TY - JOUR
T1 - Molecular imaging of retinoic acids in live cells using single-chain bioluminescence probes
AU - Kim, Sung Bae
AU - Fujii, Rika
AU - Nishihara, Ryo
AU - Bose, Rajendran J.C.
AU - Citterio, Daniel
AU - Suzuki, Koji
AU - Massoud, Tarik F.
AU - Paulmurugan, Ramasamy
N1 - Funding Information:
This work was partly supported by JSPS KAKENHI Grants: 26288088, 15KK0029, 16K14051, 17H01215, and 24225001.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/6/10
Y1 - 2019/6/10
N2 - Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was 10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.
AB - Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was 10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.
KW - Artificial luciferase
KW - Bioluminescence
KW - Protein-protein interactions
KW - Retinoic acid receptor
KW - Single-chain probe
KW - retinoic acids
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U2 - 10.1021/acscombsci.9b00035
DO - 10.1021/acscombsci.9b00035
M3 - Article
C2 - 31034200
AN - SCOPUS:85065848152
SN - 2156-8952
VL - 21
SP - 473
EP - 481
JO - Journal of Combinatorial Chemistry
JF - Journal of Combinatorial Chemistry
IS - 6
ER -