A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice

Abstract

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH(2)-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

Figure 1. Split luciferin ligation reaction in live cells

(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

Figure 2. Split luciferin ligation reaction 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.

Figure 3. Caspase-3 activity imaging using luciferin ligation reaction in living transgenic reporter 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.

Figure 4. Overall representation of the dual imaging concept for luciferin ligation

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.