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

Due to its high efficiency and selectivity, cell-free biosynthesis has found broad utility in the fields of bioproduction, environment monitoring, and disease diagnostics. However, the practical application is limited by its low productivity. Here, we introduce the entropy-driven assembly of transcription templates as dynamic amplifying modules to accelerate the cell-free transcription process. The catalytic DNA circuit with high sensitivity and enzyme-free format contributes to the production of large amounts of transcription templates, drastically accelerating the as-designed cell-free transcription system without interference from multiple enzymes. The proposed approach was successfully applied to the ultrasensitive detection of SARS-CoV-2, improving the sensitivity by 3 orders of magnitude. Thanks to the high programmability and diverse light-up RNA pairs, the method can be adapted to multiplexing detection, successfully demonstrated by the analysis of two different sites of the SARS-CoV-2 gene in parallel. Further, the flexibility of the entropy-driven circuit enables a dynamic responding range by tuning the circuit layers, which is beneficial for responding to targets with different concentration ranges. The strategy was also applied to the analysis of clinical samples, providing an alternative for sensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

A functional RNA/DNA circuit for one-pot detection of SARS-CoV-2 RNA.

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a functional RNA/DNA circuit. By ingeniously integrating the nucleic acid circuit technology and CRISPR/cas12a system, this method can achieve femtomolar detection of the target RNA in one step and successfully distinguish COVID-19 positive cases from clinical samples, proving its great potential for clinical application.

A simple and rapid method to assay SARS-CoV-2 RNA based on a primer exchange reaction.

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a primer exchange reaction (PER). By ingeniously integrating the PER cascade and CRISPR/cas12a system, this method can achieve convenient detection of the target RNA in 40 min and distinguish a single-base mutation from the target sequence, demonstrating its superior analytical performance.