A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen

Abstract

Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD‐CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD‐CD1 was derived by an in vitro selection approach where a random‐sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross‐reactivities to unintended C. difficile strains and other bacteria species. RFD‐CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain‐specific recognition of bacterial pathogens.

Integrating Deoxyribozymes into Colorimetric Sensing Platforms

“…More efforts are certainly needed to develop diversified DNAzymes for other important analytes and biomarkers. In recent years, there have been noticeable activities in developing DNAzymes for a wide range of metal ions [148,149,150,151], bacterial pathogens [152,153,154] and specific cancer cells [155]. We believe that these and future DNAzymes can be coupled with the colorimetric biosensor designs described in this review to achieve a wide range of applications.…”

In vitro selection of RNA-cleaving DNAzymes for bacterial detection

“…We term this type of DNAzyme "RNA-cleaving fluorogenic DNAzyme", or simply "RFD". Over the past decade, we have isolated many RFDs and examined their catalytic and signaling properties [38,39,[67][68][69][81][82][83][84][85][86][87][88][89][90]. More recently, we began to develop RFDs that that can be activated in the presence of a specific bacterium, such as Escherichia coli and Clostridium difficile [72,80,82,[91][92][93][94][95][96].…”

“…difficile [82]. The in vitro selection experiment was carried out using the CEM derived from a BI/027 strain of C. difficile as the positive selection target, and the CEM prepared from E. coli, B. subtilis and CD630 (a non-BI/027 strain of C. difficile) as the counter-selection target.…”

“…This means that any library with the random-sequence domain longer than 25 nucleotides would be under-sampled. We have used libraries with 40 and 70 random-sequence nucleotides and had success with both [72,82].…”

The Optimization and Characterization of an RNA-Cleaving Fluorogenic DNAzyme Probe for MDA-MB-231 Cell Detection

“…Compared to conventional approaches, the RFD system is also more time-efficient and convenient. Many detection systems with high sensitivity and selectivity based on a RFD probe have been reported in recent years, including detection of Escherichia coli ( E. coli ) [26,31], Clostridium difficile , [32], MDA-MB-231 cells [29], T47D cells [33], etc. Although these special RFDs offer an excellent opportunity for designing biosensor-based DNAzymes, only a few studies optimized the RFD probes after in vitro selection [27,34,35,36,37].…”