Design strategies of fluorescent biosensors based on biological macromolecular receptors

Abstract

Fluorescent biosensors to detect the bona fide events of biologically important molecules in living cells are increasingly demanded in the field of molecular cell biology. Recent advances in the development of fluorescent biosensors have made an outstanding contribution to elucidating not only the roles of individual biomolecules, but also the dynamic intracellular relationships between these molecules. However, rational design strategies of fluorescent biosensors are not as mature as they look. An insatiable request for the establishment of a more universal and versatile strategy continues to provide an attractive alternative, so-called modular strategy, which permits facile preparation of biosensors with tailored characteristics by a simple combination of a receptor and a signal transducer. This review describes an overview of the progress in design strategies of fluorescent biosensors, such as auto-fluorescent protein-based biosensors, protein-based biosensors covalently modified with synthetic fluorophores, and signaling aptamers, and highlights the insight into how a given receptor is converted to a fluorescent biosensor. Furthermore, we will demonstrate a significance of the modular strategy for the sensor design.

Keywords: biological macromolecular receptor; chemically modified protein based sensors; design strategy of fluorescent biosensors; genetically encoded fluorescent biosensors; signaling aptamers.

Figure 1.

Schematic illustration shows a concept of ligand sensing by dual AFP-fused FRET-based biosensors. Currently, CFP and YFP mutants are preferentially selected as FRET donor and acceptor, respectively. (a) Intramolecular FRET-based biosensors exploit the protein domains with a large structural change upon the ligand-binding event. (b) Intermolecular FRET-based biosensors accompany the dissociation or association of multiple subunits upon the ligand-binding event. The dissociation-type FRET-based biosensor is only depicted in this figure.

Figure 2.

Schematic illustration shows a fluorescent biosensor for inositol tetrakisphosphate based on the split Btk PH domain-cpGFP conjugate [56]. The original N and C termini are linked with a short peptide linker (orange), and the novel terminal of cpGFP (purple) is fused to the split Btk PH domain (blue). The conformational change of the PH domain induced by the ligand-binding event was transduced to the structural perturbation at the chromophore of conjugated GFP, and then resulted in the ratiometric fluorescence change of cpGFP.

Figure 3.

Schematic illustrations show fluorescent biosensors for (a) maltose [84] and (b) inorganic phosphate [93]. The maltose-binding protein (MBP) and phosphate-binding protein (PBP) change from an apo state (left) to a liganded state (right) by a bending-twisting motion of the N domain (green) and C domain (blue) about the hinge region (cyan). A fluorescent reporter group (orange) to monitor ligand binding has been attached to Asp 95 in MBP or Ala 197 in PBP, where was identified as an allosteric site or a peristeric site, respectively. The residues are indicated in magenta.

Figure 4.

Schematic illustration shows a fluorescent biosensor for Ins (1,3,4,5) P₄ based on the GRP1 PH domain covalently modified with a fluorescein (green) as a reporter probe [77]. The original cysteine residues (cyan) were replaced with other amino acids. The fluorophore was designed to be oriented near the binding pocket. The position labeled by the fluorescein at Glu 82 is indicated in magenta. The local environmental change of the fluorophore induced by the ligand-binding event was transduced to the fluorescence change.

Figure 5.

Schematic illustration shows a screening strategy of a tailor-made RNP fluorescent sensor [17]. Combination of the RNA subunit library of the RNP receptor and several fluorophore-labeled Rev peptide subunits generates combinatorial fluorescent RNP receptor libraries, from which RNP sensors with desired optical and/or binding properties are screened.