A simple "turn-on" fluorescent biosensor for sensitive detection of exonuclease III activity through photoinduced electron transfer and self-hybridization of a DNA probe
A simple "turn-on" fluorescent biosensor for sensitive detection of exonuclease III activity through photoinduced electron transfer and self-hybridization of a DNA probe
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一种简单的“开启”荧光生物传感器，通过光诱导电子转移和 DNA 探针的自杂交灵敏检测核酸外切酶 III 活性
DOI：
10.1039/c8ay00631h
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发表时间：
2018-05-21
影响因子：
3.1
通讯作者：
Li, Baoxin
中科院分区：
文献类型：
--
作者：
Liu, Qiang;Lian, Jinyu;Li, Baoxin
外切核酸酶III（ExoIII）在维持基因组稳定性方面起着至关重要的作用。在此，开发了用于检测Exo III活性的开启荧光策略。在该方法中，多聚（CG）单链DNA（ssDNA）探针在3′端用荧光素酰胺（FAM）标记，ssDNA可以自杂交形成双链DNA（dsDNA）。该dsDNA充当Exo III的底物。由于FAM和鸟嘌呤（G）之间的光诱导电子转移（PET），FAM标记的dsDNA探针的荧光被完全淬灭。在加入Exo III后，dsDNA将被消化，并且FAM发出非常强的荧光。因此，可以用简单的荧光读数器方便地测量Exo III活性。该方法的检测范围为5 × 10−4 U mL−1至5 U mL−1，检测限为3 × 10−4 U mL−1。本研究结果为Exo III活性检测提供了一种新的模式，具有较高的准确性和特异性，并可应用于真实的生物样品，为药物筛选和核酸外切酶相关基础研究提供了新的思路。
Exonuclease III (Exo III) plays crucial roles in maintaining the genome stability. Herein, a turn-on fluorescence strategy for detection of Exo III activity is developed. In this strategy, a poly(CG) single-stranded DNA (ssDNA) probe is labeled with fluorescein amidite (FAM) at the 3′ end, and the ssDNA can self-hybridize to form double-stranded DNA (dsDNA). This dsDNA acts as the substrate for Exo III. Due to photoinduced electron transfer (PET) between FAM and guanine (G), the fluorescence of the FAM-labeled dsDNA probe is completely quenched. Upon the addition of Exo III, the dsDNA will be digested, and FAM emits very strong fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. This method has a wide detection range from 5 × 10−4 U mL−1 to 5 U mL−1 with a detection limit of 3 × 10−4 U mL−1. The results demonstrated herein may provide a new pattern for Exo III activity detection with high accuracy and good specificity as well as satisfactory applicability in real biosamples, which holds great potential for drug screening and basic research related to exonucleases.