Targeting Insulin-like Growth Factor I with 10–23 DNAzymes: 2′-O-Methyl Modifications in the Catalytic Core Enhance mRNA Cleavage

Abstract

Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.

Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

“…7,15 Out of the two, the 10-23 DNAzyme has been widely studied for intracellular mRNA cleavage. 13,[16][17][18] This is probably because of its excellent substrate generality, it cleaves any purine/pyrimidine junctions, 7 which offers tremendous freedom to target many regions of interest within an mRNA.…”

“…For example, when an rAC junction was used, barely any cleavage was observed even with 500 mM Mg 2+ ( Figure S1, ESI). Therefore, it is also true for the [10][11][12][13][14][15][16][17][18][19][20][21][22][23] DNAzyme that its cleavage activity for every junction is not the same.…”

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…Because deoxyribozymes generally exhibit little or no catalysis at the free divalent metal ion concentrations inside cells, efforts with modified deoxyribozymes are intended to improve in vivo catalysis [80–85]. In one instance with otherwise-unmodified DNA, introducing 2′- O -methyl substitutions to improve 10–23 deoxyribozyme stability was accompanied by data supporting in vivo mRNA degradation [100]. However, broadly compelling evidence for in vivo DNA-catalyzed mRNA degradation is currently unavailable.…”

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes

“…However, a majority of work where downregulation of gene expression was studied using inactive mutant Dzs as controls have shown that biological activity of Dzs could be unequivocally ascribed to their intrinsic ability to catalyze RNA cleavage in cells (see, e.g., [12][13][14][15][16][17][18][19][20][21]). Our understanding of the intracellular mechanism(s) of action of an oligonucleotide is crucial for successful nucleic acid drug design, in particular from the point of view of possible off-target effects.…”