Translational Enhancer Based Amplification of Toehold Sensors (TacToe) for Improved Sensitivity and Speed of Viral RNA Detection.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-05-29 DOI:10.1021/acssynbio.4c00861

Tanvi Kale, Rudvi Pednekar, Séverine Marianne Cazaux, Valentina Ferrando Letelier, Justin R J Vigar, Fernán Federici, Keith Pardee, Chaitanya A Athale

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引用次数: 0

Abstract

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 Escherichia coli cell lysate-based homemade cell free extract (CFE), demonstrating the robustness and utility of these sensors in low-resource settings.

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基于翻译增强子的脚传感器（TacToe）扩增提高病毒RNA检测的灵敏度和速度。

支点开关是由互补核酸序列激活的RNA核糖开关，已显示出作为点护理（PoC）分子诊断的前景。然而，典型的实现需要额外的核酸序列特异性扩增步骤。在这里，我们描述了一种具有T7-g10翻译增强子（Tac）的新型支点开关，该支点开关可以放大由支点（Toe）传感器调节的报告基因的表达。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，与传统的支点相比，这种转录-翻译（TxTl）反应中的一步TacToe传感器对RNA的灵敏度更高，检测时间更快。用SARS-nCoV的n基因片段替换ZIVK序列，我们证明了高达皮摩尔的灵敏度。在大肠杆菌细胞裂解液为基础的自制无细胞提取物（CFE）中观察到定性可比的结果，证明了这些传感器在低资源环境下的鲁棒性和实用性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.