Development and Characterization of Novel Bioluminescence Sources

The overall objective of this project was to discover, design and demonstrate the feasibility of bioluminescent materials for use in marking, tagging, and anti-tamper applications. The results of the project were disseminated in 10 publications in peer reviewed scientific journals that acknowledged the support of AFOSR. A total of 12 funded and non-funded participants including research technicians, undergraduates, a graduate student, and 9 collaborating laboratories contributed to the project. Two major objectives were pursued: (1) to develop efficient firefly luciferase-based sources of near-Infrared (nIR) light; and (2) to develop and characterize new bioluminescence systems. Among the significant accomplishments, the following are highlighted. (1) The development of an efficient biological source of nIR light (emission maximum = 718 nm) that produced minimal visible light. This result was achieved by engineering the bioluminescent system of firefly luciferase-firefly luciferin that normally emits yellow green light. It was necessary to synthesize ~12 substrate analogs and to screen them with luciferase variants produced in our lab. Additional directed evolution trials and a systematic study of other important luciferase-analog systems resulted in the identification of our PLR3/C47T - OH-QLH2 system. (2) We investigated another nIR-producing substrate analog called iLH2. In collaborative studies, we expanded the usefulness of this compound for multianalyte monitoring applications based on its ability of iLH2 to also produce green light with different luciferases. (3) We made progress on the characterization of the bioluminescence system of the North American glow worm bioluminescence system of Orfelia fultonii. High resolution mass spectrometry and NMR results have provided a preliminary structure of the putative blue light emitter.