Subzymes: Regulating DNAzymes for point of care nucleic acid sensing

• Published in: Sensors and actuators. B, Chemical Chemical, 2019-10, Vol.297, p.126704, Article 126704
• Main Authors: Hasick, Nicole J., Ramadas, Radhika, Todd, Alison V.
• Format: Article
• Language: English
• Subjects: Acids; Catalysis; Catalytic activity; Construction costs; Construction materials; Diagnostic systems; DNAzyme; Enzymes; Fluorescence; Isothermal; Oligonucleotides; Point of care; Restoration; Sensors; Signal amplification; Substrates; Target detection
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2019.126704

•The activity of catalytic nucleic acids can be regulated by attachment and release from microparticles.•Surface-bound catalytic nucleic acids can be released and activated in response to exposure to targets.•Released catalytic nucleic acids can serve as universal signal amplifiers, enhancing the rate of DNA detection.•Subzymes serve as novel biosensors, connecting DNA target recognition with enhanced rates of signal output. Rapid, sensitive and affordable nucleic acid sensors that can operate in a point of care (POC) setting are highly desired; however, their availability and implementation remain limited. We report the development of a strategy for building sensors which use novel catalytic nucleic acid structures, herein referred to as ‘Subzymes’. We examine the effectiveness of Subzymes to mediate signal amplification in a universal manner allowing faster rates of detection. We created Subzymes by manufacturing composite oligonucleotides that contain a catalytic nucleic acid component and a substrate component. We demonstrate that the activity of some catalytic DNAzyme components can be inhibited by attaching the Subzymes to micro-particles. Subsequent cleavage of a Subzyme’s internal substrate results in the release and activation of the surface-bound DNAzyme, thus providing a mechanism to control the catalytic activity of the DNAzyme. We demonstrate that released DNAzymes are capable of cleaving fluorescent-labelled reporter substrates to generate a signal, thus confirming the restoration of their catalytic activity. The addition of Subzymes to reactions where the detection of a target is achieved by multi-component nucleic acid enzymes, known as PlexZymes, showed improved target sensitivities in a shorter amount of time (10 pM of target detected in under 60 min), demonstrating rapid detection of nucleic acid targets without the use of protein-enzymes. Subzymes are constructed from low-cost materials and operate under isothermal reaction conditions which are advantageous for on-site diagnostic testing. Thus, Subzymes offer a universal, rapid and affordable tool for nucleic acid sensing, providing new avenues for POC testing.