Turn-on and Color-changeable Fluorogenic Sensor Created by the 10BASEd-T

6-propionyl-2-(dimethylamino)naphthalene (PRODAN) is a well-known solvatochromic fluorescence reporter which shows both turn-on and color-changing effect when exposed in a hydrophobic environment [1]. Because of its extreme... [ view full abstract ]

6-propionyl-2-(dimethylamino)naphthalene (PRODAN) is a well-known solvatochromic fluorescence reporter which shows both turn-on and color-changing effect when exposed in a hydrophobic environment [1]. Because of its extreme small size with lipophilic/nonionic property, we hypothesize that it can be interacted weakly with various hydrophobic pockets of most target biomacromolecules such as proteins. A conjugation of an appropriate surrounding structure to PRODAN core would strengthen both specificity and affinity to the target along with the solvatochromism, and consequently create a target-specific fluorogenic sensor.
Meanwhile, we have established a library construction system, namely gp10 based-thioetherification (10BASEd-T), by conjugation between artificial molecules as the cores and randomized library peptides as the surroundings [2, 3]. By using this system, PRODAN core was conjugated with randomized peptide library on T7 phage, and selection against a target protein (glutathione-S-transferase; GST) was performed. After several rounds of biopanning, a consensus sequence of NTVSC*HGF (C* represents PRODAN-hybridized Cys) was found. The chemically synthesized hybrid showed intense blue-cyan fluorescence only when GST was present. After the addition of excess amounts glutathione (GSH; a natural ligand of GST), it disappeared and weak yellow fluorescence appeared instead. Detailed results such as obtaining dissociation constant, thermodynamic parameters, and epitope-mapping upon the specific interaction between the hybrid sensor and GST will also be presented [4].

[1] Weber, G., Farris, FJ. (1979) Biochemistry, 18, 776–9.
[2] Fukunaga, K., Hatanaka, T., Ito, Y., Taki, M. (2013) Mol. BioSyst., 9, 2988–91.
[3] Fukunaga, K., Hatanaka, T., Ito, Y., Minami, M, Taki, M. (2014) Chem. Commun., 50, 3921–3; inside cover article.
[4] Taki, M., Inoue, H., Mochizuki, K., Yang, J, Ito, Y. (2016) Anal. Chem., 88, 1096–99 (2016).