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.

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

“…Organs (lungs, liver, and spleen) of mice bearing respective tumors were excised and immediately after assessed for the presence of luciferase-expressing cells by ex vivo bioluminescent imaging. Earlier, by injecting mice with known numbers of 4Tluc2 cells, we have established a direct correlation between the total photon flux from the injected tissue and the number of injected luciferase-expressing 4T1luc2 cells [73]. The total flux from the organs of mice bearing tumors expressing RT_A significantly exceeded the total flux from the organs of mice bearing tumors formed by the parental 4T1luc2 cells.…”

Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications

“…Recognizing that enzyme/substrate pairs that produce nIR light are of great importance to the continued development and improvement of in vivo BLI methods, we focused on combinations that, with the exception of the quinoline analogs NH 2 -QLH 2 and OH-QLH 2 , had been successfully employed in BLI studies [ 14 , 15 , 16 , 17 , 18 , 19 , 30 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. PLR3 was matched with the quinoline-containing analogs based on the complete in vitro and live cell testing results with the seven Lucs ( Table S1 ).…”