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

✍ Scribed by Marcel Hollenstein; Christopher J. Hipolito; Curtis H. Lam; David M. Perrin

• Book ID: 101820741
• Publisher: John Wiley and Sons
• Year: 2009
• Tongue: English
• Weight: 322 KB
• Volume: 10
• Category: Article
• ISSN: 1439-4227
• DOI: 10.1002/cbic.200900314

✦ Synopsis

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] Nevertheless, this inherent lack of functionality can be offset by divalent metal cations (M 2 + ). For RNA cleavage, Mg 2 + , or other divalent cations (M 2 + ), afford k cat /K M values that approach the limits of catalytic perfection (10 8 m À1 min À1 ); however, such values are observed only at high concentrations of Mg 2 + (e.g., 10-25 mm). In contrast, at physiological Mg 2 + concentrations (0.2-0.8 mm), [29][30][31][32] even the most efficient of DNAzymes such as Dz10-23 and Dz8-17 exhibit markedly reduced k cat /K M values that fall in the range of 10 2 -10 5 m À1 min À1 . [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 temper-Scheme 1. Chemical structures of 1 (dA im TP), 2 (dU aa TP), 3 (dU ga TP) and 4 (dC aa TP).