{"title":"Small Molecule Detection with Ligation-Dependent Light-Up Aptamer Transcriptional Amplification.","authors":"Deok-Gyu Lee, Yoo-Hong Min, Ju-Young Byun, Yong-Beom Shin","doi":"10.1021/acsabm.4c00987","DOIUrl":null,"url":null,"abstract":"<p><p>ATP and NAD<sup>+</sup> are small biomolecules that participate in a variety of physiological functions and are considered as potential biomarkers for disease diagnosis. In this study, we developed a ligation-dependent light-up aptamer transcriptional amplification assay for the sensitive and selective detection of ATP and NAD<sup>+</sup>. This assay relies on a specific DNA ligase that catalyzes the ligation of a nicked DNA template in the presence of a specific small molecule. We prepared a nicked template consisting of a duplex fragment with an overhang for the T7 promoter region and a single-stranded DNA with a complementary overhang sequence for the Broccoli aptamer. The nicked template was connected using a DNA ligase in the presence of a specific small molecule. The ligation product was subjected to <i>in vitro</i> transcription to amplify the light-up aptamer-mediated fluorescence signals. By integrating the target-dependent ligation and transcription amplification, significant signal amplification was achieved with 5.9 and 142 pM detection limits for ATP and NAD<sup>+</sup>, respectively. Moreover, good selectivity to discriminate between the target and its analogues was also realized. The application of this method to biological samples was evaluated using human serum and exhibited excellent recovery values.</p>","PeriodicalId":2,"journal":{"name":"ACS Applied Bio Materials","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Bio Materials","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1021/acsabm.4c00987","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MATERIALS SCIENCE, BIOMATERIALS","Score":null,"Total":0}
引用次数: 0
Abstract
ATP and NAD+ are small biomolecules that participate in a variety of physiological functions and are considered as potential biomarkers for disease diagnosis. In this study, we developed a ligation-dependent light-up aptamer transcriptional amplification assay for the sensitive and selective detection of ATP and NAD+. This assay relies on a specific DNA ligase that catalyzes the ligation of a nicked DNA template in the presence of a specific small molecule. We prepared a nicked template consisting of a duplex fragment with an overhang for the T7 promoter region and a single-stranded DNA with a complementary overhang sequence for the Broccoli aptamer. The nicked template was connected using a DNA ligase in the presence of a specific small molecule. The ligation product was subjected to in vitro transcription to amplify the light-up aptamer-mediated fluorescence signals. By integrating the target-dependent ligation and transcription amplification, significant signal amplification was achieved with 5.9 and 142 pM detection limits for ATP and NAD+, respectively. Moreover, good selectivity to discriminate between the target and its analogues was also realized. The application of this method to biological samples was evaluated using human serum and exhibited excellent recovery values.
ATP 和 NAD+ 是参与多种生理功能的生物小分子,被认为是疾病诊断的潜在生物标志物。在这项研究中,我们开发了一种依赖于连接的发光适配体转录扩增检测方法,用于灵敏、选择性地检测 ATP 和 NAD+。这种检测方法依赖于一种特定的 DNA 连接酶,它能在特定小分子存在的情况下催化缺口 DNA 模板的连接。我们制备了一个缺口模板,它由一个带有 T7 启动子区域悬垂序列的双链片段和一个带有西兰花适配体互补悬垂序列的单链 DNA 组成。在特定小分子存在的情况下,使用 DNA 连接酶连接缺口模板。将连接产物进行体外转录,以扩增亮起的适配体介导的荧光信号。通过整合目标依赖性连接和转录扩增,实现了显著的信号扩增,ATP 和 NAD+ 的检测限分别为 5.9 和 142 pM。此外,该方法还具有良好的选择性,可以区分目标物及其类似物。利用人体血清对该方法在生物样本中的应用进行了评估,结果表明其回收率极高。