Phase I of the proposed Research Plan will investigate the design and synthesis of catalase-activated chemiluminescent substrates for future use in DNA sequencing with chemiluminescent detection. The viability of DNA sequence imaging using a novel alkaline phosphatase labile dioxetane disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2-tricyclo [3.3.1.1.3,7]decan]- 4-yl]phenyl phosphate (AMPPD), as a chemiluminescent substrate has been successfully demonstrated with significantly faster film exposure times that those obtained in side-by-side comparisons with 32P autoradiography without sacrificing sensitivity or resolution. Increasing catalytic turnover would further improve detection levels in this system. Catalase, the enzyme responsible for peroxide decomposition in mammalian and nonmammalian aerobic cells, has such potential with a turnover rate a thousand times faster than that of alkaline phosphatase. For purposes of designing a catalase-chemiluminescent detection method, we will initially develop three types of catalase-activated substrates. These compounds will be synthesized and investigated for catalase reactivity, manifested as demethylation, decarboxylation or oxygenation. If the expected enzymatic transformation occurs, a moderately stable charge transfer species will be generated which fragments to adamantanone and an excited state aryl fluorophore, subsequently emitting light upon decaying to the ground state. Once suitable catalase-labile chemiluminescent substrates have been obtained, Phase II will focus on incorporating these substrates in a chemiluminescence-based DNA sequencing method.
