(Adapted from the Applicant's Abstract). The goal is to develop a luminescent bacterial luciferase system for the nonradioactive detection of nucleic acids. Bacterial luciferase from Vibrio harveyi is an enzyme that catalyzes the reaction of reduced flavin mononucleotide (FMNH2), molecular oxygen, and a long-chain aliphatic aldehyde to yield the oxidize flavin mononucleotide (FMN), the corresponding long-chain carboxylic acid, and light. The investigators have designed an analogue of FMN that consists of a 2'- deoxyuridine molecule covalently bound to a flavin mononucleotide through a polymethylene linker arm. This analogue has the following desirable properties: 1) it can be easily prepared using existing methodologies; 2) it can be conveniently linked to a oligonucleotide; and most importantly 3) the analogue (in its reduced form) should be an effective substrate for luciferase. This technology has applications in DNA sequencing and nucleic acid hybridization procedures. This flavin-luciferase system is expected to be comparable to existing non radioactive DNA detection methods in sensitivity, yet superior in terms of the resolution obtained. In this method the luminescent species is fixed to the oligonucleotide, while in other methods the luminescent species is released from the oligonucleotide at a high turnover number. This rapid turnover of luminescent molecules compromises the resolution obtained.
Thesaurus Terms:bioassay, chemical synthesis, flavin mononucleotide, luciferin monooxygenase, method development, nucleotide analog analytical method, nucleic acidNational Institute of General Medical Sciences (NIGMS)
