Evaluation of cyclic luciferin as a substrate for luminescence measurements in in vitro and in vivo applications

Abstract

Bioluminescence imaging (BLI) is a powerful tool for cell tracking, monitoring of gene delivery and expression in small laboratory animals. An alternative luciferase (Luc) substrate cyclic luciferin (Cycluc) was recently advanced for BLI applications as providing a stronger, more stable signal at significantly lower doses than the classical substrate D-luciferin (D-Luc) increasing sensitivity of Luc detection 10 to 100 times. We evaluated benefits of using Cycluc in in vivo studies in mice injected with murine adenocarcinoma 4T1 cells expressing Luc, and in single-cell organisms, the oocytes of Xenopus laevis. No significant increase in the efficacy of detection of the luminescent signal was recorded in either of the systems. Kinetic studies demonstrated that Km for Cycluc was 10000 higher, whereas Vmax was 100 lower than that of D-Luc. Cycluc efficiently bound to the active center of luciferase, but its turnover was extremely low, leading to actual inhibition of bioluminescence. This compromises Cycluc as a substrate for measurement of the activity of the wild-type luciferases, still widely used as reporters for in vivo monitoring microorganisms and tumor cells. It may find better applications with the development of in vivo imaging based on the genetically engineered mutant luciferases with different substrate requirements.