Selection and Characterization of an RNA‐Cleaving DNAzyme Activated by Legionella pneumophila

Abstract

Legionella pneumophila is a deadly bacterial pathogen that has caused numerous Legionnaires’ disease outbreaks, where cooling towers were the most common source of exposure. Bacterial culturing is used for L. pneumophila detection, but this method takes approximately 10 days to complete. In this work, an RNA‐cleaving fluorogenic DNAzyme, named LP1, was isolated. Extensive characterization revealed that LP1 is reactive with multiple infectious isolates of L. pneumophila but inactive with 25 other common bacterial species. LP1 is likely activated by a protein target, capable of generating a detectable signal in the presence of as few as 10 colony‐forming units of L. pneumophila, and able to maintain its activity in cooling tower water from diverse sources. Given that similar DNAzymes have been incorporated into many sensitive assays for bacterial detection, LP1 holds the potential for the development of biosensors for monitoring the contamination of L. pneumophila in exposure sources.

A Syringe‐Based DNAzyme Sensor for Bacterial Detection

“…More importantly, F‐EC1 retained the recognition specificity, as showed by its lack of cleavage in the presence of Bacillus subtilis as the control bacteria (more specificity tests will be presented later). The increased activity was attributed to the removal of the F and Q moieties originally located near the cleavage site, as the same observation was made with RFD‐LP1, a DNAzyme derived for the recognition of the waterborne bacterial pathogen Legionella pneumophila [5f] …”

Recent Advances on DNAzyme‐Based Sensing

“…This is the first demonstration of a DNAzyme can be selectively activated by a specific strain of pathogenic bacteria. The Li group subsequently selected a series of RFDs for other pathogenic bacteria, such as DHp3T4 DNAzyme for Helicobacter pylori , [24] LP1 for Legionella pneumophila [25] . Other groups also successfully selected several DNAzymes for pathogenic bacteria.…”

A highly sensitive and selective fluoride sensor based on a riboswitch-regulated transcription coupled with CRISPR-Cas13a tandem reaction

“…Functional nucleic acids, 1,2 such as aptamers, [3][4][5][6][7] riboswitches, 8,9 ribozymes, [10][11][12][13] and DNAzymes, [14][15][16][17][18] have emerged as a major class of sensors for on-site and real-time detections in applications such as environmental monitoring 19,20 and point-of-care diagnostics [21][22][23][24] of a wide range of targets with high selectivity. [25][26][27][28][29][30] Despite the progress, these functional nucleic acid sensors work well mainly for positively charged or neutral molecules. Although anions are important analytes, 31 only one nucleic acid sensor has been reported for chloride imaging, which utilizes a nucleic acid strand conjugated with an anion-specic organic molecule for sensing.…”