UC Riverside

The bioluminescence from the luciferase-luciferin reaction has been extensively used in biological assays for the detection and visualization of analytes and cell viability in vitro and in vivo. Compared to fluorescence, bioluminescence imaging has recently started to explore the engineering of novel pairs of luciferase and luciferin that could broaden the scope of bioluminescence assays. This dissertation describes the work with three luciferin analogues: d2-luciferin, S-luciferin, and F2-luciferin, designed to target: 1. The production of inhibitory by-products. 2. A thiol/disulfide redox bioluminescence probe for the detection and visualization in intact cells. 3. The pH dependence of the bioluminescence reaction. Our results with d2-luciferin showed that the formation of inhibitory by-product, dehydroluciferin, was reduced from 16%, with luciferin, to 8% with d2-luciferin. However, these results did not reflect a higher bioluminescence intensity as was expected from the reduction of dehydroluciferin formation. Work with S-luciferin showed that the substitution of a thiol for the hydroxyl group in luciferin decreases the electron donating capacity needed for bioluminescence. Furthermore, results showed that S-luciferin is a poor substrate of luciferase with a Ki of 0.07 μM. We target the pH dependence of luciferin with F2-luciferin, which has a lower pKA at the 6’-hydroxyl group allowing the molecule to be ionized around pH 7. Our results showed that F2-luciferin is a better chromophore at pH 7 with 75% more fluorescence emission compared to luciferin. As a substrate of luciferase, F2-luciferin showed a bioluminescence intensity optimum at pH 6.5 with a KM of 0.73 μM. However, the bioluminescence emission intensity is only 12% of that of luciferin. Preliminary docking of S-luciferin and F2-luciferin into luciferase showed the potential of in silico experiments that could lead to luciferase engineering. These results suggested that further work is needed in order to create novel luciferase-luciferin pairs that could broaden the scope of bioluminescence imaging. Therefore, our next work focused on studying oxyluciferin analogues before developing luciferin analogues with a desired property. Iodinated oxyluciferin analogues were studied for the production of singlet oxygen. From the analogues studied Me2-I2-oxyluciferin showed the highest production of singlet oxygen. This preliminary work shows that iodinated luciferin analogues have the potential to produce singlet oxygen through the luciferase reaction.