DNA-templated catalysis

The first example of DNA-templated metal catalysis was reported by the gronp of Kramer [136]. hi this approach, metal-catalyzed hydrolysis of an ester attached to a DNA template was performed. The catalytic system consisted of three parts a DNA template strand, a ligand for Ctf attached to a PNA strand complementary to half of the template, and an ester substrate connected to a PNA strand complementary to the other half of the DNA template (Fig. 10). Complexation of both PNA strands to the template brings the catalytic center and the substrate in close proximity, and hydrolysis of the ester group is accelerated approximately 150-fold relative to the background hydrolysis rate, which makes it suitable as a detection method for DNA sequences [137]. 

Using an excess of the PNA-bound substrate, turnover numbers of up to 35 could be reached, although the background rate was substantial. Mismatches in the PNA/DNA combinations led to a considerable decrease in reaction rates, especially if the mismatch was near the site where the reaction occurred. This method was used to detect as little as 10 fmol of template DNA from a mixture of four similar DNAs that differed at a single position. 

The first examples required a 2-picolyl ester for efficient cleavage because of its chelation of copper. By introducing a Cu -binding group in the linker between the PNA strand and the target ester, it was shown that (in principle) any ester could be 

A related system using DNA-templated organocatalysis rather than metal catalysis was reported in 2000, with an imidazole group linked to a DNA strand that cleaved an ester moiety on another DNA strand when both were annealed to a template DNA sequence [91]. Many other DNA-templated reactions have been described, mostly not using metal ions for catalysis [140-142], 

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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. 

Fig. 8 Schematic representation of the concepts of DNA-directed catalysis (a) and DNA-templated catalysis (b)...

Roelfes s supramolecular assembly is one of the most efficient enan-tioselective catalysts for aqueous Michael additions. The DNA template approach has also been used for enantioselective Friedel-Crafts reactions in water, with outstanding results in terms of conversion and enantioselectivities (110). All these results confirm the impressive potential of DNA-based enantioselective catalysis. 

DNA-dependent DNA polymerases are responsible for directing the synthesis of new DNA from deoxyribo-nucleotide triphosphates (dNTPs) opposite an existing DNA template, which contains the genetic information critical to an organism s survival. To properly preserve this information, during each round of catalysis, a polymerase must accurately select and catalyze the insertion of a complementary nucleotide (dNTP) substrate, from a pool of four structurally similar molecules, into a nascent DNA strand. Present across all three domains of life, including Archaea, Bacteria, and Eukaryota, polymerases are necessarily and diversely utilized during DNA replication, recombination, repair, and translesion synthesis (TLS). 

Figure 10.16 Deoxyribozymes. An approach to selection of deoxyribozymes that ligate RNA. DNA template is represented by dark grey line. The DNA loop contains random sequence variations to allow for the possibility of catalysis. Step A complementary RNA (yellow) is ligated to a DNA template with a 5 -terminal overhang Step B a second RNA (red) is Watson-Crick base pair associated with the same DNA template but with a 3 -terminal overhang. Ligation reaction is then promoted. Where ligation is possible, the PAGE electrophoretic mobility of the product will differ from the un-ligated situation Step C PCR is then used to determine the DNA sequence(s) responsible for RNA ligation catalysis. Further rounds of maturation are also possible [illustration adapted from Flynn-Charlebois et al., 2003, Fig. IB).

This chapter has presented an overview of applications of DNA in metal ion catalysis. Three general approaches were outlined metal-dependent DNAzymes, DNA-directed and templated catalysis, and DNA-based asymmetric catalysis. 

The silicatein a subunit alone proved sufficient to catalyze the acceleration of TEOS condensation at neutral pH [7], For these experiments, we purified and reconstituted the silicatein a subunit that we produced from a recombinant DNA that we cloned in bacteria — cells which normally would make no silicatein without the introduced recombinant DNA template (Fig. 5). This method allowed us to be sure that the catalysis of silica synthesis we observed was due solely to the silicatein a protein produced from the cloned DNA and purified from the bacteria, since no other proteins from the sponge could be present. 

Catalytic action on replication is the origin of the second factor (x.) in the individual terms of equation (16). RNA molecules are lousy catalysts, otherwise it would be very hard to understand that almost all catalysis in biochemistry is done by proteins. The role of RNA (or DNA) in catalysis through the action of proteins is to be seen in its function as template for translation. In case of Qg RNA the plus strand codes for one of the four subunits of Q -replicase. Positive catalytic feedback of RNA on RNA replication thus occurs via translation. 

Orotic acid readily forms dimers even when irradiated in liquid medium [582, 583]. 5-Bromouracil (5-BrU) in DNA is dehalogenated, rather than forming cyclobutane-type dimers. Such DNA derivatives are more sensitive to ultraviolet irradiation than normal DNAs [584-594], Irradiation of 5-bromo-uracil and derivatives in aqueous medium produces 5,5 -diuracil [590, 591]. However, derivatives such as 3-sbutyl-5-bromo-6-methyluracil have been reported to yield cyclobutane dimers either by irradiation of frozen aqueous solutions, or by catalysis with free radical initiators, such as aluminium chloride, ferric chloride, peroxides or azonitriles [595]. 5-Hydroxymethyluracil is reported to dimerize very slowly in frozen water at 2537 A [596]. The fundamental research in the photochemistry of the nucleic acids, the monomeric bases, and their analogues has stimulated new experiments in certain micro-organisms and approaches in such diverse fields as template coding and genetic recombination [597-616]. 

Protein is formed mainly of polymerised amino-acids. The primary structure, unlike that of synthetic polymers, is non-repetitive and, for its production, requires a chemical template stored in the structure of the DNA molecule. The sizes of proteins vary considerably (in a range of molecular weights from 6000 to 1,000,000). Proteins fulfill many roles within the cell, the most important of which is that of catalysis. Proteins which have catalytic activity are called enzymes whilst other proteins have important roles in storage, transport, protection (antibodies), as chemical messengers (hormones) and in structure 17,, 8). 

DNA polymerases catalyze DNA synthesis in a template-directed manner (Box 16). For most known DNA polymerases a short DNA strand hybridized to the template strand is required to serve as a primer for initiation of DNA synthesis. Nascent DNA synthesis is promoted by DNA polymerases by catalysis of nucleophilic attack of the 3 -hydroxyl group of the 3 -terminal nucleotide of the primer strand on the a-phosphate of an incoming nucleoside triphosphate (dNTP), leading to substitution of pyrophosphate. This phosphoryl transfer step is promoted by two magnesium ions that stabilize a pentacoordinated transition state by complex-ation of the phosphate groups and essential carboxylate moieties in the active site (Figure 4.1.1) [2],... 

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