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DNAzymes: Enzyme-mimicking DNA architectures

Deoxyribozymes, so-called “DNAzymes”, are functional single-stranded DNA molecules exhibiting catalytic activity (Breaker & Joyce, 1994). They pose a wide range of advantages over their biological counterparts (protein-based enzymes), ranging from thermal stability to commercial availability and ease of manipulation, for which they are exploited across biology, medicine, nanotechnology, and material sciences.

Similar to ribozymes, several DNAzymes have been devised to carry out specific catalytic reactions. For instance, some of them perform enzymatic activity comparable to ribonucleases, RNA ligases, and undertake DNA cleavage. Similarly, other DNAzyme devices can achieve chemical functionalisation of nucleic acids such as DNA phosphorylation, DNA adenylation, DNA deglycosylation, or even result in thymine dimer-photoreversion and porphyrin metallation (Morrison et al., 2018).

For instance, hemin - a cofactor involved in peroxidase reactions - has been shown to bind strongly to external guanines arranged in quadruplex conformations (Travascio et al., 1998). Therefore, these DNA nanostructures display peroxidase-like activity, as first reported in the 1990s. Moreover, DNAzymes are readily interfaced with aptamers due to their nucleic acid nature; in that sense, aptamer-DNAzyme conjugates - in which the recognition elements trigger the generation of an analytical signal - pose a specific, sensitive, and modular assaying platform. In particular, the ease of synthesis and thermal stability makes these devices the ideal candidate for the engineering of a breadth of sensing systems responsive to a range of chemical and molecular cues.

Notably, DNAzymes have also been adopted as constituents within synthetic biological systems proposed in iGEM projects. Team Heidelberg, in 2015, showcased a peroxidase-mimicking moiety to replace the conventionally-used horseradish peroxidase (HRP) enzyme in Western blotting assays. The latter was achieved via coupling the DNAzyme to an aptamer domain with a linker, dubbed “apta-body”, which in tandem pose a functional and modular platform that circumvents the need for secondary antibodies in blotting and ELISA techniques.

Figure 5: DNAzymes. a) A typical G-quadruplex structure. 5’ end coloured dark green and 3’ end coloured light green. b) The HRP-mimicking DNAzyme used by iGEM Heidelberg 2015. Hemin (green) can bind the G-quadruplex and together catalyse the oxidation of substrates, such as TMB, used in western blotting.

References

Breaker, R.R. & Joyce, G.F. (1994) A DNA enzyme that cleaves RNA. Chemistry & Biology 1(4), 223-229. https://doi.org/10.1016/1074-5521(94)90014-0

Morrison, D., Rothenbroker, M., & Li, Y. (2018). DNAzymes: Selected for Applications. Small Methods, 2, 1700319. https://doi.org/10.1002/smtd.201700319

Travascio, P., Li, Y., Sen., D. (1998). DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex. Chemistry & Biology 5(9), 505-517. https://doi.org/10.1016/S1074-5521(98)90006-0