In Vitro Selection of M-Independent, Fast-Responding Acidic Deoxyribozymes for Bacterial Detection.

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 (), at pH 5.3. Importantly, these -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 and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant () of 1.18 min, 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.