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Integration of an Expression Platform in the SELEX Cycle to Select DNA Aptamer Binding to a Disease Biomarker

中整合表達平臺以選擇與疾病生物標志物結合的 DNA 適體

Yaqi Ao, Anqi Duan, Binfen Chen, Xinmei Yu, Yaoyao Wu, Xiaojun Zhang, and Sanshu Li*
Cite this: ACS Omega 2022, 7, 12, 10804–10811
Publication Date:March 17, 2022
https://doi.org/10.1021/acsomega.2c00769

Abstract

Aptamers can be developed for biosensors, diagnostic tools, and therapeutic reagents. These applications usually require a fusion of aptamers and expression platforms. However, the fusion process is usually time-consuming and laborious. In this study, we integrated the deoxyribozyme (I-R3) as an expression platform in the SELEX cycle (called Expression-SELEX) to select aptazymes that can sense diverse molecules. We used the Maple syrup urine disease (MSUD) biomarker L-allo-isoleucine to test the selection model. After five rounds of screening, the cleavage products were sufficiently enriched to be visualized on polyacrylamide gel electrophoresis (PAGE) gel. Through high-throughput sequencing analysis, several candidates were identified. One such candidate, IR3-I-DNA, binds L-allo-isoleucine with a dissociation constant (KD) of 0.57 mM. When the ligand was present, the cleavage fraction of IR3-I-DNA increased from 0.3 to 0.5, and its Kobs value improved from 1.38 min–1 to 1.97 min–1. Our selection approach can also be applied to produce aptazymes that can bind to variable ligands and be used more directly as biosensors.

摘要 

適配體可用于生物傳感器、診斷工具和治療試劑。這些應用通常需要融合適配體和表達平臺。然而，融合過程通常既費時又費力。在這項研究中，我們整合了脫氧核酶 (I-R3) 作為 SELEX 循環(huán)（稱為 Expression-SELEX）中的表達平臺，以選擇可以感知不同分子的適體酶。我們使用楓糖漿尿病 (MSUD) 生物標志物 L-異亮氨酸來測試選擇模型。五輪篩選后，裂解產(chǎn)物充分富集，可在聚丙烯酰胺凝膠電泳 (PAGE) 凝膠上觀察。通過高通量測序分析，確定了幾個候選者。一種這樣的候選物 IR3-I-DNA 以 0.57 mM 的解離常數(shù) (KD) 結合 L-異亮氨酸。當配體存在時，IR3-I-DNA的切割分數(shù)從0.3增加到0.5，其Kobs值從1.38 min-1提高到1.97 min-1。我們的選擇方法也可用于生產(chǎn)可與可變配體結合并更直接用作生物傳感器的適體酶。

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