Chapter 6 Small molecule‐based FRET probes

• Published in: Laboratory Techniques in Biochemistry and Molecular Biology Vol. 33; pp. 225 - 288
• Main Authors: Correa, Amanda Cobos, Schultz, Carsten
• Format: Book Chapter
• Language: English
• Published: Elsevier Science & Technology 2009
• ISBN: 9780080549583; 0080549586
• ISSN: 0075-7535; 2589-2789
• DOI: 10.1016/S0075-7535(08)00006-5

The chapter describes suitable fluorophores and building blocks for the design of Förster resonance energy transfer (FRET) probes. In examples, the strategy and the application of small molecule-based FRET probes for the detection of enzymes involved in bond formation and hydrolysis including proteases and lipases, protein–substrate interaction, as well as sensors based on conformational and environmental changes are discussed. This chapter focuses on sensors and reporters based on small molecules, specifically on those bearing non-protein fluorophores. The main building blocks and the different strategies for probe design are described in the chapter. The success of the probe for a specific application will depend on the selection of the right fluorophores and for this reason the most commonly used types of fluorophores and quenchers are overviewed. The optical properties and some of the synthetic needs to prepare fluorescent sensors are discussed. The chapter discusses potential technical problems in their application. A significant number of fluorogenic sensors are based purely on an increase or decrease of fluorescence. The introduction of FRET pairs with improved properties allows the generation of probes suitable for the measurement of spatiotemporal activities in living cells and in vivo.