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Protein-inspired modified DNAzymes: dramatic effects of shortening side-chain length of 8-imidazolyl modified deoxyadenosines in selecting RNaseA mimicking DNAzymes.

The discovery of imidazole/amine-functionalized DNAzymes that efficiently cleave RNA independently of divalent metal cations (M(2+)) and cofactors underscores the importance of expanding the catalytic repertoire with modified nucleosides. Considerable effort has gone into defining polymerase tolerances of various modified dNTPs for synthesizing and amplifying modified DNA. While long linkers are generally found to enhance incorporation and therefore increase sequence space, shorter linkers may reduce the entropic penalty paid for orienting catalytic functionality. Catalytic enhancement ultimately depends on both the functional group and appropriate linkage to the nucleobase. Whether a shorter linker provides enough catalytic enhancement to outweigh the cost of reduced polymerizability can only be determined by the outcome of the selection. Herein, we report the selection of DNAzyme 20-49 (Dz20-49), which depends on amine, guanidine, and imidazole-modified dNTPs. In contrast to previous selections where we used dA(ime)TP (8-(4-imidazolyl)ethylamino-2'-dATP), here we used dA(imm)TP (8-(4-imidazolyl)methylamino-2'-dATP), in which the linker arm is shortened by one methylene group. Although the most active clone, Dz20-49, was absolutely dependent on the incorporation of either dA(imm)p or dA(ime)p, it catalyzed cofactor independent self-cleavage with a rate constant of 3.1 ± 0.3 × 10(-3) min(-1), a value not dissimilar from unmodified catalysts and strikingly inferior to modified catalysts selected with dA(ime)TP. These results demonstrate that very subtle differences in modified nucleotide composition may dramatically effect DNAzyme selection.