Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum

Abstract

Vibrio anguillarum is a bacterial pathogen that causes serious damage to aquatic fish, and its rapid detection and prevention are critical. DNAzymes are DNA-based catalysts with excellent stability. In this study, in vitro selection of DNAzymes was performed using the crude extracellular matrix (CEM) of V. Anguillarum as the target. Different from previous selections targeting bacterial CEM, this work used an unmodified DNA library, allowing easier adoption of the technology. After seven rounds of selection, a DNAzyme named VAE-2 with high activity and specificity was obtained. It showed the highest activity toward V. Anguillarum among eight types of tested bacterial strains. Polyvalent metal ions are needed for its activity. Protease treatment of the CEM and filtration studies indicated that the target is a protein with a molecular weight between 50 k and 100 k Da. A fluorescent biosensor was designed for V. anguillarum with a detection limit down to 4000 cfu/mL, and detection was demonstrated for real fish tissue and feeding water samples. Being the first work of DNAzyme-based sensing of aquatic bacteria, this study indicates that unmodified DNA can be used for targeting bacterial CEM, and it provides a new framework for developing other RNAcleaving DNAzymes for rapid detection of pathogenic bacteria and water pollution.

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

“…Although all Legionella species have the potential to be pathogenic, L. pneumophila is responsible for the large majority of cases of Legionnaires’ disease [7, 35, 36] . Encouraged by the high selectivity demonstrated by the previously reported DNAzymes for Clostridium difficile and Vibrio anguillarum , [19, 37] we were interested in investigating the specificity of LP1 towards other Legionella species. LP1F5′ was used for these experiments.…”

“…Although all Legionella species have the potential to be pathogenic, L. pneumophila is responsible for the large majority of cases of Legionnaires disease. [7,35,36] Encouraged by the high selectivity demonstrated by the previously reported DNAzymes for Clostridium difficile and Vibrio anguillarum, [19,37] we were interested in investigating the specificity of LP1 towards other Legionella species.L P1F5' was used for these experiments.W ee xamined the following Legionella species: Legionella micdadei, Legionella dumofii, and Legionella longbeachae ( Figure S7). After a1 -h incubation at room temperature,L P1F5' was only cleaved in the presence of L. pneumophila but not by the other three Legionella species ( Figure 4A).…”

Catalytic Nucleic Acids: Biochemistry, Chemical Biology, Biosensors, and Nanotechnology

“…The fluorophore/quencher not only provides signal, but also likely directly participates in the molecular recognition/ catalysis since the activity is often lost after removing them. Using the same method, Gu et al recently selected an unmodified DNAzyme against an aquatic bacterium, Vibrio anguillarum ( Figure 8D), and the target is also likely to be a protein (Gu et al, 2019). Most of these DNAzymes still require metal ions for catalysis, but they also need another molecule at the same time.…”

Selection and applications of functional nucleic acids for infectious disease detection and prevention

“…a CoV2-RBD-4C aptamer against the receptor-binding domain of the SARS-CoV-2 Spike glycoprotein [2]. b The RNA-cleaving DNAzyme VEA-2 [3]. c The peroxidase DNAzyme PS2.M [4] SELEX usually includes the following steps: a random library of approximately 10 13 -10 15 oligonucleotide sequences is incubated with the target (whole cells, cell debris, or biomarkers) and the binding sequences are collected for amplification followed by increased selection pressure or negative selection to exclude nonspecific binding aptamers.…”

“…After sequencing, the candidate sequences are selected. The target of DNAzymes can be RNA [48], mixture derived from the bacteria [49], potential cell target mixture [50], and crude extracellular matrix (CEM) of bacteria [3]. For instance, Li's group [51] developed a selection method that incubated a DNA library with a bacterial culture supernatant for both counter selection and positive selection.…”