Structure and Reactivity of Metal Complexes Bound to DNA

Citation

Bhattacharya, Pratip K. (2004) Structure and Reactivity of Metal Complexes Bound to DNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ZANY-J082. https://resolver.caltech.edu/CaltechETD:etd-05122004-052751

Abstract

Establishing correlations among structure, dynamics and reactivity is a fundamental problem in biological chemistry. Here, this problem is explored in the context of the design and reactivity of different metallointercalators bound to DNA.

First, the effects of intervening mismatches on DNA structure, dynamics and DNA charge transport reactivity is examined. The π-stacked DNA base pairs mediate charge transport chemistry over long molecular distances in a reaction that is exquisitely sensitive to DNA sequence dependent conformation and dynamics. To examine the long-range charge transport as a function of intervening base mismatches, a series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator, [Ru(phen)(bpy')(dppz)]²⁺ (phen = 1,10 phenanthroline; bpy' = 4-butyric acid-4'-methylbipyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine) linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC ~ GG ~ GT ~ GA > AA > CC ~ TT ~ CA ~ CT. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was then compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from ¹H NMR measurements of the imino proton exchange rates. The exchange kinetics of the imino protons were measured from selective longitudinal relaxation times, and the effect of the mismatch was observed on the base pair lifetime up to a distance of two neighboring base pairs. The overall order of base-pair lifetimes in the selected sequence context of the base pair was as follows: GC > GG > AA > CC > TT. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, guanine damage ratios was found to correlate most closely with base-pair lifetimes. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.

In a related ¹H NMR structural study of the ruthenium intercalator, [Ru(phen)(bpy')(dppz)]²⁺ covalently tethered to a short eightmer DNA duplex, d(ACGAGCAC)•d(GTICTCGT) with a nine carbon linker, the type of construct used in charge transport experiments, a very fast exchange was observed. Comparison of the NOESY data obtained from the NMR study of this system and control samples comprising of the duplex with only linker and the duplex alone, led to the conclusion that the nine carbon linker is positioned between the second and fourth bases from the point of its origin. The absence of any site specificity of the metal complex in the oligonucleotide complicates the structural characterization by NMR study. This led us to conceive of a more general strategy of obtaining structural information of metal complexes that bind non-specifically to DNA based on paramagnetic NMR.

The selective paramagnetic relaxation of oligonucleotide proton resonances of two short self-complementary oligonucleotides; d(GTCGAC)₂ and d(GTGCAC)₂ by Ni(phen)₂(L)²⁺ where L= dipyridophenazine (dppz), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and phenanthrenequinone (phi) was examined to obtain structural insight into the non-covalent binding of these metal complexes to DNA. In the oligonucleotide d(GTCGAC)₂, preferential broadening of the G1H8, G4H8, T2H6, and C3H6 proton resonances was observed with Ni(phen)₂(dppz)²⁺, Ni(phen)₂(dpq)²⁺ and Ni(phen)₂(phi)²⁺. In the case of the sequence d(GTGCAC)₂, where the central two bases are juxtaposed from the previous one, preferential broadening was observed instead for the A5H2 proton resonance. Thus, a subtle change in the sequence of the oligonucleotide can cause significant change in the binding location of the metal complex in the oligonucleotide. Owing to comparable changes for all metal complexes and sequences in broadening of the thymine methyl proton resonances, the switch in preferential broadening was attributed to a change in site location within the oligomer rather than to an alteration of groove location. Therefore, even for DNA-binding complexes of low sequence-specificity, distinct variations in binding as a function of sequence are apparent and can be monitored using paramagnetic probes.

Finally, ¹H NMR spectroscopy was employed to study the binding of [Rh(bpy)₂chrysi]³⁺ (chrysi = 5,6-chrysenquinone diimine), a metal complex which specifically targets mismatches, to a ninemer oligonucleotide d(GCCTCAGGC)₂ containing centrally placed CC mismatch. Evidence supports intercalation by the metal complex within the mismatch site (i) upfield chemical shifts and significant broadening of the chrysi resonances and (ii) an increase in duplex melting temperature in the presence of the metal complex. To simplify the NMR spectra, the Δ isomer of [Rh(d₈₋bpy)₂chrysi]³⁺ was employed in NMR experiments with DNA. A break in the connectivity in the NOE walk is observed between T₄ and C₅, thereby marking the binding site of the metal complex at the CC mismatch. Intermolecular NOE's place the metal complex in the major groove of the oligonucleotide.

Thus through a series of experiments in this thesis, attempts have been made to correlate the structure and dynamics of metal complexes bound to DNA. Truly, metal complexes bound to DNA provide an interesting system to study structure, function and dynamics in a single package.

Thesis Files