DNAzymes (deoxyribozymes) are single-stranded DNA molecules endowed with catalytic activity, obtained by in vitro selection. In the past 25 years, dozens of DNAzymes have been identified and employed for applicative purposes, yet our knowledge of the structural and mechanistic basis of DNA catalysis remains very limited. The RNA-cleaving 8-17 DNAzyme, which depends on divalent metal ions for function, is possibly the most studied catalytic DNA in terms of mechanism. It is very efficient, implying that it adopts a combination of distinct catalytic strategies, but until recently it was uncertain which strategies are at play and how they are implemented. Recently, however, new functional studies and the attainment of high-resolution X-ray structures of an 8-17 construct, have offered a great opportunity for a more detailed understanding of its mechanism. This review examines the functional information gathered on 8-17, in the light of the available crystal structures, pointing out the congruences and possible inconsistencies between the functional and structural data. We will analyze separately three aspects of the DNAzyme function: the structural requirements for catalysis, the role of metal ions and the influence of pH on activity. Ultimately, we will contrast the experimental data with a model for the 8-17 mechanism proposed in the crystallographic study, whereby one specific G residue (G14) acts as a general base and a metal-coordinated water molecule acts as a general acid. Throughout this analysis we will signal the most outstanding mechanistic issues that remain to be addressed, with implications for the broader field of DNA catalysis.
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