Master of Science in Engineering Science (MSES)


Engineering Science (Interdepartmental Program)

Document Type



“Cage” molecules reversibly block the bioactivity of a target substrate molecule by a photolyzable covalent bond formed at a functional site of the target molecule. The attachment of cage molecules to DNA oligodeoxynucleotides (ODNs) to transiently block bioactivity, and site-specific restoration of bioactivity using targeted light exposure, would enable a new method of control for use in gene therapy, molecular/DNA computing, molecular biology, and drug delivery. The reaction of the cage molecule 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) with DNA ODNs in an batch reaction yields a mixture of products with varying degrees of caging. Purification and verification of the hypothesized site of DMNPE attachment are necessary for future applications of this technology to control DNA bioactivity with light. Size exclusion chromatography, high pressure liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), and nuclear magnetic resonance (NMR) were performed on caged DNA samples. Alternatives to manganese dioxide (MnO2) as a DMNPE activator were investigated because MnO2 was found to interfere with NMR. Nickel peroxide (NiO2) was found to be an effective alternative. Increased caging was found to correspond with a broadening and small upfield shifts of 1-D ³¹P NMR resonances. 2-D heteronuclear multiple bond correlation (HMBC) NMR experiments successfully matched previous characterizations of the DMNPE site of attachment on caged ATP, and show crosspeaks between the ribose ring and phosphate moiety of ATP and DNA structures, but did not show a crosspeak between the DMNPE benzyl proton and DNA phosphate moiety. This may be due to bond angle or relaxation effects of the cage adduct. Because no phosphate attachment was discovered, base alkylation was evaluated by reaction of deoxynucleosides and DNA dimers with DMNPE. 2′-deoxynucleosides showed no caging under similar reaction conditions (pH 5.5). DNA dimers dTpT and dApA in those reaction conditions showed a caged product on thin layer chromatography plates, and dGpG and dCpC results also suggested some minimal product formation. Thus, the initial hypothesized site of DMNPE attachment at the phosphate backbone was retained. These results demonstrate useful techniques for future efforts in purification and characterization of caged nucleic acid species.



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Committee Chair

William Todd Monroe