（触发）探针带有一个三芳基膦基团，设计用于还原叠氮化物并切断连接子。数据表明，在 pH5.6 下，这些探针在添加互补同聚嘌呤双链 DNA 靶子时，在数分钟内产生强烈的荧光信号。信号增加了约 60 倍，并且完全依赖于靶 DNA 的存在。用假异胞嘧啶替代探针中的胞嘧啶允许模板化学在 pH7 下顺利进行。靶寡核苷酸中的单核苷酸错配大大减缓了模板反应;这证明了很高的序列选择性。以前没有报道过使用模板荧光化学来检测双链 DNA，它可以在天然和非变性条件下检测双链 DNA，至少对于同聚嘌呤-同聚嘧啶靶位点是如此。
