Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

IF 10.7 2区 材料科学 Q1 CHEMISTRY, PHYSICAL
Stefen Stangherlin, Yuzhe Ding, Juewen Liu
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引用次数: 0

Abstract

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

小分子结合适配体的解离常数 (Kd) 测量:均相化验方法和关键评估。
自 1990 年以来,已经分离和发现了许多适配体,可用于各种分析、生物医学和环境应用。这一趋势至今仍在继续。鉴定适配体结合特性的关键步骤是测量其与目标分子和非目标分子的结合亲和力。解离常数(Kd)是表征适配体结合的最常用值。本文综述了可与小分子靶标结合的适配体的同源检测方法。所综述的方法包括无标记方法(如等温滴定量热法、靶分子的内在荧光、DNA 染色染料和核酸酶消化测定法)和标记方法(如链置换反应)。有些方法不推荐使用,如基于金纳米粒子聚集和纳米材料中荧光团标记 DNA 的解吸的方法。测量到的表观 Kd 与真正的适配体结合 Kd 之间的差异值得强调。此外,还讨论了避免滴定机制和注意达到平衡所需的时间。最后,将突变的非结合序列作为对照非常重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Small Methods
Small Methods Materials Science-General Materials Science
CiteScore
17.40
自引率
1.60%
发文量
347
期刊介绍: Small Methods is a multidisciplinary journal that publishes groundbreaking research on methods relevant to nano- and microscale research. It welcomes contributions from the fields of materials science, biomedical science, chemistry, and physics, showcasing the latest advancements in experimental techniques. With a notable 2022 Impact Factor of 12.4 (Journal Citation Reports, Clarivate Analytics, 2023), Small Methods is recognized for its significant impact on the scientific community. The online ISSN for Small Methods is 2366-9608.
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