The interaction between short oligodeoxynucleotides of specific sequence with cisplatin, cis-Pt(NH₃)₂Cl₂, has been examined under single stranded and duplexing conditions by ¹H nuclear magnetic resonance (NMR) spectroscopy. The oligonucleotides were synthesized by a modified phosphotriester technique adapting procedures initially used in oligoribonucleotide synthesis. The oligo sequences were designed to favour the intrastrand cis-Pt(NH₃)₂[d(-GpXₙpG-)-N7(l),N7(n+l)] crosslink, where X was up to three thymine units. Variable pH NMR was used to determine the site of platination on the oligomer. Variable temperature NMR was used to determine changes in base stacking and duplex formation resulting from oligomer platination.
In the short oligomers, the intrastrand cisplatin crosslink formed between terminal guanines resulted in the internal section of the sequence being bulged out of a normal stacking orientation. The chemical shifts of the guanine H-8 resonances indicated that platination caused one of the guanine bases to flip from the normal anti geometry into a more syn-like orientation. This was observed for the three oligomers examined (X=thymidine, n=1,2,3). Variable temperature NMR of exchangeable imino protons in the oligomers with larger loops (n=2,3), indicated that the loop region of the complex was a random coil and that water access to the loop was restricted.
i i i
Longer sequences were examined which contained the cis-Pt(NH₃)₂[d(GpXₙpG)-] complex as well as external nucleotide units, 3' and 5' to the platinated region. Platination disrupted intrastrand base stacking of nucleotides externally adjacent to the platinum complex.
Complementary and partially complementary sequences were examined under duplexing conditions. Stabilities, reflected in the Tₘ of the duplex, of fully complementary hexamer, heptamer and octamer oligomer mixtures were obtained by variable temperature NMR. Changes in duplex stability resulting from oligomer sequence and length along with the nature of the duplex to coil transition were discussed.
Imperfect duplexes, designed to mimic the loop structures formed in platinated oligomers, indicated that the duplex containing one extra core thymine formed a helix resembling the fully complementary hexamer duplex with the extra base stacked out of the helix. With the core of the duplex containing two thymine units and one opposing complementary adenine, both thymines stacked into the helix with the adenine oscillating between the two thymines. When the oligomer mixture contained two extra thymine units with no opposing complements, duplex formation was not observed. A comparison between the duplex to coil transition in normal and imperfect duplexes was discussed.
Similar duplex experiments were carried out using the platinated oligomer sequences. In each case examined, aggregation of the platinated strand, coupled with the oligomer sequences employed contributed to the failure of duplex formation. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22767 |
| Date | 11 1900 |
| Creators | Allore, Brian |
| Contributors | Lock, C. J. L., Neilson, T., Chemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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