Photoactivable Bioluminescent Probes for Imaging Luciferase Activity
Qing Shao, Tingting Jiang, Gang Ren, Zhen Cheng and Bengang Xing
The firefly luciferase (fLuc) has been widely utilized for optical
reporter gene imaging in biochemical assays, cell culture and
living animals. Normally, all the cellular structures exhibit highly
complex spatiotemporal organization. How to precisely track the
dynamic properties of cellular functions and repetitively monitor
the reporter gene expression at a desired time or location in intact
cells, tissues or living animals will be of great importance in many
biomedical applications. Here, we present a set of photoactivable
bioluminescent probes and report the first-time study of real-time
imaging firefly luciferase expression by using photocage
technology in living mice.[1]
(A)
(B)
(C)
(D)
(E)
(F)
Fig. 3 : Fluorescence imaging of C6 glioma cells loaded with (A), (D); D-luciferin
only (25 µM); (B), (E); NPE-luciferin only (25 µM) but no UV excitation; (C), (F);
NPE-luciferin (25 µM) and followed by 1 min UV excitation.
Fig. 1: Illustration of luminescence emission upon light irradiation
To demonstrate proof of concept, we prepared our photocaged
bioluminescent probes by simply masking the 6-hydroxy group of
fLuc substrate, D-luciferin with different cage groups. Their further
inherent fluorescent and bioluminescent properties were
investigated by using fLuc as reporter enzyme in buffer, cell and
living animals (Figure 1).
Fig. 4 : (A) Bioluminescence change of various “caged” luciferin before and after
photolysis the presence of fLuc, ATP and MgCl2. (B) Dependence of
Bioluminescence emission on cell number.
Fig. 5: Imaging of fLuc activity in living mice (n=4). The tumors were implanted in
mice by injection of C6-fLuc cells in the left ear and right shoulder. A). 3 mg of
NPE-luciferin injection without UV excitation; B). 3 mg of NPE-luciferin injection
and 4 min UV excitation of left ear tumor only; C). 3 mg of D-luciferin injection.
Fig. 2: (A) Fluorescence change of caged luciferin upon photolysis; (B) Time
course of integrated fluorescent intensity of caged luciferins (1 µM) upon light
illumination.
Acknowledgement
We gratefully thank Mr. Shao Qing and Miss Jiang Tingting for
their hard working and great contributions.
As expected, all the caged D-Luciferins showed weak
fluorescence signals due to the photoinduced electron transfer
(PET) process. However, upon the brief photolysis, the
photoactivation would switch on fluorescent activity and generate
the strong fluorescent signals (Figure 2). All these photocaged
bioluminescent probes could cross the cell membrane with high
efficiency and therefore were used for fluorescent imaging in living
cells (Figure 3). More importantly, all these stable, specific and
cell-permeable photocaged bioluminescent probes exhibit the
rapid photorelease of D-luciferin and confer robust bioluminescent
signals with minimum background upon brief UV illumination and
thus could be used for real-time imaging the dynamics of firefly
luciferase expression in vitro and living mice (Figure 4 and 5).
Reference
[1] Shao, Q.; Jiang T. T.; Ren, G.; Cheng, Z.; Xing, B. G. Chem.
Comm. 2009, 15, 4028. Hot article in Chem. Comm and
highlighted in Analytica-World and Bionity.
Dr. Xing Bengang is an Assistant Professor in
the Division of Chemistry and Biological
Chemistry. He obtained his Ph.D. from
Nanjing University and joined NTU in 2006.
Email: bengang@ntu.edu.sg