Abstract：
APOBEC3A (A3A) is a cytidine deaminase with critical roles in molecular diagnostics. Herein, we demonstrate the enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of A3A activity in cancer cells. Target A3A can convert cytosine (C) in substrate probe to uracil (U), and then the template binds with substrate probe to form a dsDNA containing U/A base pairs. Uracil DNA glycosylase (UDG) excises the U base to produce an apurinic/apyrimidinic (AP) site that can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1) to obtain the substrate fragment with 3\'-OH end. Subsequently, the substrate fragment initiates cyclic enzymatic repairing amplification (ERA), releasing trigger-1 and trigger-2. The resultant trigger-1 can act as the primer to induce multiple cycles of cyclic ERA, producing numerous trigger-1 and trigger-2. The hybridization of trigger-2 with signal probe forms the dsDNA duplexes with an AP site, inducing the cyclic cleavage of signal probes by APE1 to release abundant Cy5 molecules from the AuNPs. Released Cy5 molecules can be easily quantified by single-molecule imaging. This nanosensor allows for specific and sensitive detection of A3A activity with a detection limit of 0.855 aM, and it can further measure kinetic parameters, screen inhibitors, and quantify endogenous A3A activity at the single-cell level, with prospect application in disease diagnostics and therapy.
摘要：
APOBEC3A（A3A）是一种胞苷脱氨酶，在分子诊断中具有关键作用。在这里，我们展示了基于Au纳米颗粒的纳米传感器的酶促DNA修复扩增驱动的构建，用于单分子监测癌细胞中的A3A活性。目标A3A可以将底物探针中的胞嘧啶（C）转化为尿嘧啶（U），然后模板与底物探针结合以形成含有U/A碱基对的dsDNA。尿嘧啶DNA糖基化酶（UDG）切除U碱基以产生一个嘌呤/嘧啶（AP）位点，该位点可以被嘌呤/嘧啶核酸内切酶1（APE1）切割以获得具有3'-OH末端的底物片段。随后，底物片段启动循环酶修复扩增（ERA），释放触发器-1和触发器-2。所得的触发因子-1可以作为引物诱导多个循环ERA，产生许多触发器-1和触发器-2。触发器-2与信号探针的杂交形成具有AP位点的dsDNA双链体,通过APE1诱导信号探针的循环裂解，以从AuNP释放大量的Cy5分子。释放的Cy5分子可以通过单分子成像容易地定量。这种纳米传感器可以对A3A活性进行特异性和灵敏的检测，检测极限为0.855aM，它可以进一步测量动力学参数，筛选抑制剂，并在单细胞水平上量化内源性A3A活性，在疾病诊断和治疗中的应用前景。
