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We report a \u201cone stone, two birds\u201d strategy that uses one selected DNA molecule to develop two DNAzymes for targeting two bacterial species. Through in vitro selection, we isolated an RNA\u2010cleaving fluorogenic DNAzyme, brRFD2, that can be activated by\n                    <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n                    (KP) and\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    (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\u2010tolerant, while the EC RNase is heat\u2010tolerant. By exploiting these differences, brRFD2 exhibited a strong response to KP at pH 3.6, with a\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>obs<\/jats:sub>\n                    \u223c6600\u2010fold higher than that for EC, while showing high activity to EC at pH 4.5 using pre\u2010heated bacteria, with a\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>obs<\/jats:sub>\n                    \u223c520\u2010fold higher than that for KP. brRFD2 also demonstrated outstanding performance in the diagnosis of hospital\u2010acquired 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\u2010responsive DNAzymes by simply regulating the reaction conditions.\n                  <\/jats:p>","DOI":"10.1002\/anie.7611103","type":"journal-article","created":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T16:48:07Z","timestamp":1778863687000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Converting One In Vitro Selected DNA Molecule Into Two Bacteria\u2010Responsive DNAzymes by Regulation of Reaction Conditions"],"prefix":"10.1002","volume":"65","author":[{"given":"Xiaoqian","family":"Li","sequence":"first","affiliation":[{"name":"Dalian POCT Laboratory Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education) School of Environmental Science and Technology Dalian University of Technology  Dalian 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