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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 ampli?cation 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：核酸適體的截短優(yōu)化可以提高其特異性和魯棒性。目前，核酸適體的截短主要是基于分子對(duì)接模擬的預(yù)測(cè)，但這通常需要繁瑣的試錯(cuò)，并可能導(dǎo)致錯(cuò)誤的預(yù)測(cè)。本文以PD-L1結(jié)合適體Apt80為例，提出了一種基于S1核酸酶消化的策略來(lái)合理截?cái)噙m體。由于空間位阻，Apt80和PD-L1之間的識(shí)別和結(jié)合區(qū)域不能被酶消化。與Apt80相比，截短的Apt38在結(jié)合后表現(xiàn)出更高的親和力和更大的構(gòu)象變化。截短的Apt38被用作開發(fā)信號(hào)雙擴(kuò)增熒光aptasensor的平臺(tái)，利用核酸外切酶I和qRT-PCR分析輔助靶向回收。該傳感器對(duì)PD-L1具有較高的靈敏度，在緩沖體系中的檢出限為0.076ng/mL，在人血清中的檢出限為0.3625ng/mL。該傳感器具有靈敏度高、特異性強(qiáng)、操作簡(jiǎn)便、檢測(cè)成本低等優(yōu)點(diǎn)，有助于臨床醫(yī)生對(duì)腫瘤患者病情的評(píng)估及是否需要使用抑制劑。

關(guān)鍵詞：適體截短；S1核酸酶消化；PD-L1；信號(hào)雙放大自適應(yīng)傳感器

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