Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
Methods. 2013 Dec 15;64(3):218-23. doi: 10.1016/j.ymeth.2013.08.011. Epub 2013 Aug 21.
A luminescent iridium(III) complex has been discovered to be selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for 3'→5' exonuclease activity in aqueous solution. A proof-of-concept of this assay has been demonstrated by using prokaryotic exonuclease III (ExoIII) as a model enzyme. In this assay, a G-quadruplex-forming hairpin oligonucleotide (hairpin-G4 DNA, 5'-GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-3') initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the iridium(III) complex and duplex DNA. Upon digestion by ExoIII, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for 3'→5' exonuclease over other DNA-modifying enzymes.
研究发现，一种发光明亮的铱（III）配合物对 G-四链体 DNA 具有选择性，并被用于在水溶液中基于无标记 G-四链体的 3'→5'外切核酸酶活性检测。该测定法的概念验证已通过使用原核外切核酸酶 III（ExoIII）作为模型酶来证明。在该测定中，形成 G-四链体的发夹寡核苷酸（发夹 G4 DNA，5'-GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-3'）最初以双链构象存在，由于铱（III）配合物与双链 DNA 之间的弱相互作用，导致发光信号较弱。经 ExoIII 消化后，富含鸟嘌呤的序列被释放并折叠成 G-四链体，大大增强了铱（III）探针的发光发射。与其他 DNA 修饰酶相比，该方法对 3'→5'外切核酸酶具有高度的敏感性。
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