Citrate inhibition of cisplatin reaction with DNA studied using fluorescently labeled oligonucleotides: implication for selectivity towards guanine

Abstract

The reaction between cisplatin and DNA is conveniently studied using fluorescently labeled oligonucleotides and gel electrophoresis; as an example of application, the inhibition of this reaction by citrate is demonstrated, which might 10 increase selectivity of cisplatin towards guanine over adenine.Cisplatin is one of the most successful and important anticancer drugs. [1][2][3][4] It is generally accepted that DNA is the molecular target of cisplatin, forming intrastrand crosslinked guanines, 1 although the exact mechanism is still under 15 debate. 5 A lot has already been learned about the reaction between DNA and cisplatin. 6 The difference in the Clconcentration outside a cell (~100 mM) and inside (~4-12 mM) might facilitate dissociation of Cl -and adding water inside the cell. 1 The aquated product is trapped in the cell to 20 react with various nucleophilic species including DNA. 7,8 Mechanisms related to electron transfer have also been proposed. 9 Due to the lack of appropriate analytical tools to follow cisplatin inside live cells, 5 most studies were carried out in simple buffers. The cellular environment, however, is 25 much more complex containing numerous small molecules, nucleic acids and proteins that compete for cisplatin binding. 10,11 The cisplatin concentration inside cells is estimated to be just nanomolar to low micromolar. 5 Many cellular compounds can tightly bind to cisplatin, leaving little 30 free cisplatin for DNA binding. Examples of such competitors include sulfer containing proteins, 12,13 glutathione (GSH), 14,15 and even inorganic anions. 16 Before cisplatin can react with DNA, it has to be released from these competing ligands. 17 Many an initial test, we employed FAM-labeled 15-mer DNA homopolymers. The DNAs were mixed with increasing concentrations of cisplain for 16 h and the samples were then loaded into a non-denaturing polyacrylamide gel. A gradual shift of the FAM-A15 band with reduced mobility was 70 observed with increasing cisplatin concentration ( Figure 1A), suggesting reaction between this DNA and cisplatin. The Pt-DNA adduct did not migrate as a single band, suggesting the presence of a broad range of products, possibly due to different levels and positions of platination. Fluorescence 75 quenching was also observed, especially at high Pt concentrations. On the other hand, no shift was observed with FAM-T15 and its fluorescence just dropped in intensity with increasing cisplatin concentration ( Figure 1B). Reactions also occurred with FAM-C15 and its product distribution pattern 80 was quite different, where discrete bands were observed at low Pt concentrations and the gel smeared at high Pt concentrations ( Figure 1C). Finally, FAM-G15 showed slightly smeared gel even for the initial free DNA, possibly due to its tendency to form various secondary structures such as inter- 85 and intra-molecular quadruplexes ( Figure 1D). Mass spectrum of FAM-G15 showed a few high molecular weight species, consistent with the smeared gel ( Figure S1, ESI). Addition of cispla...

Metal Sensing by DNA

“…For example, nitrogen and sulfur based ligands are very difficult to dissociate once they coordinate with Pt. Even relatively weak oxygen-based ligands, such as citrate, can effectively impede the association between cisplatin and DNA . Since Pt binds DNA tightly, this binding gives rise to metalated DNA.…”

“…Even relatively weak oxygen-based ligands, such as citrate, can effectively impede the association between cisplatin and DNA. 496 Since Pt binds DNA tightly, this binding gives rise to metalated DNA. Platinated DNA carries a positive charge at each binding site, and it has been used to increase the binding affinity between DNA and graphene oxide (GO).…”

Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

“…The Pt-DNA adducts migrate more slowly in the presence of higher concentration of cisplatin, suggesting that more cisplatin molecules are associated with the DNA. 31 We emphasize that the cisplatin adduct is stable enough to survive the denaturing gel conditions (e.g. 8 M urea and strong electric eld).…”

A platinum shell for ultraslow ligand exchange: unmodified DNA adsorbing more stably on platinum than thiol and dithiol on gold

“…13 Citrate is a good ligand for Pt, 14 allowing charge stabilization. Dynamic light scattering (DLS) indicated an average hydrodynamic size of ~5 nm ( Figure 1A) and this was confirmed by TEM (inset).…”