Li Bingzhi, Zhang Peng, Zhou Bin, Xie Siying, Xia Anqi, Suo Tiying, Feng Shuang, Zhang Xing
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210023, China.
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210023, China; Jiangsu Conat Biological Products Co., Ltd., Shugang Road 20, Taixing, 225400, China.
Anal Chim Acta. 2021 Mar 1;1148:238194. doi: 10.1016/j.aca.2020.12.069. Epub 2021 Jan 4.
Flap endonuclease 1 (FEN1), a ubiquitous enzyme involved in DNA repair and replication, is overexpressed in highly proliferative cancer cells. FEN1 has been recognized as a promising diagnostic marker of cancers; however, very few analytical techniques have been developed for the convenient detection of FEN1. To realize the simplified quantification of FEN1, we developed a FEN1-responsive fluorescent nanoprobe based on DNA-silver nanoclusters (DNA-AgNCs). The nanoprobe was rationally designed with a double-flapped dumbbell conformation, where its 5' flap was produced with DNA-AgNCs, and the 3' flap was elongated by a guanine-rich enhancer sequence (GRS). Rigidified by the DNA scaffold, DNA-AgNCs and the GRS are in close proximity, resulting in high fluorescence because of the GRS-induced activation of DNA-AgNCs. Upon the addition of FEN1, the 5' flap of the nanoprobe is cleaved due to the structure-specific endonuclease activity of FEN1. This cleavage released the DNA-AgNCs from the nanoprobe, broke the proximity between DNA-AgNCs and the GRS, and caused decreased fluorescence. This nanoprobe can be applied in the sensitive detection of FEN1 with a detection limit of 40 fM, and it showed high specificity for the monitoring of FEN1 in clinical samples. As the first attempt to develop biosensors targeting FEN1 based on DNA-AgNCs, this work provided a potent platform for monitoring FEN1 and screening FEN1 inhibitors.
翼端核酸内切酶1（FEN1）是一种参与DNA修复和复制的普遍存在的酶，在高增殖性癌细胞中过度表达。FEN1已被公认为一种有前景的癌症诊断标志物；然而，针对FEN1便捷检测所开发的分析技术却非常少。为了实现FEN1的简化定量，我们基于DNA银纳米簇（DNA-AgNCs）开发了一种对FEN1有响应的荧光纳米探针。该纳米探针采用双瓣哑铃构象进行合理设计，其5'瓣由DNA-AgNCs产生，3'瓣由富含鸟嘌呤的增强序列（GRS）延长。DNA-AgNCs和GRS通过DNA支架刚性化而紧密靠近，由于GRS诱导的DNA-AgNCs激活而产生高荧光。加入FEN1后，由于FEN1的结构特异性核酸内切酶活性，纳米探针的5'瓣被切割。这种切割将DNA-AgNCs从纳米探针中释放出来，打破了DNA-AgNCs与GRS之间的接近性，导致荧光降低。这种纳米探针可用于FEN1的灵敏检测，检测限为40 fM，并且在临床样品中对FEN1的监测显示出高特异性。作为基于DNA-AgNCs开发靶向FEN1生物传感器的首次尝试，这项工作为监测FEN1和筛选FEN1抑制剂提供了一个有力的平台。
