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

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