DNA‐based enzymes, also known as deoxyribozymes or DNAzymes, are single‐stranded DNA molecules with catalytic activity. DNAzymes do not exist in nature but can be isolated from random‐sequence DNA pools using in vitro selection. To date, many DNAzymes that collectively catalyze a diverse range of chemical transformations have been reported. Here, examples of new DNAzymes engineered to mimic some intriguing functions of naturally occurring protein‐based enzymes are discussed. This is followed by discussions of recent examples of a particular class of DNAzymes, known as “RNA‐cleaving DNAzymes”, that have been derived specifically so that their activity is strictly dependent on a given chemical or biological stimulus. Some unique ways to employ ligand‐responsive DNAzymes for the design of bioanalytical assays and biosensors are then highlighted. Being DNA molecules, DNAzymes have proven to be entirely compatible with DNA amplification. Several approaches are then discussed, which relay the activity of an analyte‐activated DNAzyme into the production of massive amounts of DNA amplicons, via “rolling circle amplification”, in biosensing applications designed to deliver very high levels of detection sensitivity.

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.

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.

Structural DNA nanotechnology enables user-prescribed design of DNA nanostructures (DNs) for biological applications, but how DN design determines their bio-distribution and cellular interactions remain poorly understood. One challenge is that current methods for tracking DN fates in situ, including fluorescent-dye labeling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction (HCR) probes to achieve thousandfold signal amplification. We identify cell-type and shape-specific spatiotemporal uptake patterns within 1 minute of uptake and at picomolar DN concentrations, 10,000x lower than field standards. We additionally optimized compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo/vibratome-sections, slice cultures, and whole-mount organoids. Together, origamiFISH enables faithful mapping of DN interactions across subcellular and tissue barriers for guiding the development of DN-based therapeutics.

Structural DNA nanotechnology enables user-prescribed design of DNA nanostructures (DNs) for biological applications, but how DN design determines their bio-distribution and cellular interactions remain poorly understood. One challenge is that current methods for tracking DN fates in situ, including fluorescent-dye labeling, suffer from low sensitivity and dye-induced artifacts. Here we present origamiFISH, a label-free and universal method for single-molecule fluorescence detection of DNA origami nanostructures in cells and tissues. origamiFISH targets pan-DN scaffold sequences with hybridization chain reaction (HCR) probes to achieve thousand-fold signal amplification. We identify cell-type and shape-specific spatiotemporal uptake patterns within 1 minute of uptake and at picomolar DN concentrations, 10,000x lower than field standards. We additionally optimized compatibility with immunofluorescence and tissue clearing to visualize DN distribution within tissue cryo/vibratome-sections, slice cultures, and whole-mount organoids. Together, origamiFISH enables faithful mapping of DN interactions across subcellular and tissue barriers for guiding the development of DN-based therapeutics.