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Hongwei Hu, Yujing Ding, Zihan Gao, Hao Li,
S1 nuclease digestion-based rational truncation of PD-L1 aptamer and establishment of a signal dual amplification aptasensor,
Sensors and Actuators B: Chemical,
Volume 331,
2021,
129442,
ISSN 0925-4005,
https://doi.org/10.1016/j.snb.2021.129442.
(http://www.sciencedirect.com.group21-s.aronip.com/science/article/pii/S0925400521000101)

Abstract: Truncation optimization of aptamers can improve both specificity and robustness. At present, aptamer truncation is mainly performed based on predictions from molecular docking simulations, but this usually requires tedious trial-and-error and may result in false predictions. Here, we propose a strategy based on digestion by S1 nuclease to rationally truncate the aptamer, using the PD-L1-binding aptamer Apt80 as an example. Due to steric hindrance, recognition and binding regions between Apt80 and PD-L1 are not digested by the enzyme. The truncated form, Apt38, shows higher affinity and larger conformational change after binding, when compared to Apt80. The truncated Apt38 was used as a platform for developing a signal dual amplification fluorescence aptasensor using targeted recycling assisted by exonuclease I and qRT-PCR analysis. This aptasensor exhibited a high sensitivity toward PD-L1 with a limit of detection of 0.076 ng/mL in buffer system and 0.3625 ng/mL in human serum. Owing to its high sensitivity, specificity, ease operation and low cost for detection of PD-L1, this aptasensor should be useful in assisting clinicians to evaluate the status of cancer patient and to decide whether inhibitor drugs are needed.
Keywords: Aptamer truncation; S1 nuclease digestion; PD-L1; Signal dual amplification aptasensor

摘要：核酸适体的截短优化可以提高其特异性和鲁棒性。目前，核酸适体的截短主要是基于分子对接模拟的预测，但这通常需要繁琐的试错，并可能导致错误的预测。本文以PD-L1结合适体Apt80为例，提出了一种基于S1核酸酶消化的策略来合理截断适体。由于空间位阻，Apt80和PD-L1之间的识别和结合区域不能被酶消化。与Apt80相比，截短的Apt38在结合后表现出更高的亲和力和更大的构象变化。截短的Apt38被用作开发信号双扩增荧光aptasensor的平台，利用核酸外切酶I和qRT-PCR分析辅助靶向回收。该传感器对PD-L1具有较高的灵敏度，在缓冲体系中的检出限为0.076ng/mL，在人血清中的检出限为0.3625ng/mL。该传感器具有灵敏度高、特异性强、操作简便、检测成本低等优点，有助于临床医生对肿瘤患者病情的评估及是否需要使用抑制剂。

关键词：适体截短；S1核酸酶消化；PD-L1；信号双放大自适应传感器

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