Converting One In Vitro Selected DNA Molecule Into Two Bacteria‐Responsive DNAzymes by Regulation of Reaction Conditions

ABSTRACT Bacteria‐responsive DNAzymes have been widely used in pathogen detection. We report a “one stone, two birds” strategy that uses one selected DNA molecule to develop two DNAzymes for targeting two bacterial species. Through in vitro selection, we isolated an RNA‐cleaving fluorogenic DNAzyme, brRFD2, that can be activated by Klebsiella pneumoniae (KP) and Escherichia coli (EC) under different reaction conditions. Target assessing experiments indicated that RNases from KP and EC function as the activating components for brRFD2, but they differed in properties: the KP RNase is acid‐tolerant, while the EC RNase is heat‐tolerant. By exploiting these differences, brRFD2 exhibited a strong response to KP at pH 3.6, with a k obs ∼6600‐fold higher than that for EC, while showing high activity to EC at pH 4.5 using pre‐heated bacteria, with a k obs ∼520‐fold higher than that for KP. brRFD2 also demonstrated outstanding performance in the diagnosis of hospital‐acquired pneumonia (HAP) caused by KP and EC, achieving an 88.9% sensitivity and 96.7% specificity for KP and 100% sensitivity and specificity for EC across 48 clinical samples. This work demonstrates that a single DNAzyme sequence can function as two different bacteria‐responsive DNAzymes by simply regulating the reaction conditions.