A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage

Abstract

[a] Catalytically efficient, sequence-specific RNA cleavage holds great therapeutic value for selective gene inactivation against viral infection and cancer. Towards that end, SELEX and related combinatorial methods of in vitro selection [1][2][3] have been used to discover RNA-cleaving DNAzymes that have received considerable attention. [4][5][6][7][8] In addition to therapeutic use in catalysing sequence-specific destruction of mRNA for targeted gene deactivation, applications of catalysis to sensing have also been suggested. [4,[9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] Like ribozymes, DNAzymes present a limited chemical repertoire compared to proteins. [27,28] [33] These findings highlight an inextricable link between high concentrations of Mg 2 + and catalytic efficiency. [34,35] By the same token, one must hypothesise that the scarcity of intracellular Mg 2 + limits the efficacy of DNAzymes in vivo.This catalytic shortcoming might be circumvented by selecting for DNAzymes with synthetically appended chemical functionalities that enhance the chemical repertoire of nucleic acid enzymes, which is otherwise impaired by a low physiological concentration of M 2 + . [36][37][38][39] The generation of functionalized nucleic acids for in vitro selection involves the enzymatic polymerization of synthetically functionalized nucleoside triphosphates (dXTPs in which X is any given nucleobase).[40] An abiding interest in this approach is highlighted in numerous studies in which synthetic nucleotides have been successfully incorporated for the discovery of highly functionalized nucleic acids, [41][42][43][44][45] aptamers, [46][47][48][49][50][51] RNA-based enzymes [52,53] and DNAzymes. [54][55][56][57] Our initial attempt at enhancing the chemical repertoire of DNAzymes resulted in the DNAzyme Dz9 25 -11, which required both the imidazole and amine-bearing modified nucleotides 1 and 2 (Scheme 1) for activity.[28] Nevertheless, catalytic properties remained modest: whereas a reasonably fast rate of selfcleavage was observed (k obs~0 .3 min À1 ), the optimum temperature for self cleavage remained depressed at 13 8C; further, although the cis-cleaving DNAzyme was engineered to cleave at a single ribonucleoside linkage with both multiple turnover and high sequence specificity, [58,59] both optimal temperature (25 8C) and k cat (0.03 min À1) also remained rather low. Notably, attempts to reselect 2nd-generation catalysts from either 20 or 40 degenerate positions failed to provide any improvement and instead Dz9 25 -11 and closely related sequence variants embedded within the N40 region were selected.[60] This "null result" led to an important conclusion: modified dNTPs 1 and 2 found in Dz9 25 -11 failed to provide more efficient catalysts, even when selections were attempted from longer libraries composed of 40 nucleotides (N40). Consequently, we hypothesised that further increasing chemical diversity by introducing a third modified nucleotide bearing a cationic guanidinium ion would increase...

Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents

“…29,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66 Early reports with SELEX using DNA libraries with a hydrophobic pentynyl group at the 5-position of deoxyuridine and thrombin as the target resulted in aptamers with quite poor (close to micromolar) affinities. 57 The incorporation of positively charged primary amines through flexible linkers at the 5-position of uracil in the context of DNA 58,61 or RNA 64 has also been shown to be compatible with SELEX and has led to the identification of aptamers against several small molecule targets, some negatively charged 56,58,59,64 and with affinities in the low micromolar to millimolar range (typical for small molecules).…”

DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides

“…The demand for polymerases capable of incorporating unnatural nucleotides is certain to grow as the interest to build modified DNA structures continues, including alternative genetic alphabets (Geyer et al, 2003), highly tagged substrates (Hollenstein et al, 2009), modified backbones (Pinheiro et al, 2012), and other unusual structures (Fa et al, 2004; Leconte et al, 2005; Hirao et al, 2007). …”

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…(A) M 2+ -independent RNA-cleaving Dz10–66 deoxyribozyme, with three kinds of modified nucleotide [84]. The substrate has a single RNA nucleotide embedded within a DNA sequence.…”