Authors

Komatsu, N., Aoki, K., Yamada, M., Yukinaga, H., Fujita, Y., Kamioka, Y., & Matsuda, M.

Affiliations

Laboratory of Bioimaging and Cell Signalling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan, PREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan, Department of Pathology and Biology of Diseases, Graduate School of Medicine, and dInnovative Techno-Hub for Integrated Medical Bio-Imaging, Kyoto University, Kyoto 606-8501, Japan.

Application Area

General Biology

FRET

Abstract

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signalling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the "orientation-dependent" FRET and to render the biosensors completely "distance-dependent" with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.

Extract

… “an LED illumination system (CoolLED precisExcite; Molecular Devices, Sunnyvale, CA)”…

Product Associated Features

pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.

Product Type

pE-100

Journal

Oncogene

Year of Publication

2015

Country of Publication

Japan