Whilst there are a number of reports describing the cytotoxic nature of certain oligodeoxynucleotides, in particular G-rich oligodeoxynucleotides (see Introduction Section), there is little known about the motif or sequence requirements for cytotoxicity. The current study attempted to define further the exact requirements for cytotoxicity and identify proteins involved in binding to these oligodeoxynucleotides, thereby unravelling the mechanisms involved in the eventual cytotoxicity of the molecules.
We determined that the 5′ sequence, the core or the tail of Dz13 alone are insufficient to regenerate the cytotoxic activity of Dz13 against HMEC-1 cells, thereby indicating a length and/or sequence requirement. We used the novel strategy of testing pools of oligodeoxynucleotides comprised of random sequences along with the 5′ G-rich elements of Dz13 and Rs6 in various position and length contexts to demonstrate that the G-rich element needs to be present at the 5′ extremity and be followed by a tail component that is preferably composed of mixed nucleotides. A comparison of the active oligodeoxynucleotides, in combination with the 5′-motif reduction experiment, leads us to conclude that the required 5′ sequence is G-rich, composed of 6–9
nt with at least four consecutive purines. The presence of a triple G motif (G-G-G) provides for the greatest potency and the 5′ extremity of the triple G motif needs to be positioned no more than 3
nt from the 5′ end of the ODN. We have previously shown by CD that Dz13 does not assemble into stable secondary structures in vitro
). Nevertheless, substitution of any of the guanosines in the triple G motif of Dz13 abrogated its cytotoxic activity (15
). This suggests that the hydrogen bonding activity of these guanosines does not lead to G-quadruplex formation, but is nevertheless required for cytotoxicity, perhaps by enhancing binding of the oligodeoxynucleotides to intracellular proteins. The tail sequence requirements were intriguing in that most sequences with some degree of ‘complexity’ supported cytotoxic activity, but some individual tail sequences, in particular those free of guanosines, had reduced activity. It is possible that the less complex tail sequences such as the polydA tail are being sequestered by polydA-binding proteins thereby abrogating cytotoxicity. Wu et al
) have previously demonstrated that tail sequence and complexity encourages the multimeric aggregation of the oligodeoxynucleotides, thereby activating TLR9 and cell uptake. However, we have previously demonstrated a need for transfection and ruled out endosomal TLR engagement in the mechanism of action of Dz13 and Rs6 against HMEC-1 cells (15
). Therefore, although multimeric assembly remains a possibility, it is unlikely to relate to uptake and TLR engagement.
We previously compared the activity of Dz13, a prototypic G-rich oligodeoxynucleotide, with that of other published sequences including NT36, an ATM-inducing oligodeoxynucleotide (23
) and 20AG, an oligodeoxynucleotide originally designed to be a triplex-forming inhibitor of K-ras (17
). Both of these oligodeoxynucleotides have purine tracts in the 5′ sequence and NT36 is of comparable length to the active ODNs described in the present study. As shown for Dz13, the potency of the shorter 20AG ODN was length dependent. That is, the cytotoxicity of the AG molecule increased as the length of the molecule increased.
ODNs have been proposed to exert their cytotoxicity through specific interaction with cellular proteins such as nucleolin (24
) and eEF1A (7
). Nucleolin was identified as one of the proteins that bound to Dz13 and Dz13scr. The absence of an effect on HMEC-1 proliferation by the nucleolin-binding oligodeoxynucleotide GRO29A provided further evidence for the lack of direct involvement of nucleolin in Dz13-mediated cytotoxicity. Likewise, a representative from a class of GT-rich ODNs, which reportedly binds eEF1A and is cytotoxic to human T-lymphoblasts (11
), was not cytotoxic to HMEC-1 cells. Furthermore in our experiments, the GT oligodeoxynucleotide did not displace eEF1A from Dz13 capture beads as potently as Dz13 or the other cytotoxic ODNs tested, indicating that it has less binding affinity to the eEF1A found in this cell line. The ODNs described in the present study therefore represent a novel class of potently cytotoxic molecules.
The ability of several of the proteins, including eEF1A to bind competitively to the cytotoxic oligodeoxynucleotides and not be eluted by the non-cytotoxic ODNs in vitro
suggests that the ODNs may competitively bind to proteins within the cells and that this is mechanistically relevant to the cytotoxicity of the molecule. eEF1A is an extremely abundant protein with a multitude of roles including protein synthesis, stress-sensing, apoptosis and cellular proliferation (25
). Scaggiante et al
) recently reported a correlation between eEF1A binding and cytotoxicity of G-rich oligodeoxynucleotides that is independent of secondary structure formation. They also reported that some non-cytotoxic G-rich oligodeoxynucleotides bind eEF1A to a small degree in the absence of competitor (7
). The fact that both Dz13 and Dz13scr were able to bind eEF1A in vitro
and in cells indicates that some of the captured eEF1A is also binding via a non-specific mechanism in HMEC-1 cells. However, the selective release of eEF1A from Dz13 capture-beads with those ODNs that are cytotoxic suggests that there is a relationship between eEF1A binding and cytotoxicity.
eEF1A has been proposed to act as a signalling molecule in proliferating cells through the binding of other nucleic-acid-binding proteins such as ZPR1 (27
) followed by translocation to the nucleus. Once inside the nucleus eEF1A has the capacity to bind DNA, RNA and RNA polymerase and therefore potentially plays a role in transcriptional regulation. Binding of eEF1A to Dz13 was observed in nuclear, cytoplasmic, membrane/organelle and cytoskeletal fractions; cytoplasmic eEF1A eluted most strongly from Dz13 beads. This could reflect a greater concentration of DNA-binding proteins in the nuclear fraction and consequently a greater number of proteins eluting in general or that nuclear eEF1A has a higher affinity for Dz13. It is possible that the higher affinity cytotoxic oligodeoxynucleotides displace eEF1A from nuclear or cytoplasmic protein complexes such as the nuclear cytotoxicity-related complex (CRC; 7), thereby inhibiting the normal functioning of eEF1A in the cell, resulting in a reduction in cellular proliferation and eventually cell death.
Vimentin was also identified in the pull-down assay. Vimentin has been shown to bind to G-rich ODNs, causing the translocation of the vimentin–DNA complex to the nucleus (28
). However, this was not evident in HMEC-1 cells transfected with Dz13. Collectively the protein pull-down experiments indicate that binding of cytotoxic ODNs to eEF1A and other proteins is potentially a key event in the mechanism of action of the molecules.
In conclusion, we have determined that ODNs such as Dz13 and Rs6 which were initially designed as catalytic DNAzymes, belong to a group of cytotoxic G-rich ODNs with novel sequence and length requirements. Although the mechanism is unclear, it is likely to be mediated through binding to eEF1A. These molecules are preferentially active against proliferating cells and therefore constitute part of a new class of potentially useful molecules for the treatment or investigation of diseases characterized by abnormal cell proliferation.