Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes

Conspectus DNA-based enzymes, or DNAzymes, are not known to exist in Nature but can be isolated from random-sequence DNA pools using test tube selection techniques. Since the report of the first DNAzyme in 1994, many catalytic DNA molecules for catalyzing wide-ranging chemical transformations have been isolated and studied. Our laboratory has a keen interest in searching for diverse DNAzymes capable of cleaving RNA-containing substrates, determining their sequence requirements and structural properties, and examining their potential as biosensors. This Account begins with the description of an accidental discovery on the sequence adaptability of a small DNAzyme known as “8-17”, when we performed 16 parallel selections to search for DNAzymes that targeted each and every possible dinucleotide junction of RNA for cleavage. DNAzyme 8-17 dominated all the selection pools targeting purine-containing junctions. In-depth sequence analysis revealed that 8-17 could manifest itself in many sequence options defined by the requirement of four absolutely conserved nucleotides. This study also exposed the fact that 8-17 had poor activity toward pyrimidine–pyrimidine junctions. With this information in hand, we proceeded to the discovery of diverse non-8-17 DNAzymes that exhibited robust catalytic activity under physiological conditions. These DNAzymes were found to universally interact with their substrates through two Watson–Crick binding arms and have a catalytic core of varying length and secondary-structure complexity. RNA-cleaving DNAzymes were also isolated to function at acidic conditions (pH 3–5), and these molecules exhibited intriguing pH profiles, with the highest activity precisely matching the pH used for their selection. Interestingly, these DNAzymes appear to use non-Watson–Crick interactions in defining their structures. More recently, we have embarked on the development of ligand-responsive RNA-cleaving fluorogenic DNAzymes that can recognize specific bacterial pathogens, such as Escherichia coli and Clostridium difficile, using a method that does not require a priori identification of a specific biomarker. Instead, the crude extracellular mixture as a whole is used as the target to drive the DNAzyme isolation. High recognition specificity can be achieved with a double-selection approach in which a DNA library is negatively selected against the cellular mixture prepared from unintended bacteria, followed by positive selection against the same mixture derived from a specific species or strain of bacterial pathogen. Finally, we have shown that DNAzymes’ compatibility with DNA replication can benefit the design of amplification mechanisms that uniquely link the action of RNA-cleaving DNAzymes to rolling circle amplification, an isothermal DNA amplification technique. These methods are well suited for translating the target-binding and cleavage activity of an analyte-activated RNA-cleaving DNAzyme into the production of massive amounts of DNA amplicons to achieve ultrahigh detection sensitivity. Given the high chemical stability of DNA, our ability to discover catalytic DNA sequences by simultaneously evaluating as many as 10 16 different DNA sequences, the accessibility to diverse RNA-cleaving DNAzymes in a single DNA pool, and the availability of methods for designing simple biosensors that incorporate RNA-cleaving DNAzymes, we believe we are moving closer to employing RNA-cleaving DNAzymes for exciting applications, such as point of care diagnostics or field detection of environmental toxins.

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