Accelerating Cell-Free Biosensors by Entropy-Driven Assembly of Transcription Templates

摘要

Dueto its high efficiency and selectivity, cell-free biosynthesishas found broad utility in the fields of bioproduction, environmentmonitoring, and disease diagnostics. However, the practical applicationis limited by its low productivity. Here, we introduce the entropy-drivenassembly of transcription templates as dynamic amplifying modulesto accelerate the cell-free transcription process. The catalytic DNAcircuit with high sensitivity and enzyme-free format contributes tothe production of large amounts of transcription templates, drasticallyaccelerating the as-designed cell-free transcription system withoutinterference from multiple enzymes. The proposed approach was successfullyapplied to the ultrasensitive detection of SARS-CoV-2, improving thesensitivity by 3 orders of magnitude. Thanks to the high programmabilityand diverse light-up RNA pairs, the method can be adapted to multiplexingdetection, successfully demonstrated by the analysis of two differentsites of the SARS-CoV-2 gene in parallel. Further, the flexibilityof the entropy-driven circuit enables a dynamic responding range bytuning the circuit layers, which is beneficial for responding to targetswith different concentration ranges. The strategy was also appliedto the analysis of clinical samples, providing an alternative forsensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

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