Results: 4

1.
Figure 4

Figure 4. Overall representation of the dual imaging concept for luciferin ligation. From: A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice.

Both luciferin ligation precursors could be caged as sensors for two different biomolecules. Only when both become uncaged D-luciferin or D-aminoluciferin is formed as the result of split luciferin ligation reaction, allowing the production of light by luciferase.

Aurélien Godinat, et al. ACS Chem Biol. ;8(5):10.1021/cb3007314.
2.
Figure 2

Figure 2. Split luciferin ligation reaction in living mice. From: A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice.

(a) Overall schematic of in situ formation of D-luciferin or D-aminoluciferin in living transgenic reporter animals. (b) Observed luminescence from luciferase transgenic mice as a function of time after IP injection of OH-CBT; D-cysteine and OH-CBT in equimolar concentrations (1:1); D-cysteine and OH-CBT in 1:10 ratio; and D-luciferin (all equivalent to 75mg/kg concentration of D-luciferin in 100 μL of PBS). Error bars are ± SD for five measurements. (c) Representative image of mice 15 min post-injection of OH-CBT, OH-CBT + D-cysteine (equimolar concentration), OH-CBT + D-cysteine (10:1 respective concentration ratio), OH-CBT + D-cysteine (1:10 respective concentration ratio) and D-luciferin. (d) Total luminescence over 50 min from resulting bioluminescent signal after IP injection of corresponding reagents (D-cysteine and NH2-CBT in 1:1, 10:1 and 1:10 ratio, D-cysteine and OH-CBT in 1:1, 10:1 and 1:10 ratios as well as D-luciferin). Error bars are ± SD for five measurements.

Aurélien Godinat, et al. ACS Chem Biol. ;8(5):10.1021/cb3007314.
3.
Figure 1

Figure 1. Split luciferin ligation reaction in live cells. From: A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice.

(a) Overall schematic of the split luciferin ligation reaction between D- or L-cysteine and hydroxy- or amino-cyanobenzothiazole derivatives (OH-CBT and NH2-CBT) in various biological environments. (b) Observed bioluminescence produced as a function of time from SKOV3-Luc-D3 live cells, incubated with following reagents: D-cysteine; OH-CBT; OH-CBT plus L-cysteine; OH-CBT plus D-cysteine; and D-luciferin (all at 75 μM in PBS pH=7.4). Error bars are ± SD for three independent measurements. (c) Total luminescence produced in 1 h from live SKOV3-Luc-D3 cells incubated with corresponding reagents, calculated by integrating the area under corresponding kinetic curves in Fig. 1b. SKOV3-Luc-D3 cells were incubated for 1 h with either D-cysteine; OH-CBT; OH-CBT and L-cysteine (added simultaneously); D-luciferin, or OH-CBT and D-cysteine (added simultaneously) at 75 μM in PBS (wells 1 5). Cell first pretreated with D-cysteine for 20 min, followed by wash and 1 h incubation with OH-CBT and cell first pretreated with OH-CBT for 20 min, followed by wash and 1 h incubation with D-cysteine (all at 75 μM in PBS, wells 7-8). Error bars are ± SD for three independent measurements

Aurélien Godinat, et al. ACS Chem Biol. ;8(5):10.1021/cb3007314.
4.
Figure 3

Figure 3. Caspase-3 activity imaging using luciferin ligation reaction in living transgenic reporter mice. From: A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice.

(a) Overall representation of caspase-3 activity imaging with DEVD-(D-Cys) peptide and NH2-CBT in living animals. (b) Test tube assay ofcaspase 3 activity imaging with DEVD-(D-Cys) peptide and NH2-CBT. Total luminescent signal over 2 h from DEVD-(D-Cys) peptide or D-cysteine control (200 μM) after incubation with increasing Caspase 3 concentrations (25, 50 and 100 nM) over 3 h at 37 °C before addition of NH2-CBT (400 μM) followed by 1 h incubation at 37°C and subsequent imaging after addition of luciferase buffer. Error bars are ± SD of three measurements. (c) Total luminescence over 1 h from transgenic reporter mice treated with either PBS (control group) or combination of LPS (100 μg/kg in 50 μL of PBS) and D-GalN (267 mg/kg in 50 μL of PBS). Six hours post-treatment, the animals received IP injections of either DEVD-aminoluciferin (34 mg/kg in 100 μL of PBS) or a combination of DEVD-(D-Cys) peptide (22.6 mg/kg in 100 μL of PBS) and NH2-CBT (6.8 mg/kg in 20 μL of DMSO). Statistical analyses were performed with a two-tailed Student’s t test. **P < 0.01 (n=8 for DEVD-aminoluciferin groups and n=4 for combination of DEVD-(D-Cys) and NH2-CBT reagents). Error bars are ± SD for 8 and 4 measurements respectively. (d) Representative image of mice, 15 min post-injection of DEVD-aminoluciferin or a combination treatment with DEVD-(D-Cys) and NH2-CBT reagents.

Aurélien Godinat, et al. ACS Chem Biol. ;8(5):10.1021/cb3007314.

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