Yan Zhaoqing, Tang Anli A, Eshed Amit, Ticktin Zackary M, Chaudhary Soma, Ma Duo, McCutcheon Griffin, Li Yudan, Wu Kaiyue, Saha Sanchari, Alcantar-Fernandez Jonathan, Moreno-Camacho Jose L, Campos-Romero Abraham, Collins James J, Yin Peng, Green Alexander A
Department of Biomedical Engineering, Boston University, Boston, MA, USA.
Molecular Biology, Cell Biology & Biochemistry Program, Graduate School of Arts and Sciences, Boston University, Boston, MA, USA.
medRxiv. 2023 Jun 6:2023.06.02.23290873. doi: 10.1101/2023.06.02.23290873.
Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. Here, we describe a class of aptamer-based RNA switches called aptaswitches that recognize specific target nucleic acid molecules and respond by initiating folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide a fast and intense fluorescent readout, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. We demonstrate that aptaswitches can be used to regulate folding of six different fluorescent aptamer/fluorogen pairs, providing a general means of controlling aptamer activity and an array of different reporter colors for multiplexing. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/L in one-pot reactions. Application of multiplexed one-pot reactions against RNA extracted from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that can be readily integrated into rapid diagnostic assays.
快速、简单且低成本的诊断技术是对抗传染病的关键工具。在此，我们描述了一类基于适配体的RNA开关，称为适配体开关，它能识别特定的靶核酸分子，并通过启动报告适配体的折叠做出响应。适配体开关几乎可以检测任何序列，并提供快速且强烈的荧光读数，在短短5分钟内产生信号，且只需最少的设备就能肉眼检测。我们证明适配体开关可用于调节六种不同荧光适配体/荧光团对的折叠，提供了一种控制适配体活性的通用方法以及一系列用于多重检测的不同报告颜色。通过将等温扩增反应与适配体开关相结合，我们在一锅反应中实现了低至1个RNA拷贝/升的灵敏度。针对从临床唾液样本中提取的RNA进行多重一锅反应，在30分钟内检测SARS-CoV-2的总体准确率为96.67%。因此，适配体开关是用于核酸检测的多功能工具，可轻松整合到快速诊断检测中。
