a Department of Chemical and Biomolecular Engineering (BK21+ Program) , KAIST , Daejeon , Republic of Korea.
b Department of Biological Engineering, College of Engineering , Konkuk University , Seoul , Republic of Korea.
Artif Cells Nanomed Biotechnol. 2019 Dec;47(1):256-259. doi: 10.1080/21691401.2018.1552593.
We herein describe a simple but efficient method for the determination of terminal transferase (TdT) activity, which relies on our finding that Fe(III)-quenched boron-dipyrromethene (BODIPY)-ATP is utilized as a switch-on monomer for polymerization and enables the facile synthesis of fluorescence oligonucleotides without additional, post-processing steps. As TdT carries out the synthesis of DNA by adding the monomers into growing chains, Fe(III) is displaced from BODIPY with the release of pyrophosphate group, which consequently leads to the generation of highly fluorescent long oligonucleotides. With this strategy, we selectively detected the TdT activity with high sensitivity. In addition, its practical applicability was successfully demonstrated by determining TdT activities in human serum.
我们在此描述了一种简单但高效的末端转移酶（TdT）活性测定方法，该方法基于我们的发现，即三价铁（Fe(III)）猝灭的硼二吡咯甲川（BODIPY）-ATP 可被用作聚合的开关单体，并且能够在无需额外后处理步骤的情况下轻松合成荧光寡核苷酸。由于 TdT 通过将单体添加到生长链中来合成 DNA，因此三价铁（Fe(III)）会从 BODIPY 中被取代，同时释放焦磷酸基团，从而导致产生高度荧光的长寡核苷酸。通过这种策略，我们可以选择性地高灵敏度检测 TdT 活性。此外，通过测定人血清中的 TdT 活性，成功地证明了其实际应用。
