Artificial Ribonucleases

Metal-free catalysts for the hydrolysis of RNA derived from 2-aminobenzimidazoles were reported. The most active catalysts, tris derivatives (99 R = H, CC Me) built upon a framework of tris(2-aminoethyl)amine, were shown by fluorescence correlation spectroscopy to aggregate with oligonucleotides. However, at very low concentrations the compounds were still active in the non-aggregated state. Conjugates of the ester (99 CC Me) with antisense oligonucleotides or RNA binding peptides will, it was claimed, be promising candidates as site specific artificial ribonucleases.98... 

The above-mentioned mechanism suggests that positioning the two histidines appropriately would lead to artificial ribonuclease under optimized pH conditions. Figure 6.13 shows an example of an artificial ribonuclease created in this way, which has a cyclodextrin core as the hydrophobic pocket and two histidine residues as catalytic sites. This artificial enzyme catalyzed the second step of the phosphodiester cleavage. The hydrophobic part of the cyclic phosphodiester (substrate) was accommodated into the core of the cyclodextrin and the phosphodiester was exposed between the two histidines. The water molecule was activated through proton removal (performed by the neutral histidine, left), and the activated water performed a nucleophilic attack on the phosphate atom. The protonated histidine (right) assisted this nucleophilic attack by protonating of the phosphodiester. Because of the cooperation between... 

Other 2 -0-modifications include phenanthroline linked oligonucleotides (39) which have been prepared as artificial ribonucleases. " When targeted towards complementary RNA in the presence of zinc ions, cleavage of the RNA was achieved, albeit with low selectivity. ODNs containing 2 -0-p-D-ribofuranosyladenosine were prepared and used as modified primers in RNA-templated DNA synthesis catalysed by HIV RT. It was shown that the additional 2 -0-ribofuranosyl group within the first seven positions of the 3 -end of the primer prevents its elongation. ... 

By the covalent addition of two imidazole groups to the cyclodextrin torus, artificial ribonucleases have been formed, and these have notable specificity with respect to both substrates and products (Breslow, 1983). Similar accelerations and specificities were found by coupling pyridoxamine covalently to a cyclodextrin to give an artificial transaminase. The accelerations produced by these models are only about 200-fold, but work in progress holds out hope for obtaining far greater velocities (Breslow, 1983). 

In contrast, completely artificial ribonucleases are designed and synthesized according to the functions and properties we need, and thus factors (l)-(3) can be minimized. Furthermore, even non-natural functional groups can be easily incorporated in these man-made catalysts. 

In the second-generation artificial ribonucleases, molecular scissors are incorporated into the inside of DNA strand in order to achieve efficient catalytic turnover. Upon scission, the RNA fragment used for the binding to DNA is divided into two small fiagments. The stability of the RNA/DNA hetero-duplex decreases as the RNA becomes shorter, so that the small RNA fragments are spontaneously removed fixnn the artificial enzymes. 

Fig. 24. Schematic view of sequence-selective RNA scission by artificial ribonucleases involving bulge-forming DNA. The black ribbons show the oligonucleotides used for the artificial enzymes to recognize the target sites.

A series of water-soluble calix[n]arenes (n = 4 - 8) with different conformational abilities were investigated as artificial ribonucleases that hydrolyze cytidine-2, 3 -cyclophosphate at pH 2 and 30°C [56]. Best results were obtained with calix[4]arene 12, which inaeased the rate by 16 times (Scheme 4.9). 

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