PDF(Pubmed)
Abstract：
SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.
摘要：
SELEX(通过指数富集的配体的系统进化)过程旨在进化高亲和力适体作为诊断和生物传感中的结合实体。适体可以代表游戏规则改变者，作为用于管理即时发生的传染病或其他健康威胁的诊断测定的成分。如果没有过程中的质量控制措施，SELEX的总体成功率较低。我们提出了一种定量PCR方法，用于快速简便地定量与其靶标结合的适体。每个SELEX轮次的解链温度(Tm)的同时测定通过增加GC含量和单独的Tm与适体对相应靶标的亲和力的逐次增加的相关性来提供关于进化成功的信息。基于九项成功并已发布的SELEX流程，其中证明了适体亲和力/特异性的进化/选择，我们在这里显示了IMPATIENT-qPCR对多克隆适体文库和产生的单个适体的功能。基于这种新的进化质量控制的便利性，我们希望把它作为一个有价值的工具来加速SELEX流程。IMPATIENT-qPCRSELEX成功监测。使用SELEX技术和直接适体进化监测的高亲和力适体的选择和进化使用解链曲线移动分析通过使用荧光染料SYBRGreenI的定量PCR至更高的Tm。关键点:•快速和容易的分析。•一系列真正成功的项目的普遍适用性。
