ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling,Angewandte Chemie International Edition

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ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2016-01-06 , DOI: 10.1002/anie.201509131
Debin Ji ₁ , Michael G. Mohsen ₁ , Emily M. Harcourt ₁ , Eric T. Kool ₁

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A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP‐releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with K_m values averaging 13‐fold higher than those of natural dNTPs, and k_cat values within 1.5‐fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

中文翻译：

ATP释放核苷酸：将DNA合成与荧光素酶信号转导联系起来

据报道，通过使用嵌合核苷四磷酸二聚体从DNA合成产生发光信号的新策略，其中ATP而不是焦磷酸是离去基团。ATP释放核苷酸（ARN）被合成为四个规范核苷酸的衍生物。这四种衍生物都是DNA聚合酶的良好底物，K _m值平均比天然dNTP和k _cat高13倍。值是天然核苷酸的1.5倍以内。重要的是，发现使用荧光素酶的ARN产生的背景信号非常少。DNA合成实验表明，在荧光素酶存在下，可以利用ATP副产物引发化学发光信号。当将聚合酶与嵌合核苷酸一起使用时，靶标DNA / RNA触发化学计量上大量ATP的释放，从而允许对核酸进行灵敏的等温发光检测，这些核酸的种类包括噬菌体DNA和短miRNA。

更新日期：2016-01-06

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