Cole Kyle H, Lupták Andrej
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States.
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, United States; Department of Pharmaceutical Sciences, University of California, Irvine, CA, United States; Department of Chemistry, University of California, Irvine, CA, United States.
Methods Enzymol. 2019;621:329-346. doi: 10.1016/bs.mie.2019.02.009. Epub 2019 Mar 12.
Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers.
适体是一类小型功能性核酸,它们能与多种靶标结合,通常具有高度特异性和亲和力。基因组适体构成核糖开关的配体结合结构域,而合成适体则作为诊断和治疗工具,以及合成生物学中调控RNA的配体结合结构域发挥作用。由于缺乏能够揭示靶标结合结构域和单个序列生化特性的高通量方法,适体的发现和表征受到了限制。随着高通量测序的出现,对体外筛选的适体群体(以及催化核酸,如核酶和脱氧核酶)进行大规模分析成为可能。近年来,新实验方法和软件工具的发展极大地简化了筛选池分析。本文概述了筛选后数据分析,并描述了有助于快速发现适体及其生化表征的高通量方法。