A Multi‐component All‐DNA Biosensing System Controlled by a DNAzyme

Abstract We report on a programmable all‐DNA biosensing system that centers on the use of a 4‐way junction (4WJ) to transduce a DNAzyme reaction into an amplified signal output. A target acts as a primary input to activate an RNA‐cleaving DNAzyme, which then cleaves an RNA‐containing DNA substrate that is designed to be a component of a 4WJ. The formation of the 4WJ controls the release of a DNA output that becomes an input to initiate catalytic hairpin assembly (CHA), which produces a second DNA output that controls assembly of a split G‐quadruplex as a fluorescence signal generator. The 4WJ can be configured to produce either a turn‐off or turn‐on switch to control the degree of CHA, allowing target concentration to be determined in a quantitative manner. We demonstrate this approach by creating a sensor for E. coli that could detect as low as 50 E. coli cells mL −1 within 85 min and offers an amplified bacterial detection method that does not require a protein enzyme.