Lee Jihee, Han Jueun, Song Yejin, Gu Boram, Kim Eunjung
Department of Bioengineering and Nano-Bioengineering, Research Center for Bio Materials and Process Development, Incheon National University, Incheon 22012, Republic of Korea.
School of Chemical Engineering, Chonnam National University, Gwangju 61186, Republic of Korea.
ACS Meas Sci Au. 2024 Nov 8;4(6):737-750. doi: 10.1021/acsmeasuresciau.4c00063. eCollection 2024 Dec 18.
Thermal cycling-based quantitative polymerase chain reaction (qPCR) represents the gold standard method for accurate and sensitive nucleic acid quantification in laboratory settings. However, its reliance on costly thermal cyclers limits the implementation of this technique for rapid point-of-care (POC) diagnostics. To address this, isothermal amplification techniques such as rolling circle amplification (RCA) have been developed, offering a simpler alternative that can operate without the need for sophisticated instrumentation. This study focuses on the development and optimization of toehold-mediated RCA (TRCA), which employs a conformationally switchable dumbbell DNA template for the sensitive and selective detection of cancer-associated miRNAs, specifically miR-21. In addition, we developed variants of hyperbranched TRCA (HTRCA), nicking-assisted TRCA (NTRCA), and hyperbranched NTRCA (HNTRCA) to facilitate exponential amplification by enhancing TRCA through the sequential incorporation of reverse primer (Pr) and nicking endonuclease (nE). By conducting a systematic kinetic analysis of the initial rate and end point signals for varying concentrations of key reaction components, we could identify optimal conditions that markedly enhanced the sensitivity and specificity of the TRCA variants. In particular, HNTRCA, which exploits the synergistic effect of Pr and nE, demonstrated an approximately 3000-fold improvement in the detection limit (260 fM) and a wider dynamic range of more than 4 log orders of magnitude compared to TRCA, thereby evidencing its superior performance. Also, we established a mechanistic model for TRCA that includes the roles of Pr and nE in different amplification processes. Model parameters were fitted to the experimental data, and additional simulations were conducted to compare the four amplification methods. Further tests with real biological samples revealed that this technique showed a good correlation with qPCR in quantifying miR-21 expression in various cell lines (0.9510 of Pearson's ), confirming its potential as a robust and rapid tool for nucleic acid detection. Therefore, the simplicity, high sensitivity, and potential for integration with POC diagnostic platforms make the HNTRCA system suitable for field deployment in resource-limited environments.
基于热循环的定量聚合酶链反应(qPCR)是实验室环境中进行准确且灵敏核酸定量的金标准方法。然而,其对昂贵热循环仪的依赖限制了该技术在即时检测(POC)诊断中的应用。为解决这一问题,已开发出诸如滚环扩增(RCA)等等温扩增技术,提供了一种更简单的替代方法,无需复杂仪器即可运行。本研究聚焦于支点介导的RCA(TRCA)的开发与优化,该技术采用构象可切换的哑铃状DNA模板对癌症相关的微小RNA(miRNA),特别是miR - 21进行灵敏且选择性的检测。此外,我们开发了超支化TRCA(HTRCA)、切口辅助TRCA(NTRCA)和超支化NTRCA(HNTRCA)变体,通过依次掺入反向引物(Pr)和切口内切酶(nE)来增强TRCA,从而促进指数扩增。通过对不同浓度关键反应组分的初始速率和终点信号进行系统动力学分析,我们能够确定显著提高TRCA变体灵敏度和特异性的最佳条件。特别是,利用Pr和nE协同效应的HNTRCA与TRCA相比,检测限提高了约3000倍(260 fM),动态范围更宽,超过4个数量级,从而证明了其卓越性能。此外,我们建立了一个TRCA的机制模型,其中包括Pr和nE在不同扩增过程中的作用。将模型参数拟合到实验数据,并进行额外模拟以比较四种扩增方法。对真实生物样本的进一步测试表明,该技术在定量各种细胞系中miR - 21表达时与qPCR具有良好的相关性(Pearson相关系数为0.9510),证实了其作为一种强大且快速的核酸检测工具的潜力。因此,HNTRCA系统的简单性、高灵敏度以及与POC诊断平台集成的潜力使其适用于资源有限环境中的现场部署。