Kale Tanvi, Pednekar Rudvi, Cazaux Séverine Marianne, Letelier Valentina Ferrando, Vigar Justin R J, Federici Fernán, Pardee Keith, Athale Chaitanya A
Div. of Biology, IISER Pune, Maharashtra 411008, India.
ANID - Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio), Santiago 7820436, Chile.
ACS Synth Biol. 2025 Jun 20;14(6):1920-1930. doi: 10.1021/acssynbio.4c00861. Epub 2025 May 29.
Toehold switches are RNA riboswitches activated by complementary nucleic acid sequences that have shown promise as point-of-care (PoC) molecular diagnostics. However, typical implementations require an additional nucleic-acid-sequence-specific amplification step. Here, we describe a novel toehold switch with a T7-g10 translational enhancer (Tac) that amplifies the expression of the reporter gene regulated by toehold (Toe) sensors. We compare such a TacToe sensor to a previously developed sensor for detecting short RNA sequences from the Zika virus (ZIKV). We demonstrate that this one-step TacToe sensor in a transcription-translation (TxTl) reaction has an improved sensitivity to RNA and a faster time of detection, compared to the conventional toehold. Replacing the ZIVK sequence with a segment of the N-gene of SARS-nCoV, we demonstrate up to a picomolar sensitivity. Qualitatively comparable results are observed in cell lysate-based homemade cell free extract (CFE), demonstrating the robustness and utility of these sensors in low-resource settings.
触发开关是由互补核酸序列激活的RNA核糖开关，已显示出作为即时护理（PoC）分子诊断的潜力。然而，典型的实施方案需要额外的核酸序列特异性扩增步骤。在这里，我们描述了一种新型的带有T7-g10翻译增强子（Tac）的触发开关，它能放大由触发（Toe）传感器调控的报告基因的表达。我们将这种TacToe传感器与先前开发的用于检测寨卡病毒（ZIKV）短RNA序列的传感器进行了比较。我们证明，与传统的触发开关相比，这种在转录-翻译（TxTl）反应中的一步式TacToe传感器对RNA具有更高的灵敏度和更快的检测时间。用严重急性呼吸综合征冠状病毒（SARS-nCoV）N基因的一段序列替换ZIVK序列，我们证明了其高达皮摩尔的灵敏度。在基于细胞裂解物的自制无细胞提取物（CFE）中观察到了定性可比的结果，证明了这些传感器在资源匮乏环境中的稳健性和实用性。
