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Fundamental mechanisms and biological applications of DNA-mediated charge transport


Augustyn, Katherine Emily (2007) Fundamental mechanisms and biological applications of DNA-mediated charge transport. Dissertation (Ph.D.), California Institute of Technology.


The Pi-stacked array of heterocylic aromatic DNA base pairs provides an intriguing medium for facilitating the transport of migrating charges. The mechanism of hole transport through this dynamic molecule has been extensively investigated using a wide range of techniques. In particular, our group has taken advantage of the octahedral metal complexes of rhodium (III) and ruthenium (II) to probe charge transport reactions through DNA at long range. These intercalating photooxidants, which are extremely well coupled to the DNA ?-stack, can provide us with mechanistic information through a variety of biochemical and spectroscopic techniques. Here we continue to investigate the mechanism of DNA-mediated charge transport on fast time scales using a variety of hole traps and photooxidants and examine this interesting chemistry in a biological context. DNA-mediated charge transport across three different adenine tracts lengths is monitored using a probe interior to the bridge, N6-cyclopropyladenine, CPA. Upon oxidation, the cyclopropylamine-subsituted deoxynucleoside decomposes rapidly, and the efficiency of decomposition can be used as a kinetically fast measurement of hole occupancy. This trap, incorporated serially across the bridge, can be oxidized by a distally bound photooxidant, [Rh(phi)2(bpy’)]3+ (phi = 9,10-phenanthrenequinone diimmine) without significant attenuation in yield over a distance of 5 nm. These results are consistent with complete delocalization across the DNA bridge. Photooxidation of N2-cyclopropylguanine, CPG, within duplex DNA is used to probe DNA charge transport reactions initiated by the covalently bound photooxidants, [Rh(phi)2(bpy’)]3+ and anthraquinone. Duplexes containing the photooxidant separated from the CPG trap by an increasing number of intervening bases are examined in order to probe DNA charge transport reactions with this kinetically fast hole trap as a function of distance and sequence. Charge transport events through sequences containing various length adenine tracts as well as most mixed sequence bridges do not simply decay exponentially nor geometrically as a function of distance. In particular, for variable-length A-tracts, decomposition decreases in a periodic fashion with increasing distance between the photooxidant and the trap; the period is ~4-5 base pairs. Results obtained from charge injection studies using 2-aminopurine as a fluorescent probe have shown a similar periodic distance dependence. These periodicities are not observed in measurements of oxidative DNA damage using double guanine sites as a slow, irreversible hole trap. Thus, CT through DNA must be probed on multiple time scales to provide mechanistic information. These results are consistent with our model for DNA CT through transient delocalized DNA domains defined by sequence-dependent base pair dynamics. While mechanistic investigations are critical for a fundamental understanding of how charges migrate through DNA, it is important to consider the biological consequences of this process. A biological role for DNA-mediated CT has been investigated in the context of the transcription factor, p53, a tumor suppressor protein involved in myriad cellular pathways such as apoptosis and growth arrest. DNA assemblies containing an anthraquinone photooxidant tethered to the 5’ end of sequences containing p53 binding sites were constructed to examine the binding affinity as a function of photooxidation. We demonstrate that through photoinduced DNA-mediated CT, the p53 protein becomes oxidized and exhibits differential binding for various promoter sequence including Gadd45, p21, and Mdm2. Additionally, insertion of a mismatch intervening between the photooxidant and the p53 binding site serves to attenuate this change in binding affinity associated with photooxidation. MALDI-TOF mass spectrometric analysis of p53 tryptic digests following irradiation of the DNA bound protein provides further evidence that a chemical change occurs, consistent with oxidation of a cysteine residue in the DNA binding domain. Dipyridophenazine complexes of ruthenium (II) have been used extensively to spectroscopically investigate DNA-mediated charge transport. A novel tris heteroleptic dipyridophenazine complex of ruthenium (II), [{Ru(phen)(dppz)(bpy’-his)}{Ru(NH3)5}]5+, containing a covalently tethered ruthenium pentaammine quencher coordinated through a bridging histadine has been synthesized and characterized spectroscopically and biochemically in a DNA environment and in organic solvent. Capable of undergoing intramolecular photoinduced electron transfer, the steady-state and time-resolved luminescence measurements indicate that the tethered-quencher complex is quenched relative to the parent complexes [Ru(phen)(dppz)(bpy’]2+ and [Ru(phen)(dppz)(bpy’-his)]2+ in DNA and acetonitrile. Intercalated into guanine containing DNA, [{Ru(phen)(dppz)(bpy’-his)}{Ru(NH3)5}]5+, upon excitation and intramolecular quenching, is capable of injecting charge into the duplex as evidenced by EPR detection of guanine radicals. DNA-mediated charge transport is also evidenced using a kinetically fast cyclopropylamine-substituted base as a hole trap that undergoes irreversible oxidative ring opening on the picosecond time scale. Guanine oxidation is not observed in measurements using guanine radical as a slow, irreversible hole trap indicating that back electron transfer reactions are competitive with hole injection into the duplex. Moreover, transient absorption measurements reveal a novel photophysical reaction pathway for [{Ru(phen)(dppz)(bpy’-his)}{Ru(NH3)5}]5+ in the presence of DNA, competitive with the intermolecular flash-quench process. These results illustrate the remarkable redox chemistry occurring within a bimolecular ruthenium complex intercalated in duplex DNA.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:A-tracts; anthraquinone; cyclopropyladenine; cyclopropylguanine; p53; periodic distance dependence
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Barton, Jacqueline K.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Newman, Dianne K.
  • Barton, Jacqueline K.
  • Collier, C. Patrick
Defense Date:22 May 2007
Record Number:CaltechETD:etd-05282007-225441
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2205
Deposited By: Imported from ETD-db
Deposited On:31 May 2007
Last Modified:26 Dec 2012 02:48

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