In Vitro Selection of M<sup>2+</sup>-Independent, Fast-Responding Acidic Deoxyribozymes for Bacterial Detection
Qinbin Zhou, Guangxiao Zhang, Yunping Wu, Qiang Zhang, Yi Liu, Yangyang Chang, Meng Liu
Abstract
We report on the first efforts to isolate acidic RNA-cleaving DNAzymes (aRCDs) from a random-sequence DNA pool by in vitro selection that are activated by a microbe Escherichia coli ( E. coli ), at pH 5.3. Importantly, these E. coli -responsive aRCDs only require monovalent metal ions as cofactors for cleaving a fluorogenic chimeric DNA/RNA substrate. Such characteristics can be used to efficiently protect RCDs from both intrinsic chemical instability and external enzymatic degradation. One remarkable DNAzyme, aRCD-EC1, is specific for E. coli, and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant ( k obs ) of 1.18 min –1, making it the fastest bacteria-responding RCD reported to date. Clinical evaluation of this aRCD-based fluorescent assay using 40 patient urine samples demonstrated a diagnostic sensitivity of 100% and a specificity of 100% at a total analysis time of 50 min without a bacterial culture. This work can expand the repertoire of DNAzymes that are active under nonphysiological conditions, thus facilitating the development of diverse DNAzyme-based biosensors in clinical diagnosis.