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Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.
Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes. ACS combinatorial science Kim, S. B., Fujii, R. n., Nishihara, R. n., Bose, R. J., Citterio, D. n., Suzuki, K. n., Massoud, T. F., Paulmurugan, R. n. 2019Abstract
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 a (RARa 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.
View details for PubMedID 31034200