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