DNAzymes

The very special properties of DNA, one of the icons of modem science, make it one of the most versatile molecules in chemistry. In nature, it serves as the carrier of genetic information and as such is one of the cornerstones of life [1]. In vitro, a very diverse set of applications have been explored, ranging from programmable building blocks in bionanotechnology [2] to scaffolds for catalysis. In this review, we will focus on this last aspect, with a particular emphasis on metal catalysis. Three approaches will be discussed DNAzymes, DNA-templated catalysis, and DNA-based asymmetric catalysis (Fig. 1). Artificial DNA-metal base pairing [3] will not be covered, as no catalysis using these systems has been reported to date. 

The discovery [4, 5] of naturally occurring ribozymes - RNA molecules with catalytic activity - made it clear that proteins are not the only biopolymers capable of catalysis in nature. At present, a large number of natural RNAzymes are known. 

Although no catalytically active DNAs are known in nature, the chemical similarity between DNA and RNA led to the fundamental scientific question whether DNA could catalyze reactions. Furthermore, from a practical point of view, DNA has several advantages over RNA. These include a higher chemical stability and its availability at low cost by means of automated DNA synthesis. 

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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... 

Sioud, M. and Iversen, R 2005. Ribozymes, DNAzymes and small interfering RNAs as therapeutics. Current... 

A rather new approach for detecting metal ions with very high sensitivity and selectivity utilizes DNAzymes. DNAzymes are a special class of enzymes formed from DNA nucleotides. Compared to proteins and ribozymes, they are more stable, structurally simpler, and therefore cheaper. As DNAzymes often require metal ion cofactors, they are interesting sensing platforms for these metal ions [149]. 

Fig. 23 (a) DNAzyme-based sensor design with two dabcyl quenchers and a FAM fluorophore (top) and mechanism of operation (bottom), (b) Fluorescence response before and after complete cleavage through Pb2+ inset contains the corresponding image of the DNAzyme probe in the absence (left) and presence of Pb2+ (after 2 min of reaction time, right). (Reprinted with permission from [150]. Copyright 2003 American Chemical Society)... 

The preparation of DNAzyme sensors is usually done in a combinatorial way by producing a random set of DNA strands and selecting the analyte-responsive strands on an affinity column followed by elimination of strands that are sensitive to a broader group of metal ions. In case of success, a DNAzyme with high selectivity and sensitivity is obtained after several iterative cycles. DNAzymes have been found by this process for Cu2+, Pb2+, and U02+ [151-153]. 

Liu J, Lu Y (2003) Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers. Anal Chem 75 6666-6672... 

Liu J, Lu Y (2007) A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivity. J Am Chem Soc 129 9838-9839... 

Yim TJ, Liu JW, Lu Y, Kane RS, Dordick JS (2005) Highly active and stable DNAzyme - Carbon nanotube hybrids. Journal of the American Chemical Society 127 12200-12201. 

The controlled aggregation of Au NPs stabilized with nucleic acids has been used as an optical probe for the detection of Pb2+ ions or adenosine by DNAzymes [201]. 

Using whole-body imaging techniques, Cy3-labeled DNAzymes were seen associating with the actin cytoskeleton. The highest levels of sustained fluorescence... 

Ribozyme cleavage DNAzyme cleavage RNase H cleavage... 

Cis-ribozyme S Trans-ribozyme 0, . f. . DNAzyme muITTtOtTTI mT Tfflm Chimeric 2 -0-me RNA/DNA targeting oligo r y c llllllllffllllllllll Illlllllfflilllllll... 

RNA can be extended with (a) a sequence that encodes one of small ribozymes (Ferre-D Amare and Doudna, 1996 Price el al., 1995) (b) a recognition sequence for a DNAzyme (Santoro and Joyce, 1997) or (c) with a sequence complementary to a DNA oligo with subsequent cleavage of the hybrid by RNase H (Stone el al., 2007, see Akiyama and Stone, Chapter 2, this volume). 

Small ribozymes and DNAzymes leave 2 —3 cyclic phosphate at the site of cleavage, and this group must be removed before the ligation. This task can be accomplished by treating cleaved products with polynucleotide kinase (PNK) in the absence of ATP (Schurer et al., 2002). 

Deoxyribozymes (also called DNA enzymes or DNAzymes) are specific sequences of DNA that have catalytic activity. All currently known deoxyribozymes have been identified by in vitro selection from large random-sequence DNA pools (Joyce, 2004 Silverman, 2009). The catalytic range of DNA encompasses both oligonucleotide and nonoligonucleotide substrates (Baum and Silverman, 2008 Silverman, 2008). This report focuses on deoxyribozymes that are useful for reactions of RNA substrates, especially to assist studies of RNA structure, folding, and catalysis. 

Tack, F., Bakker, A., Maes, S., Dekeyser, N., Braining, M., Elissen-Roman, C., Janicot, M., Brewster, M. et al. (2006) Modified polypropylene inline) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes). Journal of Drug Targeting 14 69-86. 

Tian Y, He Y, Chen Y, Yin P, Mao C (2005), A DNAzyme that walks processively and autonomously along a one-dimensional track. Angew Chem Int Ed 44 4355-4358... 

Roelfes, Feringa, and Kraemer (104,105) developed a similar hybrid supramolecular catalyst, which they named DNAzyme, relating to DNA as the biomacromolecular chiral host. 9-Aminoacridine-modified Cu(II) complexes were employed as metal fragments with high DNA affinities (Figure 29). The acridine unit is likely to intercalate into the double helix of the DNA. The Cu(II) catalytic center is thus brought into close proximity to the DNA, which creates a chiral environment around the Cu(II) site. Stereoinduction can be expected in any transformation mediated by this Cu(II) center. 

Roelfes and coworkers first assessed the catalytic performance of their DNAzyme in asymmetric Diels-Alder reactions of cyclopentadienes with a dienophile that binds to the Cu(II) center through a pyridyl group. The length of the polymethylene spacer and the R substituent proved to be crucial, both for the enantioselectivity as such and also for the sense of stereoinduction (Figure 29). 

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