Cisplatin-adduct-dependent DNA bending reporter with spacing controls
Biochemical assay · Synthetic 20-base-pair DNA duplexes containing a central GG cisplatin adduct and opposing 4-mercaptobutyl reporter groups; matched non-adducted GG-5, GG-6 and GG-7 controls. [1]
At six-base-pair reporter spacing, cisplatin-adducted DNA formed a reducible disulfide reporter much faster than non-adducted DNA, consistent with bending that brings the reporter groups closer. Five- and seven-base-pair spacing failed to discriminate adducted from non-adducted DNA. This is a defined DNA model, not a cytotoxicity assay or a direct bend-angle measurement.
| Endpoint | Reported result | Reference |
|---|---|---|
| Formation of a DTT-reversible interstrand disulfide reporter product in thiol-tethered DNA; this reporter bond is distinct from the cisplatin intrastrand adduct. | Qualitative HPLC product formation Six-base-pair insert: rapid reporter formation with the cisplatin adduct, extremely slow without it. Five-base-pair insert: both adducted and non-adducted duplexes converted within 30 min. Seven-base-pair insert: neither formed the reporter efficiently. | [1] |
Literature-Reported Treatment Conditions
- Treatment concentration
- Adduct preparation: 360 nmol oligonucleotide with 700 nmol cisplatin in 300 μl water; the purified adduct, rather than free cisplatin, was used in the reporter assay.
- Exposure duration
- Adduct formation: 3 days at 16°C. Reporter time courses: Fig. 3, from 0 h through 1 day, depending on duplex.
- Method
- HPLC-purified cisplatin-modified oligonucleotides annealed to complementary reporter strands; reporter analysis at 23°C under oxygen.
- Vehicle
- Water for cisplatin adduct preparation; reporter buffer: 100 mM NaCl and 10 mM sodium phosphate, pH 7.0.
- Reported limits
- No Adooq lot identity or free-drug cellular potency established; reporter readout does not quantify the platinum cross-link yield or an absolute bend angle.
Methods and source locations
Compound reported in the paper: Cisplatin; Compound label in the paper: cis-Diamminedichloroplatinum(II); DNA adducts labeled Pt-5, Pt-6 and Pt-7
Compound-level biochemical evidence only; no supplier or Adooq lot equivalence inferred.
Materials and Methods: oligonucleotide synthesis (final paragraph) and disulfide-bond analysis; Results: Disulfide bond formation in cisplatin-adducted duplexes; Fig. 3A–G and caption; Discussion (first three paragraphs)
Formation of a DTT-reversible interstrand disulfide reporter product in thiol-tethered DNA; this reporter bond is distinct from the cisplatin intrastrand adduct.: Reversed-phase HPLC under heat-denaturing conditions; DTT reduction identifies the reporter. Experiments repeated three times for reproducibility; no exact yields digitized. — Materials and Methods: oligonucleotide synthesis (final paragraph) and disulfide-bond analysis; Results: Disulfide bond formation in cisplatin-adducted duplexes; Fig. 3A–G and caption; Discussion (first three paragraphs)