1. Zdenka Dudová-Vychodilová1, Hana Pivoňková1, and Miroslav Fojta1
Cis-diamminedichloroplatinum(II) as a chemical probe for study of guanine substitution by 7-deazaguanine in PCR amplicons
Zdenka Dudová-Vychodilová1, Hana Pivoňková1, and Miroslav Fojta1
1 Institute of Biophysics and Biochemistry of the AS CR, v.v.i., Královopolská 135, Brno, Czech Republic 5 4
Measurement
2, 3 1 6
introduction
In addition to direct electrochemical detection, we used DNA modification with cis-diamminedichloroplatinum(II) (cisplatin) to measure approximate level of G* incorporated into DNA during PCR. Platinum agents react primarily with the N7 atoms of guanine, forming 1,2-intrastrand and 1,3-intrastrand cross-links as the major adducts [3]. We incubated PCR products containing various levels of 7-deazapurines with cisplatin at rb = 0,05 (rb is the number of platinum atoms per DNA nucleotide). After incubation we applied adsorptive transfer stripping voltammetry (AdTS) squarewave voltammetry (SWV) measurements on Hanging Mercury Drop Electrode (HMDE) and well-developed, symmetrical signal (peak P) was obtained for the efficiently modified DNA [4]. Intensity of the peak P responded to the level of G* residues incorporated in PCR product by remarkable decrease as the amount of G* (which does not react with cisplatin) increased. In case of DNA with A* incorporated, no specific effects were observed.
Electrochemical labelling of genomic DNA sequences is a popular field of study in recent times. In this work we analysed DNA with incorporated 7-deazaguanines (G*) and 7-deazaadenines (A*) in place of standard purine residues. It was shown and confirmed that incorporation of 7-deazaguanines into DNA by polymerase chain reaction (PCR) is facile and that G* deoxynucleotide triphosphate (dNTP) is a good substrate for DNA polymerases [1]. Enzymatic incorporation of A* was shown to be facile too. Both 7-deazapurines have recently been used to construct base-modified dNTPs for DNA labelling [2].
Conclusion
the cisplatination-specific peak P clearly indicate DNA cisplatination at rb = 0,05
intensity of the peak P respond to the level of G* residues incorporated in PCR product - decreased as the amount of G* increased
DNA with incorporated A* interact with cisplatin complexes as unmodified DNA
Experimental
PCR – 500 ng template DNA; 0,5 μM forward and reverse primer; 3 U Pfu DNA polymerase; 125 μM dNTPs
DNA labeling with cisplatin – DNA incubated with cisplatin in 0,01 M NaClO4 in 37 °C for 48 hours, rb = 0,05
– competiting plasmid DNA (linear pBSK) presented during labeling
Electrochemistry – three-electrode setup: Ag/AgCl/3 M KCl electrode as a reference electrode, platinum wire as an auxiliary electrode, HMDE as a working electrode
– AdTSV SWV settings: initial potential, -1,85 V; end potential, 0,0 V; frequency, 200 Hz; amplitude 50 mV; electrolyte, 0.3 M ammonium formate pH 6,97
– acumulation time, 60 s;
PCR as a tool for enzymatic incorporation of dG*TP/dA*TP into DNA molecules
Purification of PCR amplicons
Cisplatination of amplified DNA in the presence of competitor plasmid DNA
Capture of biotinylated DNA on streptavidine magnetic beads
Separation of cisplatinated amplicons using magnetic beads, washing
Denaturation and separation
Biotinylated primer
Cisplatin
Competitor plasmid DNA
Streptavidine magnetic bead
Results
Modified DNA molecules with different levels of incorporated deazapurines (0 %, 50 % and 100 %) were measured by AdTS SWV after interaction with cisplatin complexes (graphs below). Specifity of interaction of cisplatin complexes with modified DNA was studied (column graphs).
Peak P
acknowledgment
Financial support from The Czech Science Foundation GACR (project P206/12/G151) and GA ASCR (IAA ) is gratefully acknowledged.
References
[1] H. Pivonkova, P. Horakova, M. Fojtova and M. Fojta. Analytical Chemistry 82 (2010)
[2] M. Hocek, M. Fojta, Chem. Soc. Rev., 40 (2011)
[3] Y. W. Jung and S. J. Lippard. Journal of Biological Chemistry 278 (2003)
[4] P. Horakova, L. Tesnohlidkova, L. Havran, P. Vidlakova, H. Pivonkova and M. Fojta. Analytical Chemistry 82 (2010)
Dependence of DNA cisplatination degree in the presence of competiting plasmid DNA
Peak P without plasmid competitor
Peak P with plasmid competitor