Year

2010

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

Electrospray ionisation mass spectrometry (ESI-MS) was used to screen the relative binding affinities of berberine and its derivatives (oSS14, mSS14, pSS14, SS104, BT80 and KG01) for double-stranded (ds) and quadruplex (q) DNA, and to compare this against the intercalating anti-cancer drug, daunomycin. The DNA sequences used in this study were the dsDNA sequences: D1 (CCTCTCTGGACCTTCC), D2 (GCTGCCAAATACCTCC) (only one strand shown) and F10 (TGCTGGACGAAAAAAAAAA/CGTCCAGC), and three quadruplex sequences: the four-stranded Q4 (TTGGGGGT)4, the single-stranded Q1 GGG(TTAGGG)3 and the two-stranded Q2 (GGGGTTTTGGGG)2. Circular dichroism spectroscopy was used to confirm that the qDNA sequences were folded in solution conditions compatible with ESI-MS, and consistent with CD spectra observed in other laboratories. ESI ion mobility mass spectrometry using a mass spectrometer with a travelling wave ion mobility cell, combined with experiments in which solution conditions were altered to destabilise the qDNA structures, provided evidence that the qDNA secondary structure was maintained in the gas phase under the conditions of the ESI-MS experiments.

Berberine had a high affinity for dsDNA and bound with multiple stoichiometries. Berberine also had a relatively high affinity for Q1 and Q4 with lower levels of binding (abundant free DNA was present in mixtures analysed by ESI-MS) to Q2. Daunomycin bound substantially to both ds- and qDNA with a higher affinity for dsDNA. In preliminary work oSS14 showed selectivity for Q4 DNA over dsDNA. The derivatives mSS14, pSS14, SS104 and KG01 retained this selectivity but bound more tightly to Q4 DNA (smaller amounts of free DNA remaining in mixtures). KG01 also had moderate binding affinity towards Q1. BT80 had a high affinity for all the qDNA types and retained substantial selectivity for qDNA over dsDNA. When pSS14 was left to bind to Q4, ions appeared in the mass spectra that were consistent with the formation of qDNA dimers with bound pSS14. The binding of ligands to Q2 increased its stability.

The selectivity of the berberine derivatives, especially BT80, for qDNA over dsDNA may lead to their applications as leads for quadruplex DNA-specific chemotherapeutic (anti-cancer) agents or as molecular probes for qDNA structures in cells.

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