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It has recently been shown that certain oligodeoxynucleotides (ODNs) designed as catalytic DNA molecules (DNAzymes) exhibit potent cytotoxicity independent of RNA-cleavage activity in a number of cell lines. These cytotoxic ODNs all featured a 5' G-rich sequence and induced cell death by a TLR9-independent mechanism. In this study, we examined the sequence and length dependence of ODNs for cytotoxicity. A G-rich sequence at the 5' terminus of the molecule was necessary for cytotoxicity and the potency of ODNs with active 5' sequences was length dependent. Cytotoxicity appeared to be generally independent of 3' sequence composition, although 3' sequences totally lacking G-nucleotides were mostly inactive. Nucleolin, elongation factor 1-alpha (eEF1A) and vimentin were identified as binding to a cytotoxic ODN (Dz13) using protein pull-down assays and LC-MS/MS. Although these proteins have previously been described to bind G-rich ODNs, the binding of eEF1A correlated with cytotoxicity, whereas binding of nucleolin and vimentin did not. Quiescent non-proliferating cells were resistant to cytotoxicity, indicating cytotoxicity may be cell cycle dependent. Although the exact mechanism of cytotoxicity remains unknown, marked potency of the longer (&gt; or =25 nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.

Cytotoxic G-rich oligodeoxynucleotides: putative protein targets and required sequence motif