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

中整合表达平台以选择与疾病生物标志物结合的 DNA 适体

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 

适配体可用于生物传感器、诊断工具和治疗试剂。这些应用通常需要融合适配体和表达平台。然而，融合过程通常既费时又费力。在这项研究中，我们整合了脱氧核酶 (I-R3) 作为 SELEX 循环（称为 Expression-SELEX）中的表达平台，以选择可以感知不同分子的适体酶。我们使用枫糖浆尿病 (MSUD) 生物标志物 L-异亮氨酸来测试选择模型。五轮筛选后，裂解产物充分富集，可在聚丙烯酰胺凝胶电泳 (PAGE) 凝胶上观察。通过高通量测序分析，确定了几个候选者。一种这样的候选物 IR3-I-DNA 以 0.57 mM 的解离常数 (KD) 结合 L-异亮氨酸。当配体存在时，IR3-I-DNA的切割分数从0.3增加到0.5，其Kobs值从1.38 min-1提高到1.97 min-1。我们的选择方法也可用于生产可与可变配体结合并更直接用作生物传感器的适体酶。

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