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

It has recently been shown that certain oligodeoxy-nucleotides (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 I-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 (>= 25nt) ODNs in particular, indicates the potential of these molecules for treatment of diseases associated with abnormal cell proliferation.