Hierarchical Hybridization Chain Reaction for Amplified Signal Output and Cascade DNA Logic Circuits

In this work, we propose a three-layer hierarchical hybridization chain reaction (3L hHCR) composed of ^1stHCR, ^2ndHCR, and ^3rdHCR to achieve robust signal amplification efficiency and broaden the applied range of HCR-based systems. In principle, the execution of superior HCR generates the formation of the initiator (named as ^2ndI or ^3rdI) of the subordinate HCR that relies on the introduction of the target sequence (^1stI). To avoid the high background signal of the 3L hHCR system, a strategy of "splitting reconstruction" was adopted. The initiator of the subordinate HCR was designed as two separate fragments (splitting) that are obtained together (reconstruction) for the motivation of the subordinate HCR after the completion of the superior HCR. The implementation of the entire 3L hHCR system generates significant fluorescence recovery that derives from the impediment of Förster resonance energy transfer between fluorophore and quencher; thus, ultrasensitive detection of ^1stI in the range of 50 pM to 10 nM can be achieved. Surprisingly, when the concentration of ^1stI is lower than 1 nM, the 3L hHCR shows excellent ability to discriminate against various concentrations of ^1stI, which is better than that of the 2L hHCR I system. Due to the hierarchical self-assembly mechanism, the 3L hHCR can also be reliably operated as a cascade AND logic gate with a high specificity and molecular keypad lock with a prompt error-reporting function. Furthermore, the 3L hHCR-based molecular keypad lock also shows potential application in the accurate diagnosis of cancer. The 3 L hHCR shows visionary prospects in biosensing and the fabrication of advanced biocomputing networks.