Hart, Jonathan Ross (2006) Synthesis and applications of bulky rhodium(III) intercalators for the recognition of DNA mismatches. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06092006-062410
The recognition of DNA base mismatches is of considerable interest for both the diagnosis and treatment of mismatch repair-deficient cancers. Two new mismatch recognition complexes have been synthesized. The first, [Rh(bpy)2(phzi)]3+ (phzi=benzo[a]phenazine-5,6-quinone diimine), recognizes DNA mismatches with high specificity and affinity, 1 x 107 Mm-1, two orders of magnitude stronger than [Rh(bpy)2(chrysi)]3+ (chrysi=chrysene-5,6-quinone diimine), the parent complex that binds single thermodynamically-destabilized base-mismatch sites in duplex DNA. The second, [Rh(bqdi)2(chrysi)]3+, is able to recognize more stable mismatches such as the G-G mismatch.
These complexes have been applied in a variety of ways. A method has been developed for the discovery of new single nucleotide polymorphisms, SNPs, within a sequence of interest amplified from pooled genomic DNA. SNPs are readily detected using these mismatch selective molecules without false positives; allele frequencies as low as 0.05 can be detected.
Upon photoexcitation, the rhodium(III) diimine complexes cleave DNA by hydrogen atom abstraction from the sugar to yield 3'-phosphate terminated DNA that is inactive for enzymatic modification. This 3'-phosphate can be removed using T4-polynucleotide kinase opening up the possibility of enzymatic modification at the site of rhodium cleavage. The cleavage site can be fluorescently labeled. Terminal transferase can also be used to attach a homopolymer tail tagging the damage site, allowing the amplification of the DNA up to the damaged site.
This assay can also be employed towards the development of early cancer diagnostics. Some cancers are deficient in the repair of DNA base mismatches. As a consequence, these cells have an increased number of mismatches within their genome. These mismatches in extracted genomic DNA were cleaved using mismatch-specific rhodium complexes. The cleavage sites were labeled with radioactivity, allowing the number of mismatch sites to be quantitated. A significant number of sites were cleaved in the mismatch repair deficient DU145 cell line, 1 base/3000 bp, while no sites were cleaved in the mismatch repair proficient cell line SW620. This method may present a new method for the detection of mismatch repair deficiency.
These mismatch-specific complexes also are shown to have an antiproliferative effect on mismatch repair deficient cell lines. Mismatch repair deficiency is a contributing factor in both hereditary and sporadic human cancers. Both [Rh(bpy)2(chrysi)]Cl3 and [Rh(bpy)2(phzi)]Cl3 show a stronger antiproliferative effect against MMR deficient cells than proficient cells. Effects of stereoisomers, incubation time, and UV irradiation are also demonstrated.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||DNA intercalators; DNA mismatch recognition; HNPCC; SNP discovery|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||24 May 2006|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||09 Jun 2006|
|Last Modified:||26 Dec 2012 02:52|
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