A Novel All-in-one Target-Powered Entropy-Driven Dynamic DNA Networks to Regulate the Activity of CRISPR/AsCas12a for Enhanced DNA Detection

Abstract

Background

The non-enzyme autonomous DNA nanodevices have been developed to detect various analytes through the programmability of Watson-Crick base pairing. Nevertheless, by comparison with enzymatic biosensors, the usage of enzyme-free DNA networks to create biosensors for testing low amounts of targets is still subject to the finite number of cycles. Besides, these biosensors still require the incorporation of other amplification strategies to improve the sensitivity, which complicates the detection workflow and lacks of a uniform compatible system to respond to the target in one pot.

Results

Here, we put forward a novel way for rapid and sensitive DNA diagnostic via EDN (entropy-driven dynamic network) coupling with CRISPR/AsCas12a-powered amplification. In the absence of the target, the autonomous hybridization among the substrate and crRNA is kinetically hindered by enclosing complementary regions, which leads to the loss of the activation function of Cas12a. On the contrary, the target initiates the EDN, reconfiguring the activator strand from a duplex to branch construction, which provides a valid means to adjust the hybridization with crRNA, thereby controlling the indiscriminate collateral cleavage activities of the CRISPR/AsCas12a. Compared with the traditional EDN, synergistic activation between the EDN and the CRISPR catalyst could dramatically enhance the detection signal of the target in one pot. Furthermore, the proposed approach provides universal platforms through the rational functional and structural design of DNA assembly modules.

Significance and Novelty

Overall, this target-triggered EDN switches the activator strand to regulate the activity of AsCas12a (called TERA), which showed more than two orders of magnitude sensitivity than the conventional Cas12a alone assay, resulting in a devisable universal CRISPR sensing platform that favours the fast, robust and one-pot detection of nucleic molecules.