RNA-cleaving DNAzymes

An intermolecular trans) version of the reaction was achieved by identification and subsequent redesign of the most active DNAzyme. This resulted in site-specific cleavage of a 19-nucleotide chimeric substrate by a 38-nucleotide Pb -dependent DNAzyme with a turnover rate of 1 min at 23 °C. However, all-RNA substrates were left untouched by this system. 

In a subsequent study, a similar in vitro selection strategy was applied towards Mg -, Mn -, and Zn -dependent DNAzymes with RNAse activity [32]. Although the reaction with the resultant DNA enzymes proceeded around 17 times slower than with the Pb -based system, focus remained on the Mg -dependent systems, as they might eventually be active under intracellular conditions. Next, a transacting catalyst capable of 17 turnovers of a chimeric substrate within 5 h was developed. The most active clone was suggested to contain a three-stem junction, based on the sequence. This makes the structure more complex than that of its Pb -dependent analog. 

Further derivatization of the 8-17 DNAzyme resulted in catalysts that could be activated [45,46] or switched on and off by light [47,48], or allosterically activated by ATP [9, 49-51] or oligonucleotides [52-55], Many more variations of RNA-cleaving DNAzymes have been reported since the original discovery by Santoro and Joyce, as recently reviewed [56], 

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Santoro and Joyce (218). The last authors cited reported the selection of multipurpose RNA-cleaving DNAzymes with potential applications in molecular biology. The elusive RNA replicases, which should be able to replicate RNA structures including their own to sustain the RNA world hypothesis, are a common target for research a recent review (219) summarized the efforts in this field. 

An RNA aptamer has been selected to activate the carboxylic acid of amino acids that mimics the formation of a mixed phosphate anhydride synthesis of aminoacyl tRNA synthetases. The optimal aptamer requires only Ca + for the reaction, and operates at low pH with Xm 50 mM and /ccat 1.1 min for the activation of leucine. This lends support to the concept of translation in an RNA-based world. An RNA-cleaving DNAzyme has been used to target a chemokine receptor required by HIV-1 for entry into susceptible cells. The DNAzyme was found to be very efficient, and specifically interfered with the fusion of cells that harboured the T-lymphocytotropic HIV-1 envelope. 

RNA-cleaving DNAzymes have found a variety of different applications. Selective targeting of mRNA in vivo, in order to suppress the expression of specific genes that cause disease, is an obvious field of application of deoxyribozymes. Efforts in this direction have been reviewed [57, 58],... 

M -lndependent RNA-Cleaving DNAzymes with Three Modified Nucleosides... 

Pun SH, Tack F, Bellocq NC, Cheng J, Grubbs BH, Jensen GS, Davis ME, Brewster M, Janicot M, Janssens B, Floren W, Bakker A (2004) Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles. Cancer Biol Ther 3 641-650... 

Upper Limits of a Degenerate DNA Synthesis - A Cap on Outcome Catalytic RNA Cleavage by Ribozymes and DNAzymes DNAzymes - Deoxyribozymes M -lndependent RNA-Cleaving DNAs... 

Figure 7 The Iterative selection of an RI IA-cleaving DNAzyme, the first example of which involved a single ribonucleotide linkage such that cleavage would cleanly target only one site, while later examples were selected to cleave an all-RNA target.

Following a very similar selection strategy, Sidorov et selected a self-cleaving DNAzyme that targeted an all-RNA sequence and produced cleavage at two different sites at approximately the same rate (feobs = O MFold, which uses... 

DNAzyme A DNA molecule that contains a catalytic motif that cleaves bound RNA in... 

Since some DNAzymes depend on specific metal ions for activity, they can be employed for the detection of those ions, hi the first example, a previously reported [59] DNAzyme was labeled with a Dabcyl fluorescence quencher at the 3 -end and the corresponding RNA substrate with a TAMRA fluorophore at the 5 -end [60]. Upon addition of Pb " ions, the substrate was cleaved, resulting in dissociation from the DNA enzyme strand. This led to spatial separation of the fluorophore-quencher pair, resulting in fluorescence (Fig. 5). The sensor system was over 80 times more responsive to Pb than to other metal ions, and had a quantifiable detection range of 10 nM to 4 pM. A similar strategy was developed for the detection of Ctf by a DNAzyme that oxidatively cleaves DNA [61]. The system showed a dynamic range of 35 nM to 20 pM and had a metal ion selectivity of a factor of 2000 for Cu over other metal ions. A comparable system was reported for the detection of the uranyl cation (UO/ ), with millionfold selectivity over other metal ions and parts-per-trillion sensitivity. 

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